CASEIN MICELLES FOR NANOENCAPSULATION OF HYDROPHOBIC COMPOUNDS

Abstract

The present invention relates to a the field of food technology and delivery of hydrophobic biologically active compounds, particularly nutrients, via food products and beverages. In particular the present invention provides isolated casein micelles useful for the encapsulation of hydrophobic nutrients, therapeutic and cosmetic compounds, compositions thereof and methods of preparing the micelles.

Claims

1. A casein micelle comprising: a hydrophobic core at least one water-insoluble biologically active compound, phosphate and calcium within the micelle, said water-insoluble biologically active compound is hydrophobically adsorbed onto a hydrophobic domain of said casein micelle, wherein said micelle has an average diameter of about 100 nm.

2. The micelle according to claim 1, wherein the water-insoluble biologically active compound is selected from the group consisting of a water-insoluble drug, a water-insoluble nutraceutical, and a water-insoluble cosmetic compound.

3. The micelle according to claim 2 wherein the water-insoluble biologically active compound is selected from the group consisting of a water-insoluble peptide, a water-insoluble protein, a water-insoluble amino acid, a water-insoluble lipid, a water-insoluble proteoglycan, a water-insoluble polysaccharide, a water-insoluble vitamin, a water-insoluble hormone, a water-insoluble drug, a water-insoluble steroid, a water-insoluble phytochemical, a water-insoluble polynucleotide, a water-insoluble flavorant, a water-insoluble sweetener, a water-insoluble anti-microbial and a water-insoluble preservative.

4. The micelle according to claim 2 wherein the water-insoluble biologically active compound is a water-insoluble nutraceutical.

5. The micelle according to claim 2 wherein the water-insoluble biologically active compound is a water-insoluble fat-soluble vitamin selected from vitamin A, vitamin E, vitamin D and vitamin K, and derivatives thereof.

6. The micelle according to claim 5 wherein the vitamin is vitamin D.

7. The micelle according to claim 2 wherein the water-insoluble biologically active compound is selected from the group consisting of water-insoluble sterol cholesterol and water-insoluble sterol derivatives thereof.

8. The micelle according to claim 2 wherein the water-insoluble biologically active compound is selected from alpha-carotene, beta-carotene, gamma-carotene, lycopene, lutein, zeaxanthin, and astaxanthin.

9. The micelle according to claim 2 wherein the water-insoluble biologically active compound is a water-insoluble unsaturated fatty acid.

10. The micelle according to claim 9 wherein the unsaturated fatty acid is selected from the group consisting of: linoleic acid, conjugated linoleic acid, linolenic acid, an omega-3 fatty acid, and a glycerol-ester of an omega-3 fatty acid.

11. The micelle according to claim 2 wherein the water-insoluble biologically active compound is selected from a phytoestrogen, a phytosterol an isoflavone, a stilbene, a lignan and a coumestan.

12. The micelle according to claim 2 wherein the water-insoluble biologically active compound is Coenzyme Q10.

13. A casein micelle comprising: a hydrophobic core at least one water-insoluble biologically active compound, phosphate and calcium within the micelle, said water-insoluble biologically active compound is hydrophobically adsorbed onto a hydrophobic domain of said casein micelle, wherein the micelle has an average diameter of about 50 nm to about 500 nm.

14. A composition comprising the casein micelle of claim 1.

15. The composition according to claim 14 wherein the composition comprises low fat dairy product or a non-fat dairy product.

16. The composition according to claim 14 wherein the composition comprises skim milk.

17. The composition according to claim 15 wherein the composition comprises low fat or non-fat yogurt.

18. A method for the preparation of a re-assembled casein micelle, the micelle comprising at least one hydrophobic biologically active compound within the micelle, the method comprising the steps of: a) preparing an aqueous solution comprising a source of casein; b) adding a cosolvent solution comprising at least one type of hydrophobic biologically active compound to the casein solution; c) adding a source of citrate ions, a source of phosphate ions and a source of calcium ions to the mixture of step (b) to form a nano-sized micelle dispersion; d) adjusting the pH of the dispersion to stabilize the nanosized micelles.

