USAGE OF EGGSHELL MEMBRANE FOR OBTAINING A FUNCTIONAL RAW MATERIAL AND FOR PURIFICATION OF NATURAL COMPOUNDS AS BIOADSORBENT

Abstract

Eggshell membranes with high collagen and hyaluronic acid content are separated from the eggshell wastes by means of a foam separation method. During the foam separation, the eggshell membranes are enriched with the extract obtained from olive leaf and olive mill wastewater, which include phenolic compounds having antioxidant and antimicrobial properties, to obtain an antimicrobial and antioxidant compound that can be used in natural supplementary food products or cosmetic products.

Claims

1. An antioxidant and antibacterial composition, comprising egg shell membranes with adsorbed phenolic compounds, wherein the adsorbed phenolic compounds are obtained from an olive leaf extract and the olive mill wastewater.

2. The antioxidant and antibacterial composition according to claim 1, comprising 99% by weight of the eggshell membranes, and 1% by weight of the adsorbed phenolic compounds.

3. the The antioxidant and antibacterial composition according to claim 1, wherein the adsorbed phenolic compounds are Oleuropein and Hydroxythyrosol.

4. The antioxidant and antibacterial composition according to claim 1, wherein the antioxidant and antibacterial composition is configured as a supplement in natural food products or as raw materials in cosmetic products.

5. The antioxidant and antibacterial composition according to claim 1, wherein the antioxidant and antibacterial composition is configured as a feed additive or for the preparation of pharmaceuticals.

6. A production method of the antioxidant and antibacterial composition according to claim 1, comprising the process steps of: sterilizing collected eggshell wastes by washing the collected eggshell wastes in a water and/or acid mixture to obtain sterilized eggshells, milling the sterilized eggshells to obtain milled eggshells having a size range of 500-5000 microns, delivering the milled eggshells into a foam separation apparatus until the milled eggshells fill up 25-35% of a column of the foam separation apparatus and adding a water and acetic acid mixture thereon, and mixing for 1 to 5 hours, delivering air to the foam separation apparatus at an end of the mixing process, adding the olive leaf extract or the olive mill wastewater such that the olive leaf extract or the olive mill wastewater constitutes 70-75% volume of the column during the delivery of the air, obtaining and collecting a foam comprising eggshell membranes separated from the milled eggshells and the adsorbed phenolic compounds in the olive leaf extract or the olive mill wastewater, wherein the adsorbed phenolic compounds bind onto a surface of the eggshell membranes, removing residual agents not bonded to the surface of the eggshell membranes by purification with an ethanol-water mixture, wherein the ethanol-water mixture is a different polarity solvent pair of the collected foam, leaving the foam to rest and removing the air therein, and obtaining the antioxidant and antimicrobial composition.

7. The production method according to claim 6, wherein a linear velocity of the air in the foam separation apparatus is 0.1-0.5 cm/s.

8. The production method according to claim 6, wherein a linear velocity of the air in the foam separation apparatus is 0.5 cm/s.

9. The production method according to claim 6, wherein a mixing time in the foam separation apparatus is 1-5 hours.

10. The production method according to claim 6, wherein a mixing time in the foam separation apparatus is 5 hours.

11. The production method according to claim 6, wherein the sterilized eggshells are milled until the milled eggshells are 2000 microns.

12. The antioxidant and antibacterial composition according to claim 2, wherein the adsorbed phenolic compounds are Oleuropein and Hydroxythyrosol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1: Process diagram of the foam separation (fractionalization) method for separating eggshell membranes.

[0024] FIG. 2: Foam separation (fractionalization) apparatus

[0025] FIG. 3: FT-IR graphic of the eggshell membrane and the eggshell membrane that has adsorbed olive leaf extract A) Eggshell membrane FT-IR spectrum B) FT-IR spectrum of the eggshell membrane after adsorption.

[0026] FIG. 4: Scanned electron microscope (SEM) view of the eggshell membrane.

[0027] FIG. 5: Scanned electron microscope (SEM) view of the eggshell membrane that has adsorbed the olive leaf extract.

