阅读说明：本技术 白及须根低聚糖及其制备方法和应用 (Bletilla striata fibrous root oligosaccharide and preparation method and application thereof ) 是由 张朝燕 易金玉 黄莹颖 马文涛 雷敏 李娜 吴文惠 郭锐华 于 2021-07-23 设计创作，主要内容包括：本发明提供了一种白及须根低聚糖及其制备方法和应用,制备方法包括：将白及须根烘干后,粉碎,并溶解在热水中得到白及须根溶液；将白及须根溶液通过提取,脱色,浓缩,醇沉,得到白及须根粗提物；将白及须根粗粗提物离心,洗涤,除蛋白,冻干即可,白及须根低聚糖的分子量为1000-1500Da,其主要由鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖和半乳糖组成；本发明的白及须根多糖具有分子量较小、无毒、体系稳定、良好的抗氧化活性及酪氨酸酶抑制活性等特点,原料廉价易得,实现了白及属植物资源的充分利用,安全无毒,具有开发美白和化妆品的潜力；本发明的制备工艺简单,耗能少,经济无毒,方便易行,适用于工业化生产。(The invention provides a bletilla striata fibrous root oligosaccharide and a preparation method and application thereof, wherein the preparation method comprises the following steps: drying rhizoma bletilla fibril, pulverizing, and dissolving in hot water to obtain rhizoma bletilla fibril solution; extracting, decoloring, concentrating and precipitating the solution of the bletilla striata fibrous roots to obtain a crude extract of the bletilla striata fibrous roots; centrifuging the crude extract of rhizoma bletilla fibril, washing, removing protein, and lyophilizing to obtain rhizoma bletilla fibril oligosaccharide with molecular weight of 1000-; the bletilla striata fibrous root polysaccharide has the characteristics of small molecular weight, no toxicity, stable system, good antioxidant activity, tyrosinase inhibitory activity and the like, is cheap and easily available in raw materials, realizes full utilization of bletilla plant resources, is safe and non-toxic, and has the potential of developing whitening and cosmetics; the preparation method has the advantages of simple preparation process, low energy consumption, economy, no toxicity, convenience, easiness and suitability for industrial production.)

1. A preparation method of bletilla striata fibril oligosaccharide is characterized by comprising the following steps: which comprises the following steps:

(1) drying the bletilla striata fibrous roots, crushing, and dissolving in hot water to obtain a bletilla striata fibrous root solution;

(2) extracting, decoloring, concentrating and precipitating the solution of the bletilla striata fibrous roots to obtain a crude extract of the bletilla striata fibrous roots;

(3) centrifuging and washing the crude extract of the bletilla striata fibrous roots to obtain bletilla striata fibrous root polysaccharide;

(4) removing protein from the bletilla striata fibrous root polysaccharide, centrifuging, and freeze-drying to obtain bletilla striata fibrous root oligosaccharide;

the molecular weight of the bletilla striata fibril oligosaccharide is 1000-1500 Da;

the bletilla striata fibril oligosaccharide mainly comprises rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose.

2. The method of preparing a bletilla striata fibril oligosaccharide according to claim 1 wherein: in the step (1), the drying temperature is 40-70 ℃.

3. The method of preparing a bletilla striata fibril oligosaccharide according to claim 1 wherein: in the step (1), the mass-volume ratio of the bletilla striata fibrous roots to the hot water is 1 g: 10 mL.

4. The method of preparing a bletilla striata fibril oligosaccharide according to claim 1 wherein: in the step (2), the extraction temperature is 80-90 ℃ and the extraction time is 3 +/-0.1 h.

5. The method of preparing a bletilla striata fibril oligosaccharide according to claim 1 wherein: in the step (3), the rotation speed of the centrifugation is 3000-5000r/min, and the time is 5 +/-0.1 min.

6. The method of preparing a bletilla striata fibril oligosaccharide according to claim 1 wherein: in the step (3), the washing process comprises the following steps: the solid obtained was washed with ethanol, then with acetone solution and finally rinsed with an equal volume of ethanol solution.

7. The method of preparing a bletilla striata fibril oligosaccharide according to claim 1 wherein: in the step (4), the protein removing process comprises the following steps: dissolving the bletilla striata fibril polysaccharide in distilled water, and then adding trichloroacetic acid to adjust the pH value to 3.0.