19. The method according to claim 18, wherein step (c) comprises: i) adding a source of phosphate ions and optionally a source of citrate ions to the mixture of step (b); ii) preparing a solution of calcium ions; and iii) combining the mixture of step (i) with the calcium solution of step (ii) under a high pressure homogenization.

20. The method according to claim 18, further comprising the step of drying the micelle dispersion.

21. The method according to claim 18, wherein the solution comprises about 1% to about 20% casein.

22. The method according to claim 18, wherein the pH is adjusted to a pH in the range of about 6.0 to about 7.5.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0057] FIGS. 1A-1B presents RP-HPLC chromatograms of vitamin D2 in (A) centrifugation-pellets of rCM and of D2-rCM, and in (B) supernatants of rCM and of D2-rCM

[0058] FIGS. 2A-2C shows size distributions of (A) rCM, (B) D2-rCM and (C) naturally occurring CM in skim milk.

[0059] FIGS. 3A-3C presents Cryo-TEM images of (A) rCM, (B) D2-rCM and (C) naturally occurring CM in skim milk. The Bar on the bottom right is 100 nm long. (The dark area on the bottom is the perforated carbon film holding the sample.)

[0060] FIG. 4 shows the size distribution of vitamin D-containing CM prepared by ultra-high pressure homogenization immediately following in-line blending of vitamin D-containing caseinate solution and a calcium solution.

[0061] FIG. 5 presents absorbance spectra of 2.5% caseinate and 31.5 M Vitamin D2 solutions in the range of 210-360 nm. Dashed line Caseinate; Solid lineVitamin D2. The vertical line at 254 nm marks the wavelength of the UV lamp used during the exposure experiments.

[0062] FIG. 6 shows a cryo-TEM image of CM encapsulating vitamin D.sub.2, reassembled using ultra high pressure homogenization.

DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention provides isolated casein micelles and methods for encapsulation of hydrophobic or poorly water-soluble biologically active compounds in CM, with minimal changes to the functional properties of both the micelles and the active compound.

[0064] The present invention now discloses that adsorption of hydrophobic biologically active compounds, for example nutraceuticals, onto the hydrophobic domains of caseins, and the reformation of the micelles stabilize the hydrophobic compound in aqueous surrounding and protect them from degradation. Such casein micelles-hydrophobic compound system facilitates the enrichment of low fat and fat free dairy and other food products with the bioactive molecules, while minimizing the effect of the compound incorporation on the food properties in general and during processing. Encapsulation of biologically active compounds within casein micelles is advantageous over hitherto known encapsulation methods as the micelles are a natural component of milk products and their nanometric size minimizes their effect on the food product, including dairy as well as non dairy foods. In addition, when the active compound possesses undesirable attributes, the encapsulation in the micelles diminishes such unwanted features (e.g. in the case of omega 3 fatty acids). Another important potential benefit is the improved bioavailability of the enclosed compound due to its distribution, at a molecular level, over a very large surface area of the caseins in the nanoscopic micelles, and the fact that caseins are evolutionally optimized for ease of digestion and absorption. The open tertiary molecular structure of casein also facilitates effective proteolysis.

[0065] Specific embodiments include a method for incorporation of vitamin D2 into CM, and evaluation of the encapsulation process by: (a) evaluation of the efficiency of encapsulation, i.e. the percent of added vitamin D2 which was incorporated into the micelles, (b) preservation of micelle properties: diameter as determined by dynamic light scattering (DLS) and morphology (as determined by cryo-TEM), (c) evaluation of the protective effect of the micelles over vitamin D2 from photochemical degradation induced by UV exposure.

Definitions

[0066] For convenience and clarity certain terms employed in the specification, examples and claims are described herein.

[0067] As used herein, the term casein refers to the predominant protein in non-human mammals and human milk, comprising the subgroups .sub.s1, .sub.s2, and .

[0068] The term biologically active compound encompasses a compound having a therapeutic, nutritional and/or cosmetic activity. Biologically active compounds according to the teaching of the invention include, but are not limited to peptides, proteins, amino acids, lipids, proteoglycans, polysaccharides, vitamins, hormones, drugs, steroids, phytochemicals, polynucleotides, flavorants, sweeteners, an anti-microbials, and preservatives.