[0028] FIG. 6: High performance liquid chromatography (HPLC) profiles of olive leaf extracts (OLE) having two different concentrations A: 3% OLE, weight/volume); B: 2% OLE, weight/volume.

[0029] FIGS. 7A-7G: The 3-dimensional response surface graphics of the change of the total antioxidant capacity with parameters. FIG. 7A: The 3-dimensional response surface graphics of the change of the total antioxidant capacity with ethanol concentration (%) and liquid/solid ratio (S/K) parameters. FIG. 7B: The 3-dimensional response surface graphics of the change of the total antioxidant capacity with ethanol concentration (%) with extraction time (hour) parameters. FIG. 7C: The 3-dimensional response surface graphics of the change of the total antioxidant capacity liquid/solid ratio (S/K) parameters and extraction time (hour) parameters. FIG. 7D: The graph of the percentage proximity of the test results to the predicted values; FIG. 7E: The graph of the percentage proximity of the test results to the test results; FIG. 7F: Histogram graphic of the test results. FIG. 7G: Random distribution of the tests.

[0030] FIGS. 8A-8G: The 3-dimensional response surface graphics of the change of the total phenol amount with parameters. FIG. 8A: The 3-dimensional response surface graphics of the change of the total phenol capacity with ethanol concentration (%) with extraction time (hour) parameters.

[0031] FIG. 8B: The 3-dimensional response surface graphics of the change of the total phenol capacity with ethanol concentration (%) and liquid/solid ratio (S/K) parameters. FIG. 8C: The 3-dimensional response surface graphics of the change of the total phenol capacity liquid/solid ratio (S/K) parameters and extraction time (hour) parameters. FIG. 8D: The graph of the percentage proximity of the test results to the predicted values; FIG. 8E: The graph of the percentage proximity of the test results to the test results; FIG. 8F: Histogram graphic of the test results. FIG. 8G: Random distribution of the tests.

DEFINITIONS OF THE PARTS/ASPECTS/SECTIONS FORMING THE INVENTION

[0032] 7. Collecting the waste eggshells. [0033] 8. Sterilizing the eggshells with water and/or acetic acid. [0034] 9. Milling of the sterilized shells [0035] 10. Delivering the milled shell into the foam separation (column) [0036] 11. Delivering air through the air inlet to the feed unit [0037] 12. Adding olive leaf extract or olive mill wastewater into the column [0038] A: The antioxidant and antimicrobial composition of the invention [0039] B: Additional product which is eggshells with high calcium content that remains under [0040] the column. [0041] C: Air [0042] D: Feeding unit [0043] E: Stainless steel frit [0044] F: Foam [0045] G: Foam collector [0046] H: Slaked foam

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] The invention is related to an antioxidant and antibacterial compound comprising eggshell membranes with adsorbed phenolic compounds available in the olive leaf extract and the olive mill wastewater.

[0048] The antioxidant and antibacterial composition of the invention comprises 99% eggshell membrane and 1% phenolic compound by weight.

[0049] In the invention eggshell wastes having high collagen content and hyaluronic acid that constitute a significant waste problem in the food sector are used, the membrane of the eggshell is partially or completely separated by means of a foam separation method and during this separation process natural compounds such as olive leaf extract or olive mill wastewater with high antioxidant and antimicrobial properties are added and as a result a compound rich in antioxidants and antimicrobial agents are obtained.

[0050] When the eggshell and the membrane is being separated herbal natural compounds that are available in the herbal extract are concurrently and are selectively adsorbed on the eggshell membrane. Eggshell membranes are given functionality due to the adsorbed natural compounds, and they gain antioxidant and antimicrobial properties.

[0051] The eggshell membrane is used as a bioadsorbent for the purification of natural compounds that are functionalized with solvents having suitable polarity, where said natural compounds are selectively adsorbed onto said membrane from an herbal extract.

[0052] A foam separation unit (column) is used to separate the eggshell and the membrane from each other. Both separation and purification processes are carried out concurrently in the column. The process for obtaining antioxidant and antimicrobial compositions from eggshell wastes is carried out at room temperature.