8. A bletilla striata fibril oligosaccharide, which is characterized in that: which is obtained by the production method according to any one of claims 1 to 7.

9. Use of the bletilla striata fibril oligosaccharide of claim 8 in cosmetics.

10. Use of the bletilla striata fibril oligosaccharide of claim 8 as an antioxidant in the pharmaceutical and food fields.

Technical Field

The invention belongs to the field of medicine and food dual-purpose polysaccharide, and particularly relates to bletilla striata fibrous root oligosaccharide and a preparation method and application thereof.

Background

Bletilla striata is a traditional medicine-food dual-purpose traditional Chinese medicine widely distributed in east Asia countries, a dry tuber of the bletilla striata is widely used as a hemostatic, and a plurality of polysaccharides which are the most important active ingredients of the bacillus wenshuriensis and show various biological activities can be separated from the tuber of the bletilla striata. Is mainly used for treating hemoptysis, hematemesis, traumatic hemorrhage, gastrointestinal mucosa injury, ulcer, bruise and burn, anorectal diseases, gynecological fibroma, tumor embolism, prostate operation and the like in clinic.

The research in the literature indicates that the fibrous root of bletilla striata also contains polysaccharide with similar functions. The reported bletilla striata fibrous root polysaccharide consists of four monosaccharides, namely arabinose, galactose, glucose and mannose, and has the molecular weight of about 9.1x10⁴Da and has various biological activities, such as antibiosis, bacteriostasis, tumor resistance and the like.

Active ingredients in white and tubers are currently under investigation, compared to active ingredients in white and fibrous roots. The existing research finds that the sugar in the tuber and fibrous root of bletilla striata is mainly high molecular weight polysaccharide, but the research on the sugar with low molecular weight is less.

Patent CN111004335A discloses a method for extracting oligosaccharide from rhizoma bletillae tuber, but the components and extraction method of rhizoma bletillae and fibrous root have not been studied, and the related biological medicinal activity of the oligosaccharide has not been studied.

Disclosure of Invention

In view of the deficiencies of the prior art, one of the objectives of the present invention is to provide a method for preparing oligosaccharide from bletilla striata fibrous root. The invention takes the rhizoma bletillae which is a Chinese medicinal material used as both medicine and food as a raw material, and extracts the white and fibrous roots to obtain a novel polysaccharide with low molecular weight, and the polysaccharide shows good antioxidant activity and tyrosinase inhibitory activity.

Another object of the present invention is to provide the above bletilla striata fibril oligosaccharide.

The invention also aims to provide the application of the bletilla striata fibril oligosaccharide.

In order to achieve one of the above purposes, the solution of the invention is as follows:

a preparation method of bletilla striata fibril oligosaccharide comprises the following steps:

(1) drying the bletilla striata fibrous roots, crushing, and dissolving in hot water to obtain a bletilla striata fibrous root solution;

(2) extracting, decoloring, concentrating and precipitating the solution of the bletilla striata fibrous roots to obtain a crude extract of the bletilla striata fibrous roots;

(3) centrifuging and washing the crude extract of the bletilla striata fibrous roots to obtain bletilla striata fibrous root polysaccharide;

(4) removing protein from the bletilla striata fibrous root polysaccharide, centrifuging and freeze-drying to obtain the bletilla striata fibrous root oligosaccharide.

Wherein the molecular weight of the bletilla striata fibril oligosaccharide is 1000-;

the bletilla striata fibril oligosaccharide mainly comprises rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose.

As a preferred embodiment of the present invention, in the step (1), the temperature for drying is 40-70 ℃.

In a preferred embodiment of the present invention, in step (1), the mass-to-volume ratio of the bletilla striata fibrous roots to the hot water is 1 g: 10 mL.

As a preferred embodiment of the present invention, in the step (2), the extraction temperature is 80-90 ℃ and the extraction time is 3 + -0.1 h.

As a preferred embodiment of the present invention, in step (3), the rotation speed of centrifugation is 3000-.

As a preferred embodiment of the present invention, in the step (3), the washing process is: the solid obtained was washed with ethanol, then with acetone solution and finally rinsed with an equal volume of ethanol solution.

