Technical Field of the Invention

The invention relates to a double-layer biopolymer-based nano/micro carrier system comprising a core layer made of a natural compound and an egg shell membrane protein and a shell layer of silk fibroin and to optimization of the components of this system by changing some of their physical properties.

Background of the Invention (Prior Art)

The difficulties encountered in the application of biological activities of plant based natural compounds are that they have limited solubility, low stability, and in some cases low bioavailability values. Since the conditions encountered during processing of these valuable natural compounds in the food, cosmetic and supplementary food industry cause their biological activity to be lost or reduced, their use is restricted. For example; The trans forms of certain natural compounds (resveratrol, curcumin, catechin, carcetine, gallic acid) which can be used in application fields such as food, cosmetics, dermocosmetics and pharmaceuticals are biologically more active than their cis forms and degraded forms. However, these compounds; due to their low stability, can easily be isomerized and lose their activities by being affected from process conditions and environmental factors such as heat and sunlight. Also, as the solubility of these valuable compounds may be very low (for example, the solubility of trans resveratrol in water), they may have significantly low bioavailability. These high bioavailability decreases and low stability that are observed make it inevitable to increase the stability by means of encapsulation techniques of these compounds that can have such trans-cis conversions.

In the prior art, the stability problem of these compounds is frequently mentioned in studies carried out with natural compounds. Environmental factors such as heat, light and process conditions cause these compounds to lose their activity or cause a decrease in their efficiency and this stability problem is tried to be solved with various encapsulation methods. The encapsulation of bioactive or natural bioactive compounds can be carried out with methods such as; spray drying, liposomes, inclusion encapsulation, co-crystallization, freeze drying, emulsion and nano encapsulation. Some of these methods are not only suitable for large scale productions, but also the application of some methods is not suitable in terms of process conditions and some are not suitable in terms of biological activity.

The methods of encapsulation of natural compounds available in the prior art are as follows:

Spray drying method: Materials such as anthocionins, procyanidins, grape seed extract, apple polyphenol extract and olive leaf extract can be encapsulated with this method. However, the disadvantage of this method is that the stability of the natural compound is affected by high temperature due to the introduction of the product to be dried into the hot air stream during spray drying in order to evaporate the solution.

Microemulsion-based systems: Lipophilic bioactive compounds can be encapsulated by this method. However, the disadvantage of this method is that it requires a large amount of surfactants. Therefore this is a reason that is economically restrictive. In this method; a mixture of triglycerides oil phase, lecithin and nonionic surfactant is used as a surfactant.

Liposome -based systems: Trans resveratrol, a polyphenolic natural compound, can be encapsulated in liposome-based systems using soy lecithin and oleic acid. Different materials are used in the encapsulation of trans-resveratrol with liposomes and necessary yield comparisons are made. The most efficient approach is the extrusion method. In this method, small and stable liposomes are produced with high encapsulation yield and good antioxidant activity.

Freeze-drying method: Couldberrv extract with anthocyanin and hibiscus sabdarijfa 1. extract can be encapsulated. This method; is quite an expensive method because the drying speed is low and it requires high amounts of energy for the vacuuming process.

Inclusion method: Natural compounds such as hesperetin, hesperidine, resveratrol, oleuropein can be encapsulated with this method. Nanoencapsulation method: Natural compounds such as resveratrol, elagic acid, curcumin, quercetin, tea catechin, tannin acid, EGCG, theaflavin can be encapsulated. The compatibility of the materials used in this method is very important. If materials that are not compatible with each other are selected, the method may be insufficient to provide the stability of the natural compound.

Emulsion based systems: It shows thermodynamically unstable colloidal distribution compared to micro emulsion based systems. With this method, natural compounds are encapsulated using various oil carriers and emulsifiers. Among these oils; peanut oil is widely used because of its high solubility characteristic for natural compounds.

Biopolymer-based systems: The advantage of these systems is that they can be made from natural materials such as proteins and polysaccharides and are easier to work on a laboratory scale. However; some commonly used conventional techniques may have problems regarding scale-ups and adaptation to industry. By using biopolymer based systems, high encapsulation and long retention efficiency was obtained. Trans-resveratrol, which is a polyphenolic compound, can be encapsulated with chitosan particles by cross-linking with vanillin.

When all the systems were compared; it has been noted that a system that is sufficient for the encapsulation of natural compounds in the prior art was not available as some systems cannot be applied industrially, some are expensive, and others cause degradation of the structure of the natural compound due to process conditions. Another parameter that makes a difference between systems is retention time and loading capacity. For example; the major challenge in liposome -based systems is that the rate of entrapment of the natural compound between the phospholipid layers cannot be determined. When loading capacity is taken into consideration it is noted that the particles are much lower than the surfactant material and phospholipid content in liposome-based and micro-emulsion-based systems. The spray drying method however is not an efficient method because the stability of the natural compound is affected by high temperature and, when applied in industry, the process conditions and heat treatment applications increase the cost.