[0053] The method of obtaining the antioxidant and antimicrobial composition of the invention comprises the process steps of: [0054] Collecting the waste eggshells. [0055] Sterilizing the eggshell wastes by washing them in a water and/or acid mixture, [0056] Milling the sterilized shells to a range of 500-5000 microns, preferably until they reach the 2000 micron range size, [0057] Delivering the milled shells into the foam separation apparatus until they fill up 25-35% of the column and adding water and acetic acid mixture thereon, [0058] Mixing the milled eggshell with the added acetic acid and water solvent for 1 to 5 hours, [0059] Delivering air to the foam separation apparatus at the end of the mixing process, [0060] Adding olive leaf extract or olive mill wastewater such that they shall constitute 70-75% of the column volume during the delivery of air, [0061] Obtaining a foam comprising the phenolic compounds in the olive leaf extract or the olive mill wastewater that have bound onto the surface of the membrane that has been separated from the eggshell, [0062] Collecting the obtained foam, [0063] Removing the residual agents that have not bonded to the surface of the membrane by purification with ethanol-water mixture which is a different polarity solvent pair of the collected foam, [0064] Leaving the foam to rest and removing the air therein, and obtaining an antioxidant and antimicrobial composition (A).

[0065] A preferred embodiment of the invention is illustrated in FIG. 1. As shown in FIG. 1, in the foam fractionalization of the eggshell membrane first of all the eggshell wastes are collected from pasteurized liquid egg producers. After the eggshells are washed with a mixture of water and 1-2% acetic acid by volume, sterilization is carried out under high pressure with vapour. The obtained sterile shells are milled with a mill. The aim of this process is to bring the egg waste to 500-5000 microns, preferably to 2000 microns and to ensure that the shell-membrane separation reaches optimum level.

[0066] The milled material is delivered to the foam separation apparatus (column). Water and acetic acid mixture is added to the eggshells in the column. In order for the membrane in the eggshell to be separated from the shell in liquid, mechanical mixing is carried out for 1 to 5 hours, preferably for 1 hour. At the end of this time, air is delivered through air inlet to the feed unit. After the addition of air the partial or complete separation of the eggshell membrane from the eggshell is started. Concurrently to the delivery of air to the feed unit, the olive leaf extract and olive mill wastewater are added to column. As a result, the antioxidant and antimicrobial composition (A) that is rich in collagen and the additive product (B) which is eggshells having high calcium content, that remain below the column following the foaming process, due to its specific weight are obtained.

[0067] Foam fractionalization (Separation) apparatus is illustrated in FIG. 2. In the foam fractionalization apparatus, air (C) is delivered to the feed unit (D) that comprises the eggshell membrane comprising collagen, olive leaf extract and olive mill wastewater, and foaming (F) is established, and the obtained foam (F) is collected in a separate container with a foam collector (G), preferably with a separation funnel. The apparatus also contains a stainless steel frit (E) that enables the air (C) to be filtered and delivered homogenously to the column. Slaked foam (H) is obtained by leaving the air in the foam to wait and then removing it. The slaked foam (H) comprises olive leaf extract and olive mill wastewater that are carried together with the eggshell membrane loaded with the natural compound and the air foam.

[0068] In order to functionalize the membrane with collagen content, initially olive leaf extract and olive mill wastewater are added. The phenolic compounds in olive leaf extract and olive mill wastewater are adsorbed onto the membrane to obtain a functional high added value antioxidant and antimicrobial composition.

[0069] The eggshell membrane provides an ideal adsorption surface due to its natural porous and fibril structure. At the same time, the hydrophobic interaction of the collagen found in the eggshell membrane with phenolic compounds also increases adsorption.

[0070] After the membrane and the herbal extract within the foam are collected, they are used for further analysis. At this stage, the yield and the separation efficiency of the membrane is optimized by changing the parameters such as the amount of the waste eggshells and membranes, the ratios of the acetic acid mixture added to the milled eggshell, mixing time and speed, and air flow. The test design prepared for optimization has been given in Table 1. After the milled shells are transferred to the foam separation apparatus, the tests have been carried out by adjusting the water and acetic acid concentration to 0-100% (v/v), (the acetic acid concentration (0% (v/v) indicates that 100% pure water has been added) the average particle size of the eggshell to 0.5-5 mm, the mixing time to 1-5 hours, the linear flow rate of the air in column to 0.1-0.5 cm/s.