[0069] A nutraceutical, also known as a functional food (or its component), is generally any one of a class of dietary supplements, vitamins, minerals, herbs, healing or disease-preventative foods that have medical or pharmaceutical effects on the body. Exemplary non-polar or hydrophobic nutraceuticals include, but are not limited to fatty acids (e.g., omega-3 fatty acids, DHA and EPA); fruit and vegetable extracts; vitamins A, D, E and K; phospholipids, e.g. phosphatidyl-serine; certain proteoglycans such as chondroitin; certain amino acids (e.g., iso-leucine, leucine, methionine, phenylanine, tryptophan, and valine); various food additives, various phytonutrients, for example lycopene, lutein and zeaxanthin; certain antioxidants; plant oils; and fish and marine animal oils and algae oils. It is to be understood that certain nutraceuticals can be also referred to as therapeutics as well as cosmetic compounds.

[0070] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Materials

[0071] Sodium caseinate (Miprodan 30, 93.5% protein, MD foods ingredients amba, Videbaek, Denmark). Vitamin D2 (Sigma-Aldrich, Rehovot, Israel). Ethanol (absolute), hydrochloric acid (concentrated), (Bio-Lab, Jerusalem, Israel). Tripotassium citrate, sodium hydroxide, (Merck, Darmstadt, Germany). Calcium chloride (Carlo Erba, Rodano, Italy). Dipotassium hydrogen phosphate (Spectrum, CA, USA).

[0072] Materials for Analysis Only:

[0073] petroleum ether and diethyl ether (Bio-Lab, Jerusalem, Israel). Potassium hydroxide and pyrogallol (Merck, Darmstadt, Germany). Ethylene diamine tetraacetic acid (EDTA) (Acros, NJ, USA). Methanol and acetonitrile (both HPLC grade) (Lab Scan, Dublin, Ireland).

Methods

[0074] Non covalent binding of vitamin D2 to sodium caseinate was achieved by dropwise addition of 1-100 mM, typically 2-50 mM, more typically 5-20 mM, more typically 15 mM solution of the vitamin in absolute ethanol into a 1-20% caseinate, typically 3-8%, more typically 5% caseinate solution, while stirring, to a final concentration of about 5-50,000 M, typically about 10-5000 M, more typically about 25-1000, more typically 25-100, more typically about 65 M.

Re-Assembly of CM.

[0075] Preparation of re-assembled CM (rCM) was done based on the method described by Knoop et al. (8). However, unlike Knoop et al. the casein is provided as a rehydrated commercial sodium caseinate powder, rather than a freshly prepared caseinate, in order to extend the commercial applicability of the method. To 2-10%, more typically 5% non-enriched caseinate solution and to vitamin D2 enriched caseinate solution (200 mL each), 0.5-1.5M, more typically 1 M tri-potassium citrate (4 mL), 0.05-0.5M, more typically 0.2 M K.sub.2HPO.sub.4 (24 mL) and 0.05-0.5M, more typically 0.2 M CaCl.sub.2 (20 mL) were added. Eight consecutive additions of the K.sub.2HPO.sub.4 solution (2.5 mL) and of the CaCl.sub.2 (5 mL) were performed, at 15-minute intervals. During this process, samples were stirred in a thermostated bath at 25-42 C., more typically 37 C. The pH was maintained between 6.5-7.0, more typically between 6.7-7.0, using 0.1N HCl or 1N NaOH. The volume was then adjusted to 400 mL with water, the pH was corrected to 6.5-7.0, more typically to 6.7, and the final dispersions were stirred moderately for one hour (8; 9).

[0076] Alternatively, phosphate and optionally also citrate are added to the aqueous casein solution containing the hydrophobic molecule and the mixture is blended to make solution A. For example, this solution contains 400 mL 5% sodium caseinate, 2 mL 5 mg/mL vitamin D2 in absolute ethanol, 88 mL 0.4M K2HPO4 and 10 mL 0.8M sodium citrate. A calcium ion source solution (B) is prepared separately. The calcium solution comprises, for example, 300 mL 0.08M CaCl2. Solutions A and B are then combined during a high pressure homogenization process. Ultra-high pressure homogenization may be done by any method known in the art. For example, ultra-high pressure homogenization is performed using a Micro DeBee ultra high pressure homogenizer, at 20-25 kPSI, using a 0.1 mm orifice and 1 mm reactor cylinders, without back pressure, at a temperature of 40 C. Flow rates are adjusted so that the main stream (solution A) and the added stream (B) are processed simultaneously throughout the process.