The present invention is related to a double layer biopolymer based coaxial nano/micro carrier particle system that is obtained by applying the electro-encapsulation method to silk fibroin and egg shell membrane protein that can be obtained having different properties by pre-treatments carried out to increase the efficiency of natural compounds, to provide protection from environmental factors and to increase the stability by decreasing chemical degradability. At the same time, the silk fibroin shell layer allows the controlled release of the natural compound that encapsulated in the core layer.

Brief Description and Objectives of the Invention

In the invention a biopolymer-based nano/micro carrier particle system and its production method is disclosed.

By means of the present invention, the efficiency of natural compounds is increased, protection from environmental factors is provided and chemical stability is reduced and stability is increased.

In the invention, the egg shell membrane protein and silk fibroin pair which will be used for the first time in literature are used as a carrier particle system. By using soluble egg shell membrane protein in the core and fully biocompatible silk fibroin in the shell in this system, the effects on the natural compound from environmental factors and the stability problem is minimized, and the release profile is controlled.

By means of the electro-encapsulation method used in the present invention, particle size distribution is achieved in a narrow space (close to monodisperse) and the method ensures for the encapsulation products to be homogeneous.

As opposed to conventional electro-encapsulation methods, the use of silk fibroin solution and soluble eggshell proteins obtained from the egg shell membrane by using a coaxial nozzle for encapsulation provides cost advantage. At the same time, the biopolymers used in the invention are completely natural and free from substances that may harm human health.

The electro-encapsulation method used in the invention does not use any heat treatment, which ensures that the stability of the natural compounds is not adversely affected. Additionally, since the soluble eggshell membrane protein is obtained from egg shells which are considered as waste in the industry, it creates value for these wastes by creating an alternative field of application for the egg industry wastes. In this method, unlike other methods, lower bioactive material degradation, obtaining a product in one step, better control of particle size distribution and morphology can be achieved.

Description of the Figures Illustrating the Invention

Figure 1: Schematic representation of the electro-encapsulation method.

Figure 2: Steps of particle formation by electrospraying method.

Figure 3: A and B) The nano/micro carrier particle system of the invention.

Figure 4: Scanning electron microscopy (SEM) images of the encapsulated trans-resveratrol-containing carrier particle system. (Constant core (0.1 ml/h) and constant shell flow rate (0.3mL/h), collector plate distance (10 cm), 5% silk fibroin shell solution, 1.2% eggshell membrane protein core solution) A) 1: 10 (trans-resveratrol: eggshell protein), 12 kV; B) 1: 1 (trans-resveratrol: eggshell protein n), 12 kV; C) 2: 1 (trans-resveratrol: eggshell protein), 12 kV.

Figure 5: FT-IR spectra of the obtained particles, silk fibroin, resveratrol and eggshell membrane protein. Figure 6: Molecular weight distribution of eggshell protein produced by 3-mercaptopropionic acid treatment as measured by HPLC.

Figure 7: Molecular weight distribution of eggshell protein produced by Pepsin enzyme treatment as measured by HPLC.

Figure 8: Molecular weight distribution of silk fibroin solution measured by HPLC.

Figure 9: Molecular weight distribution of silk fibroin solution concentrated with PEG.

Figure 10: Atomic force microscopy (AFM) images of the encapsulated trans-resveratrol-containing carrier particle system. The image A) measured at AFM of the particles sprayed such that the silk and SEP (Soluble eggshell membrane protein) flow rates are O. lml/h, 20kV, d = 6 cm having a SEP (Soluble Eggshell membrane Protein) and trans resveratrol solution in the core section and having a silk fibroin solution at the shell section, and 3D topography B).

Figure 11: Time-dependent release of trans resveratrol from particles.

Description of the components / sections/ parts of the invention

1. Syringe and syringe pump that injects silk fibroin solution into the system.

2. Syringe and syringe pump that injects the natural compound and the egg shell membrane protein solution into the system.

3. External fluid nozzle.

4. Internal fluid nozzle. 5. Collector plate.

6. Voltage supply.

7. Resistor.

8. Collection plate distance adjuster.

9. Light.

10. Particle outer shell.

11. Particle core.

12. Positive electrode.

13. Earth electrode.

14. Coaxial nozzle tip portion.

15. Solutions that are to form a particle to form a conical structure at the end of the nozzle.

16. Dripping of solutions from the nozzle tip.

17. Disintegration of the droplet by means of the electric field effect.

18. Solid particles.

19. Shell portion of the nano/micro particle.

20. Core portion of the nano/micro particle.

21. Core region comprising a natural compound in the soluble eggshell membrane matrix.

22. Protected core shell portion encapsulated with silk fibroin.

23. Spherical nano / micro carrier system formed by an electro-spray method comprising the elements numbered 21 and 22.

Detailed Description of the Invention

In the present invention, a micro/nano-carrier particle system (23) is produced by means of the electro encapsulation method. The micro/nano-carrier particle system (Figure 3B); comprises a core layer (21) formed of an egg shell membrane protein carrying a natural compound and a shell layer (22) formed of silk fibroin.