As a preferred embodiment of the present invention, in the step (4), the protein removal process is: dissolving rhizoma bletilla fibril polysaccharide in a small amount of distilled water, and adding trichloroacetic acid to adjust pH to 3.0.

In order to achieve the second purpose, the solution of the invention is as follows:

the bletilla striata fibril oligosaccharide is obtained by the preparation method.

In order to achieve the third purpose, the solution of the invention is as follows:

an application of the bletilla striata fibril oligosaccharide in cosmetics.

An application of the bletilla striata fibril oligosaccharide as an antioxidant in the fields of medicines, foods and the like.

Due to the adoption of the scheme, the invention has the beneficial effects that:

firstly, the low molecular weight bletilla striata fibrous root polysaccharide can obviously inhibit the activity of tyrosinase, and is a natural whitening substance; meanwhile, the antioxidant has good antioxidant activity and is a natural antioxidant; in addition, the bletilla striata fibrous root polysaccharide has small molecular weight, no toxicity and stable system; so that it has the advantages of high activity and no pollution.

Secondly, the raw materials of the traditional rhizoma bletillae fibrous root used as both medicine and food are cheap and easy to obtain, the economic value of the rhizoma bletillae fibrous root is verified, the full utilization of rhizoma bletillae plant resources is realized, and a reference value is provided for further scientific research of the rhizoma bletillae fibrous root. In addition, the polysaccharide is derived from bletilla striata, is safe and non-toxic, and has the potential of developing whitening and cosmetics.

Thirdly, the preparation process of the invention is simple, consumes less energy, is economical, nontoxic, convenient and feasible, and is suitable for industrial production.

Drawings

FIG. 1 is a high performance gel permeation chromatogram of bletilla striata fibril oligosaccharide in an example of the present invention (Retention time on the abscissa and Electrical signal response value on the ordinate).

FIG. 2a is a schematic scanning electron microscope drawing of bletilla striata fibril oligosaccharide at a magnification of x70k in accordance with an embodiment of the present invention.

FIG. 2b is a schematic scanning electron microscope drawing showing that the bletilla striata fibril oligosaccharide of the present invention is at a magnification of x400 k.

FIG. 2c is a schematic scanning electron microscope drawing showing that the bletilla striata fibril oligosaccharide is X2.00k in the present invention.

FIG. 3 is a diagram showing the scavenging effect of bletilla striata fibril oligosaccharide on hydroxyl radicals (Concentration on abscissa) in the present invention.

FIG. 4 is a schematic diagram of the Inhibition effect of bletilla striata fibril oligosaccharide on tyrosinase according to the present invention (Concentration on the abscissa and Inhibition on the ordinate).

Detailed Description

The invention provides bletilla striata fibrous root oligosaccharide and a preparation method and application thereof. Firstly, taking fresh bletilla striata fibrous roots as a raw material, obtaining bletilla striata fibrous root oligosaccharide through water bath, alcohol precipitation, decoloration, protein removal, freeze drying and other processes, and then purifying a crude extract of the bletilla striata fibrous roots by a column separation method.

< preparation of oligosaccharide from bletilla striata fibril >

The preparation method of the bletilla striata fibril oligosaccharide comprises the following steps:

(1) drying the bletilla striata fibrous roots in an oven, crushing, weighing, and dissolving in hot water to prepare a bletilla striata fibrous root solution;

(2) extracting and filtering the solution of bletilla striata fibrous roots, re-extracting the residual residues, combining the two filtrates, decoloring for 50min by using 1.5 wt% of activated carbon, concentrating the decolored filtrate to about 0.3 time of volume under reduced pressure, and carrying out alcohol precipitation on an absolute ethyl alcohol solution with the volume of 3 times of the concentrated solution to obtain obviously layered solid and liquid substances, namely the crude extract of the bletilla striata fibrous roots;

(3) centrifuging the crude extract of the bletilla striata fibrous roots, discarding supernatant, washing the obtained solid with alcohol, then washing with acetone solution, finally rinsing with ethanol solution of the same volume, and volatilizing the alcohol to obtain bletilla striata fibrous root polysaccharide;

(4) dissolving the bletilla striata fibrous root polysaccharide in a small amount of distilled water, then adding trichloroacetic acid to adjust the pH value to 3.0, uniformly stirring, standing overnight, centrifuging the next day, taking supernatant, and freeze-drying to obtain the bletilla striata fibrous root oligosaccharide.