[0071] As a result of the tests carried out within the scope of the invention, it has been observed that the highest eggshell-membrane separation percentage was 81%, and this was obtained when the acetic acid concentration was 100%, the eggshell average particle size was 2 mm, and the mixing time was 5 hours, and the linear flow rate of the air in the column was 0.5 cm/s.

TABLE-US-00001 TABLE 1 Test design and results thereof for the separation of the eggshell from the eggshell membrane Average Mixing particle time (hour) Linear flow membrane-shell size (mm) (time kept rate of air in separation Test Ascetic acid of the in the the column efficiency, Number concentration %(v/v) shell column) (cm/s) percentage 1 0 0.5 1 0.1 45 2 100 0.5 1 0.1 66 3 0 2 1 0.1 38 4 100 2 1 0.1 68 5 0 0.5 5 0.1 42 6 100 0.5 5 0.1 77 7 0 2 5 0.1 36 8 100 2 5 0.1 65 9 0 0.5 1 0.5 40 10 100 0.5 1 0.5 69 11 0 5 1 0.5 38 12 100 5 1 0.5 71 13 0 0.5 5 0.5 42 14 100 0.5 5 0.5 70 15 0 2 5 0.5 38 16 100 2 5 0.5 81 17 0 1.25 3 0.3 34 18 100 1.25 3 0.3 74 19 50 0.5 3 0.3 43 20 50 2 3 0.3 47 21 50 1.25 1 0.3 45 22 50 1.25 5 0.3 56 23 50 1.25 3 0.1 44 24 50 1.25 3 0.5 40 25 50 1.25 3 0.3 47 26 50 1.25 3 0.3 34 27 50 1.25 3 0.3 45 28 50 1.25 3 0.3 46 29 50 1.25 3 0.3 47 30 50 1.25 3 0.3 38 31 50 1.25 3 0.3 40

[0072] The FTIR analysis of the eggshell samples that comprise collagen and that are obtained in Table 1, is illustrated in FIG. 3, where the eggshell membrane is shown for structural analysis respectively, as the eggshell membrane and the eggshell membrane that has adsorbed olive leaf extract.

[0073] The scanned electron microscope (SEM) view of eggshell membrane is shown in FIGS. 4 and 5, respectively as the eggshell membrane and the eggshell membrane that has adsorbed olive leaf extract.

[0074] The high performance liquid chromatography (HPLC) profiles of olive leaf extract (OLE) having two different concentrations are shown in FIG. 6. The phenolic compounds that correspond to each peak number in FIG. 6 have been given in Table 2.

TABLE-US-00002 TABLE 2 The phenolic compounds that correspond to each peak number in FIG. 6 Phenolic compounds Peak Number Hydroxythyrosol 1 Thyrosol 2 Catechin 3 Cafeic acid 4 Vanillic acid 5 Vanillin acid 6 Rutin 7 Luteolin 7-glucoside 8 Verbascoside 9 Apigenin 7-glucoside 10 Diosmetin 7-glucoside 11 Oleuropein 12 Luteolin 13

[0075] The change of the total antioxidant capacity with parameters (solid-liquid ratio, ethanol water mixture concentration and extraction time) is shown in FIGS. 7A-7G. As a result of the surface response analyses, the maximum total phenol amount has been obtained by the extraction process carried out in the 70% ethanol water mixture having 1:20 solid-liquid ratio as extraction conditions.

[0076] The change of the total phenol amount with parameters (solid-liquid ratio, ethanol water mixture concentration and extraction time) is shown in FIGS. 8A-8G. As a result of the surface response analyses, the maximum total phenol amount has been obtained by the extraction process carried out in the 70% ethanol water mixture having 1:20 solid-liquid ratio as extraction conditions.