Analytical Fractionation:

[0077] Micelle preparations were centrifuged at 20 C. and 25,000g for one hour and the supernatant was separated from the pellet by decantation. The supernatant was then ultra-filtered using Amicon 8050 stirred ultrafiltration cell with a 10,000 Da nominal molecular weight limit membrane (Millipore). All fractions were collected and analyzed for vitamin D2 content.

Evaluation of Micelle Protection Against UV Light Induced Degradation of Vitamin D2:

[0078] Samples containing vitamin D2-enriched rCM (D2-rCM) were placed in a wooden light-proof cabinet, and exposed to a 254 nm UV light, at 200 W/cm.sup.2 intensity for 3, 6, 12, and 24 hours. At each exposure time, three 20 mL samples were compared: a micelle dispersion preparation, a negative control (an identical sample covered with an aluminum foil to completely block the UV), and another control containing only serum from the D2-rCM preparation, which was exposed to UV. The serum samples were obtained by centrifuging D2-rCM dispersion and collecting the supernatant.

UV Spectra Determination for Caseinate and Vitamin D2.

[0079] Samples of caseinate and vitamin D2 at concentrations similar to their concentrations in the rCM suspension (2.5% and 31.75 M respectively) were prepared. UV absorbance spectra of the samples were analyzed by absorbance scan at wavelengths between 220 nm and 360 nm, using a Pharmacia Biotech Ultrospec 3000 spectrophotometer.

Saponification and Extraction:

[0080] Pellets were resuspended in a 100 mM EDTA solution of same weight as the removed supernatant, and equilibrated for 6 hours at 4 C. Both pellet, serum and permeate from each sample underwent saponification and extraction procedures based on Renken and Warthesen (10): Five mL of each sample were placed into a 25 mL glass stoppered round bottom flask wrapped with aluminum-foil. 3 mL of 5% KOH and 1.5 mL of 1% ethanol pyrogallol solutions were added. The samples were flushed with nitrogen, capped and then left to stir slowly in the dark for 12 hours at room temperature.

[0081] Each sample was then poured into a separatory funnel. The round bottomed flask was washed with 2 mL of water, then 0.75 mL of ethanol, and lastly 5 mL of petroleum ether: diethyl ether (90:10 v/v), adding each wash liquid into the separatory funnel. The mixture was gently mixed and the phases were allowed to separate. The hydrophilic phase was then poured into a second separatory funnel adding 0.75 mL ethanol and 5 mL of the ether mixture. After gentle mixing the phases were allowed to separate. The hydrophobic phase was put into the first separatory funnel. 4.5 mL water was used to wash the hydrophobic phase four times (10). The hydrophobic phase was collected and the solvents were evaporated using nitrogen. The dried sample was re-suspended in 1 mL solution of methanol:water (93:7 v/v) (11).

Determination of Vitamin D2 Content:

[0082] Vitamin D2 analysis was done by reverse phase HPLC (RP-HPLC). All samples were analyzed for vitamin D2 using a 4.6100 mm C18-C2 RP-HPLC column and a UV detector at 265 nm. The gradient used was zero to 75% acetonitrile as eluent B, while methanol:water (93:7 v/v) serve as eluent A (11). Calibration curve was prepared using vitamin D2 standard in methanol:water (93:7 v/v) solution at 7 concentrations ranging from 5 to 250 g/mL.

[0083] Vitamin D2 fractions were collected during RP-HPLC and analyzed for UV absorbance spectrum from 220 nm to 360 nm for further identity validation, using a Pharmacia Biotech Ultrospec 3000 spectrophotometer.