Wherein the molecular weight of the bletilla striata fibril oligosaccharide is 1000-;

the bletilla striata fibril oligosaccharide mainly comprises rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose.

Specifically, in step (1), the temperature of the drying may be 40 to 70 ℃, preferably 40 ℃.

In the step (1), the mass-volume ratio of the bletilla striata fibrous roots to the hot water is 1 g: 10 mL.

In the step (2), the temperature of extraction may be 80-90 ℃, preferably 80 ℃; the time is 3 +/-0.1 h.

In the step (3), the rotation speed of the centrifugation can be 3000-; the time is 5 +/-0.1 min.

The invention can extract bletilla striata fibril oligosaccharide by a hot water extraction method, and can also extract bletilla striata fibril oligosaccharide by supercritical CO₂Extracting polysaccharide by extraction method, ultrasonic extraction method, and reflux extraction method.

< bletilla striata fibril oligosaccharide >

The bletilla striata fibril oligosaccharide of the invention is obtained by the preparation method.

< application of oligosaccharide from bletilla striata fibrous root >

The bletilla striata fibril oligosaccharide can be applied to cosmetics.

In addition, the bletilla striata fibrous root oligosaccharide can be used as an antioxidant to be applied to the fields of medicines, foods and the like.

The present invention will be further described with reference to the following examples.

Example (b):

the preparation method of the bletilla striata fibril oligosaccharide of the embodiment comprises the following steps:

(1) taking fresh bletilla striata fibrous roots as raw materials, drying the raw materials in an oven at 40 ℃, crushing, weighing, and dissolving the raw materials in hot water to prepare a bletilla striata fibrous root solution; wherein the mass volume ratio of the bletilla striata fibrous roots to the hot water is 1 g: 10 mL.

(2) Extracting the solution of bletilla striata fibrous roots with water at 80 ℃ for 3h, carrying out suction filtration, carrying out re-extraction on the residual residues, combining the two filtrates, decoloring for 50min by using 1.5 wt% of activated carbon, concentrating the decolored filtrate to about 0.3 time of volume under reduced pressure, and carrying out alcohol precipitation on an absolute ethyl alcohol solution with 3 times of volume of the concentrated solution to obtain obviously layered solid and liquid substances, namely the crude extract of the bletilla striata fibrous roots.

(3) Centrifuging the crude extract of the bletilla striata fibrous roots at 3000r/min for 5min, discarding the supernatant, washing the obtained solid with alcohol, then washing with acetone solution, finally rinsing with an equal volume of ethanol solution, and volatilizing the alcohol to obtain the bletilla striata fibrous root polysaccharide.

(4) Dissolving the bletilla striata fibrous root polysaccharide in a small amount of distilled water, then adding trichloroacetic acid to adjust the pH value to 3.0, uniformly stirring, standing overnight, centrifuging the next day, taking supernatant, and freeze-drying to obtain the bletilla striata fibrous root oligosaccharide.

< experiment >

The bletilla striata fibril oligosaccharides obtained in the above examples were subjected to the following experiments. Measuring the molecular weight of the prepared bletilla striata fibril oligosaccharide by an HPGPC method; analyzing the monosaccharide composition of the product by a GC-MS method; bletilla striata fibrous root oligosaccharide is used as a raw material, the influence of polysaccharide with different concentrations on the clearance rate of hydroxyl free radicals is discussed to be measured by an enzyme-linked immunosorbent assay, and the in vitro antioxidant activity of the bletilla striata fibrous root oligosaccharide is evaluated; the inhibition effect of polysaccharides with different concentrations on tyrosinase is discussed by taking bletilla striata fibril oligosaccharide as a raw material.