A method of obtaining the biopolymer-based nano/micro carrier particle system of the present invention comprising the process steps of;

Ensuring the atomization of polymer solutions provided from injectors by a coaxial nozzle using low current and high voltage values, Delivering the internal fluid biopolymeric solution prepared for the core portion (20) of the nanoparticle to the inner part of the coaxial nozzle which is the 4 ^th aspect with the syringe pump which is the 2 ^nd aspect,

Delivering the external fluid biopolymeric solution prepared for the formation of the shell portion (19) of the nanoparticle to the external part of the coaxial nozzle which is the 3 ^rd aspect with the syringe pump which is the 1 ^st aspect,

Collecting of the nano or micro sized particles on the collector plate (5) due to surface tension and the vaporization of the solution therein, after the electric field forces overcome the surface tension forces when the biopolymer solutions received from the inner and external section of the coaxial nozzle formed of the aspects 3 and 4, enter the electric field that is established as a result of a fixed voltage that is applied via the resistor (7) between the positive electrode (12) and the earth electrode (13) of the voltage source (6),

The shrinking of the droplets due to the vaporization of the solution, while they move along (this distance can be adjusted by the collector plate distance adjuster (8)) an electrical field towards the collector plate located at the opposite side, where said droplets (16) have been charged with electricity that has been produced by the creation of a conical structure (15) at the nozzle end of the solutions that are to form the particle that is to be received at the end of the nozzle (14) with electrospraying and as a result the droplets disintegrate (17),

Roughly observing the particle formation on the collector and the spraying process with the help of the backlight (9),

Accumulation (18) on the surface of the collector plate, the micro/nanoparticles formed from the outer shell (10) and core (11) portions obtained.

The droplet size depends on the delivery distance and hence the duration, and the particle size obtained is also related to the geometry of the sprayer system.

The method of obtaining the eggshell protein used to form the interior of the nano/micro carrier particle system (23) subject to the invention comprises the following process steps;

Separating the egg shell membrane from the waste egg shell,

Leaving the eggshell membrane to dry at low temperature,

Grinding the eggshell membrane into powder form to be used in the process of preparing a soluble eggshell membrane protein solution,

Breaking off of the disulfide bonds of the protein structure of the egg shell membrane,

Dissolving of the obtained soluble powder eggshell membrane protein in an acetic acid-water solution. A method for separating the egg shell membrane from the waste egg shell;

Pre-washing with water to disinfect the waste egg shell then leaving it to rest at room temperature for 4-5 minutes in an acetic acid solution that has been prepared as 1-2% by volume,

Continuing to disinfect the waste eggshell, that has rested inside the acetic acid solution, by adding it into de-ionized water and letting ozone gas to pass through it by means of a diffuser, Separating of the disinfected waste egg shell from the egg shell membrane.

Methods for breaking off of the disulfide bonds of the protein structure included in the egg shell membrane are as follows:

a) Method 1 comprises the following process steps;

Mixing the egg shell membrane in powder form at room temperature in the presence of 1-2 M 20- 30 ml aqueous 3-mercaptopropionic acid and 10-12% acetic acid (CH3COOH),

Keeping the solution in an oven at 80-90°C for 20-22 hours,

Separating the solution which is cooled to room temperature after the waiting period from insoluble parts by centrifugation,

Adjusting the pH of the remaining transparent solution to pH 5 using 5-5,5 M sodium hydroxide (NaOH),

Washing the precipitate with methanol and drying. b) Method 2 comprises the following process steps;

Leaving a 1 gram powder eggshell membrane, in 100 ml performic acid (10 ml hydrogen peroxide, 90 ml formic acid) at 4°C for 24 hours,

Filtering and washing with water after 24 hours,

Leaving the washed membranes in an oven at 25 °C with 10 mg pepsin in 100 ml aqueous solution containing 0.5 M acetic acid for 60-72 hours,

Adding, at the end of this period, 0.2 mg pepstatin into the solution to stop the enzymatic reaction,

Dialysis of the solution that has been centrifuged and separated from the solid particles against water with a dialysis tube for 15-20 hours at 4°C,

Lyophilizing the dialyzed solution and obtaining a dried soluble eggshell membrane protein.

Different processes have been applied during the process of obtaining the egg shell membrane protein which protects the natural compound. By this means, molecular weight distribution, pH, electrical conductivity, viscosity and total protein content features can be changed to reduce the negative effects that may occur between the natural compound and the matrix.