Size and Morphology Determination of rCM:

[0084] For both rCM and D2-rCM, average size was measured by dynamic light scattering (DLS) (BIC 90Plus, Brookhaven Instruments Corp.). Morphology was determined using Cryogenic Transmission Electron Microscopy (Cryo-TEM): Specimens were prepared in a controlled environment vitrification system (CEVS) at controlled temperature and humidity to avoid loss of volatiles. The samples were brought to a desired temperature (25 C. and 35 C.) and allowed to equilibrate in the CEVS for an hour. Then, a 7 l drop of the examined dispersion was placed on a TEM copper grid covered with a perforated carbon film, and blotted with a filter paper to form a thin liquid film of the sample (100-200 nm thick). The thinned sample was immediately plunged into liquid ethane at its freezing temperature (183 C.) to form a vitrified specimen, and then transferred to liquid nitrogen (196 C.) for storage until examination. The vitrified specimens were examined in a Philips CM120 transmission electron microscope operating at an accelerating voltage of 120 kV. An Oxford Conn.3500 cryo-specimen holder was used to maintain the vitrified specimens below 175 C. during sample transfer and observation. Specimens were recorded digitally on a cooled Gatan MultiScan 791 CCD camera using the Digital Micrograph 3.1 software, in the low-dose imaging mode to minimize beam exposure and electron-beam radiation damage. Brightness and contrast adjustments were done using Photoshop 7.0 ME.

Durability of the Micelles to High Shear

[0085] Samples of rCM and D2-rCM suspensions, as well as a sample of skim milk reconstituted from powder, were homogenized using a Micro DeBee ultra high pressure homogenizer, by 1 pass at the single-reversed-flow mode at 18510 MPa, using a 0.1 mm orifice, and a back-pressure of 103 MPa. Average diameter of rCM and D2-rCM was measured before and after homogenization process by DLS (see method details above). Relative average diameter changes were then determined for each sample.

Analyses

[0086] FIG. 1 presents the results of the analysis of vitamin D2 in preparations of micelles enriched with vitamin D2 (D2-rCM) and control rCM preparations without the vitamin. Both analyses of the micelle pellets obtained by centrifugation and of their respective serum fractions are presented. In the chromatograms of the control rCM preparation fractions (pelletFIG. 1A, and serumFIG. 1B) vitamin D2 peaks were absent, while in both D2-rCM fractions those peaks were observed. UV absorbance spectra of the peaks identified as vitamin D2 indicated good matching between vitamin D2 standard and vitamin D2 eluted at the same position in the sample runs.

[0087] During the analysis, 45-95%, more typically 65-85% of total vitamin D2 added were recovered by the extraction procedure from the serum and the pellet together. 25-75%, more typically 45-60% of the recovered vitamin D2 were found to be incorporated into the micelles, which accounted for 2-15%, more typically 8% by weight of the total D2-rCM suspension prepared.

[0088] It was determined that vitamin D2 concentration in the rCM was about 2-22 times, more typically 5-10 times greater than its concentration in serum: 44-57 g/mL vs. 2-8 g/mL respectively. Therefore, fortification of milk using such vitamin D2-enriched rCM accounting for only 0.001-1%, more typically 0.1-1%, more typically 0.5-0.6% of the total milk casein, would provide about one third of the vitamin D2 recommended daily allowance (RDA) for adults in a single glass of milk (200 mL).

Size and Morphology Determination of rCM

[0089] The re-assembled micelles had average diameters of 146 and 152 nm without and with vitamin D2 respectively (FIG. 2). As mentioned hereinabove, the normal size range of CM in milk is 50-500 nm, and the average is 150 nm.

[0090] D2-rCM and rCM had similar morphology, which was also typical to naturally occurring CM, as may be judged by the available resolution of the TEM micrographs (FIG. 3). These micrographs suggest that the incorporation of vitamin D2 has minimal effect over the morphology of the CM.

Shear Stability of rCM and D2-rCM

[0091] Following an ultra-high-pressure homogenization process the average diameter of rCM was reduced to 122 nm (26% reduction) and that of D2-rCM was reduced to 125 nm (27% reduction). The reference micelles from reconstituted skim milk showed a 9% reduction in diameter during the homogenization. While this shows that the reformed micelles are expectedly somewhat weaker than the original micelles, their durability through such extreme shear suggests they could well withstand typical processing shear which is seldom that high. The similar extent of reduction in size for rCM and D2-rCM suggests that the incorporation of vitamin D2 into rCM did not weaken their structure as reflected by shear stability.