< experiment 1>

And (3) measuring the molecular weight:

the purified bletilla striata fibril oligosaccharide was subjected to molecular weight determination using High Performance Gel Permeation Chromatography (HPGPC). Bletilla striata fibril oligosaccharide was dissolved in 0.02mol/L phosphate buffer (pH 6.8) to prepare a sample solution with a concentration of 2 mg/mL. The prepared sample was centrifuged at 10000r/min for 10min, and 20. mu.L of the sample solution was pipetted through a 0.22 μm filter into the HPGPC system. Wherein the column (Shodex SB-804, 300X 8mm) was eluted with phosphate buffer at a flow rate of 0.3mL/min, and the column temperature was maintained at 25 ℃. Calibration standard curves were established using dextran standards of different molecular weights (T3, T6, T10, T40, T100, T500, and T1000). The results obtained are shown in FIG. 1.

As can be seen from FIG. 1, the retention time of the main peak of bletilla striata fibril oligosaccharide (pFSP) is 36.117min, and the molecular weight of pFSP estimated by substituting the linear regression equation is in the range of 1000-1500 Da.

< experiment 2>

Scanning Electron Microscope (SEM) observation:

after taking about 1mg of dried bletilla striata fibril oligosaccharide (pFSP) and placing the dried bletilla striata fibril oligosaccharide (pFSP) in an ion sputtering instrument to be plated with a layer of conductive gold, the appearance of the polysaccharide under the magnification of x70k (figure 2a), x400k (figure 2b) and x2.00k (figure 2c) is observed by a scanning electron microscope.

The purified bletilla striata fibril oligosaccharide is observed in microstructure through electron microscope scanning. FIG. 2 shows that the bletilla striata fibril oligosaccharide (pFSP) is an irregular lamellar structure. This indicates that the strong attraction between functional groups produces aggregation of the polysaccharide chains. In addition, the surface of bletilla striata fibril oligosaccharide (pFSP) is rough, and the smooth surface of the polysaccharide may negatively affect the water-rich property, thereby reducing the solubility of the polysaccharide itself, which is the reason for the high pFSP solubility. The shape and the form of the bletilla striata fibril oligosaccharide are changed due to the change of hydrogen bonds in the purified polysaccharide molecules. It is predicted that the interactions of the polysaccharide chains in pFSP are reduced and the polysaccharide fragments are more easily torn apart.

< experiment 3>

And (3) monosaccharide composition determination:

accurately weighing 10mg of bletilla striata fibrous root oligosaccharide, adding 2mL of 2mol/L trifluoroacetic acid solution into a round-bottom flask, sealing, hydrolyzing for 6h in a water bath at 100 ℃, and removing hydrolysate by reduced pressure rotary evaporation at 40 ℃. Adding 10mg of hydroxylamine hydrochloride and 0.5mL of pyridine into the hydrolysate, sealing, performing water bath at a constant temperature of 90 ℃ for 30min, frequently oscillating, taking out, and cooling to room temperature; adding 0.6mL of acetic anhydride, sealing, performing water bath at a constant temperature of 90 ℃ for 30min, frequently oscillating, and cooling to room temperature; drying by rotary evaporation at 50 deg.C under reduced pressure; dissolving in 4mL of chloroform, centrifuging at 4000r/min for 5min, collecting supernatant, and analyzing the product by GC-MS (gas chromatography-mass spectrometry) to obtain monosaccharide composition with column length of 150mm × 3mm and column temperature of 30 deg.C. The measurement results are shown in table 1 by taking fucose, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, fructose, GalA and GlcA11 monosaccharide standards as reference substances.

TABLE 1 pFSP results on molar ratios of individual monosaccharides

Table 1 results of the molar ratio of each monosaccharide of the bletilla striata fibril oligosaccharide (pFSP), it can be seen from table 1 that pFSP is mainly composed of the following monosaccharides, which are respectively: rhamnose, arabinose, fucose, xylose, mannose, glucose, galactose. Their molar ratio is 1: 7: 4: 20: 23: 59.

< experiment 4>

Hydroxyl radical clearance determination:

preparing 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL polysaccharide solutions 2mL in test tubes, respectively adding FeSO in equal volume₄Solution and H₂O₂Shaking the solution evenly and standing for 5-10min, adding salicylic acid solution with the same volume, mixing evenly and standing for half an hour. Preparing control solution and blank by using distilled water instead of salicylic acid and polysaccharide solution respectively in the same methodControl solution. The absorbances of the sample solution, the control solution and the blank control solution were measured at a wavelength of 510nm with distilled water as a blank, and the hydroxyl group clearance of the polysaccharide was calculated according to the formula (1), and the measurement results are shown in FIG. 3.