The method of obtaining silk fibroin that is used to form the shell portion of the nano/micro carrier particle system of the invention comprises the process steps of;

Boiling of raw silk at least once with 0.05-0.08% sodium carbonate-water (Na2C03-water) solution with 50-55 times volume of the silk weight,

After the sericin is removed from raw silk the remaining is known as the silk fibroin. Rinsing of the resulting silk fibroin with pure water and drying,

Mixing of silk fibroin in Ajisawa solution at a 70-75°C water bath for 2 - 2,5 hours and then using it to obtain a silk fibroin solution,

Filling the solution obtained into the dialysis membrane and dialyzing it for 3-4 days at +4-5 °C, by frequently changing the deionized water.

Removing the dialysis membranes from water and filtering of the solution.

The Ajisawa solution used is prepared as calcium chloride: ethanol: water (CaC12: EtOH: H20) that is 111 : 92: 144 by weight ratio. For 1.2 grams of silk, Ajisawa solution that is 20 times the weight of silk is used.

As it can be seen from the scanning electron microscopy (Figure 4) carried out, nanospheres whose shell portion comprises silk fibroin, which contain eggshell membrane protein and resveratrol that is a natural compound, are obtained between 100-250 nm successfully by means of the invention. The particles obtained as a result of the tests carried out, silk fibroin, trans resveratrol and the FT-IR spectra of the soluble eggshell membrane poteins have been illustrated together in Figure 5. The characteristic peaks in the spectrum of the nanoparticles have been compared with the characteristic peaks of the materials forming the nanoparticles. It has been observed that the obtained particles carry the characteristic peaks of silk fibroin and eggshell protein and that trans resveratrol is effectively encapsulated.

The molecular weight distribution of the eggshell membrane protein solution obtained depending on the preferred method for breaking the disulfide bonds varies. In the studies of the invention, both low yield and large molecular weight distribution was measured for the eggshell membrane protein obtained by pepsin enzyme digestion. For the determination of molecular weight, size exclusion high performance liquid chromatography (SEC-HPLC) method was used. The molecular weight distribution of protein solutions prepared according to molecular weight and retention time in the standard protein mixture was determined. When the molecular weight distribution of silk fibroin film solution was calculated, it was observed that the molecular weight distribution started at 23 kDa and ended at 1.5 kDa. Peaks indicating the presence of protein molecules at a molecular weight of 2500 kDa and 0.2 kDa were also observed, but analyzes revealed that 69% of the prepared solution had an average molecular weight of 6.6 kDa. When the molecular weight distribution of silk fibroin solution prepared with Ajisawa solution and prepared without regeneration was calculated, it was observed that the molecular weight started at 50.6 kDa and ended at 1 kDa. Peaks were also observed at molecular weights of 3500 kDa and 0.2 kDa, but 75% of the prepared solution had an average molecular weight of 0.05 kDa.

Viscosity is one of the other parameters effective in electro-encapsulation. Since the viscosity of the prepared solution directly influenced the flow through the nozzle, it was used as an encapsulation parameter. The silk fibroin solution prepared by the invention is concentrated to observe the effect of viscosity on encapsulation. To prepare samples of different viscosity, the silk fibroin solution was concentrated in a rotary vacuum evaporator. High concentration samples were also prepared using different polymers in silk fibroin dialysis. The viscosity of the prepared samples was measured by a viscosity meter.

The electrical conductivity of the prepared solutions was determined as one of the most important parameters in the electro-encapsulation. The eggshell protein prepared for this purpose was dissolved in both acetic acid and ethanol solution. Since the electrical conductivity was higher in the polymer solution prepared in acetic acid solution, better results were obtained. Electrical conductivity was measured with a conductivity meter.

In the invention, the method of preparing eggshell membrane protein involves breaking the disulfide bonds with mercaptopropionic acid solution. When pepsin digestion and mercaptopropionic acid solution and protein production results are compared, it is seen that higher yield is obtained with mercaptopropionic acid.

After the particles are obtained, release tests were conducted. Release tests were conducted in phosphate buffer prepared as pH 7 by including a certain amount of prepared particles. Samples were analyzed on the calibration curve prepared by standard trans resveratrol in HPLC. The result of the release kinetics is shown in Figure 11. As can be seen in Figure 11, the amount of trans resveratrol released into the medium increases over time. This indicates that the stability of the trans resveratrol is maintained and that trans resveratrol emits a slow release from the developed carrier system. Release starts after about one hour and continues to increase for 4 hours.

The subject matter of the invention is a biopolymer based nano/micro carrier particle system that can be used in foods, drugs, feeds, cosmetics, pharmaceuticals, and chemical and biomedical sectors.