Ultra-High Pressure Homogenization Used for Preparation of rCM

[0092] FIG. 4 shows the size distribution of CM obtained by in-line merging of two streams, just before the high pressure chamber of a high-pressure homogenizer. One stream was the aqueous casein solution containing the hydrophobic molecule(s), as well as phosphate and optionally also citrate, and the other stream was a calcium ion source solution. The average size of the CM obtained was 100 nm. FIG. 6 shows a cryo-TEM image of the vitamin D2-containing CM obtained this way. Analysis of vitamin D2 in the micelle-pellet and supernatant of the centrifuged micelle preparation showed 4-5 times higher vitamin concentration in the micelle pellet compared to the supernatant (serum). The pellet contained 30.30.4 and the supernatant contained 6.70.7 micrograms/mL of the vitamin.

Quantification of the Protective Effect of the Micelles Against UV Light-Induced Photochemical Degradation of Vitamin D2

[0093] Table 1 presents vitamin D2 degradation as the remaining percent of the initial concentration in each fraction with exposure time, in D2-rCM suspension exposed to UV light, D2-rCM suspension unexposed to UV light (control I) and in D2-rCM suspension serum exposed to UV light (control II). (UD=undetectable).

TABLE-US-00001 TABLE 1 Percent of vitamin D2 remaining in micelle preparation following UV exposure UV exposed Unexposed micelle micelle suspension suspension (control I) UV exposed Exposure Time Micelle Micelle serum (Hrs). pellet Serum pellet Serum (control II) 0 100 100 100 100 100 3 20.9 7.2 107.8 43.1 0.11 6 2.7 1.7 41.0 74.9 UD 12 2.7 0.5 78.5 77.1 UD 24 1.8 0.9 135.1 62.0 UD

[0094] The data in Table 1 merits several observations: First, the comparison of the UV exposed serum (control II) to the serum of control 1 (unexposed) shows how relatively quickly photochemical degradation of unprotected vitamin D2 occurs. The vitamin in the serum is presumably bound to residual soluble casein molecules which did not aggregate into micelles. The main interesting observation is the comparison of the rate of degradation of the vitamin within the micelles in the exposed preparation to that of the UV exposed serum (control II). This comparison demonstrates the significant relative protection conferred by the micelles to the encapsulated vitamin. The micelles also confer some protection to the vitamin in their surrounding serum, as the rate of degradation in the serum of the exposed micelle dispersion was lower than that in the exposed micelle-free serum (control II). This may be explained by a shade effect of the micelles which block and absorb much of the light. Lastly, it is observed that the degradation of the vitamin in the micelles of the unexposed preparation of control I (although slightly obscured by experimental error) was slower than in the serum of this preparation. This degradation may be due to chemical oxidation, (e.g. by dissolved oxygen) and this observation suggests that the micelles confer some protection against chemical degradation as well, however this remains to be verified by other experiments.

[0095] The nature of the protective effect of the micelles against photo-degradation of the vitamin was examined by comparing the absorbance spectra for both caseinate and vitamin D2 components in the rCM suspension

[0096] As is shown in FIG. 5, at the concentrations examined for each of the fractions (caseinate and vitamin D2) in the rCM suspension, caseinate, being a protein with aromatic side groups and double-bonds, absorbs significantly more UV light than vitamin D2. These data support the conclusion that casein micelles have protective effect for the vitamin D2 enclosed therein and to some degree also to vitamin D2 around the micelles.

[0097] Casein micelles were shown to be potential nano-vehicles for added nutraceuticals such as the fat-soluble vitamin D2 chosen here as a model. In terms of encapsulation efficiency, about 25% to about 75%, more typically 45-60% of the vitamin retrieved from the micelle suspension was found in the reformed micelleswhich contained about 2 to about 22 fold, more typically 4-10 fold higher concentration of the vitamin compared to the surrounding medium. Some vitamin D may be lost by binding to hydrophobic domains of unaggregated proteins in the serum. The extraction-based analysis method allowed the retrieval of about 45 to about 95%, more typically 65-85% of the added vitamin. The micelles' morphology and size were similar to those of naturally occurring CM, in accord with the purpose to minimize modification of micelle properties. It was also shown that apart from their effectiveness in stabilizing oil-soluble compounds in aqueous environment, the rCM have an additional protective affect against photochemical degradation of the entrapped hydrophobic compound, for example the nutraceutical vitamin D2.

[0098] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

REFERENCES

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