Hydroxyl clearance ═ 1- (sample a-control)/blank a ] x 100% (1)

In the formula: sample A, the absorbance of a sample solution; a, comparing the absorbance of the control solution; blank control A blank control absorbance of blank control solution.

As can be seen from FIG. 3, in the concentration range of 1-5mg/mL, bletilla striata fibril oligosaccharide (pFSP) has a better scavenging effect on hydroxyl radicals, and increases with increasing concentration. When the concentration of bletilla striata fibril oligosaccharide is 5mg/mL, the clearance rate reaches 81.06%, the equation obtained by linear fitting the experimental result is that y is 15.999x +10.851, and R is²0.8582, calculating the half clearance rate IC of hydroxyl radical of bletilla striata fibrous root oligosaccharide according to the equation₅₀It was 2.45 mg/mL.

< experiment 5>

Tyrosinase inhibition assay:

the enzyme inhibition effect of the bletilla striata fibrous root oligosaccharides is evaluated by measuring the tyrosinase activity inhibition rate of the extracted bletilla striata fibrous root oligosaccharides, and the tyrosinase inhibition rate of the obtained polysaccharide is measured by adopting an enzyme labeling method.

(1) Preparation of tyrosinase solution: a certain mass of peeled and cut potatoes was weighed, added with phosphate buffer (pH 6.8) at a ratio of 1: 1(m/v), and then centrifuged. The supernatant was pipetted and added to a PBS solution (pH 6.8) in a certain proportion to a constant volume in a 25mL brown bottle, and the solution was kept in an ice-water bath and used up within 1-2 h.

(2) Preparation of phosphate buffer (PBS, pH 6.8): 0.0717g/mL of Na was taken out₂HPO₄.12H₂O solution and 0.0179g/mL NaH₂PO₄·2H₂O solution 100mL in 500mL volumetric flask, with deionized water to constant volume.

(3) Preparation of 0.05 wt% L-tyrosine solution: 0.05 wt% of L-tyrosine is weighed and dissolved in a certain amount of hydrochloric acid, and then PBS buffer solution is added.

(4) Preparation of sample solution: polysaccharide solutions were prepared in test tubes at concentrations of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, and 5.0mg/mL, respectively.

(5) The experimental water was deionized water.

TABLE 2 pFSP solution formulation

(1) Blank control absorbance determination: firstly, according to the requirements of table 1, accurately weighing each solution, shaking up, preparing C₀A solution; then C is mixed₀The solution was then incubated at 37 ℃ for 10min and the absorbance was measured at 475 nm. Then according to the requirements of table 2, accurately measuring each solution, and uniformly preparing C₁A solution; then water bathing at 37 deg.C for 5-10min, adding tyrosinase solution, and heating at 37 deg.C for 5-10 min; finally, C is determined under the spectral conditions of 475nm₁Absorbance.

(2) And measuring the absorbance of the sample: similarly, each solution was measured accurately and prepared uniformly according to the specific requirements of Table 2₁Solution, T₀A solution; then according to the above measurement C₁Step (2), measuring T under the condition of normal temperature 475nm spectrum₁The absorbance and the inhibition of tyrosinase by fibrous root polysaccharide are shown in FIG. 4.

(3) Calculating the inhibition ratio according to equation 2

Inhibition rate T ═ (C1-T1)/C1 (2)

As can be seen from FIG. 4, in the concentration range of 1-5mg/mL, bletilla striata fibril oligosaccharide (pFSP) has a better inhibitory effect on tyrosinase, and increases with increasing concentration. When the concentration of bletilla striata fibril oligosaccharide is 5mg/mL, the clearance rate reaches 79.33%, and the equation obtained by linear fitting the experimental result is that y is 9.089x +36.881, R²0.9724, calculating the half inhibition rate IC of bletilla striata fibril oligosaccharide to tyrosinase according to the equation₅₀It was 1.44 mg/mL.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.