Patent Application
20070077238
The invention relates generally to the production of selenium compounds by bacteria and, more specifically, to the production of both organic and inorganic selenium compounds by lactic acid bacteria, and specifically bacteria of Pediococcus sp.
Selenium (Se) is an essential trace element discovered by the Swedish chemist, Jöns Jakob Berzelius in 1817. Similar to sulfur, selenium belongs to the group IV of the periodic table. It has six naturally occurring isotopes, namely 74Se, 76Se, 77Se, 78Se, 80Se and 82Se. The element exists in four oxidation states; −2, 0, +4 and +6. The oxidation state −2 is present in hydrogen selenide (H2Se), a highly toxic gas that is readily oxidized to elemental selenium in the presence of air. Elemental selenium (Se0) and its compounds are highly soluble in water and soil, but can be toxic to biological systems at the level of parts per million (ppm). In terms of physical appearance, elemental selenium can exist in gray-black form of metallic hexagonal selenium and in an amorphous white or as monoclinic red (Se8) forms. Inorganic selenium compounds include selenic acid (H2SeO4) and selenious acid (H2SeO3), at the +6 and +4 oxidative states, respectively. Both selenates (SeO4−2) and selenites (SeO3−2) have been reported to be more toxic than the organic selenium compounds. Organic seleno-compounds exist mainly as selenomethionine (SeMet), selenosystine (CySeSeCy) and selenocysteine (SeCys). Biologically, selenium species can be categorized at the enzymatic or genetic level. Selenocysteine is incorporated into the peptide backbone of glutathione peroxidase (GSH-Px), while SeMet replaces methionine in a random manner in amino acid translation. Seleno-amino acids, therefore, contain selenium in the form of selenocysteinyl, selenocystinal, and selenomethionyl residues.
Selenium is of fundamental importance to human and animal health. Humans can derive selenium from both plant and animal foodstuffs, while animals typically obtain the mineral via yeasts, corn and other plant sources. Absorption of selenium occurs mainly at the duodenal, caecal and colonic segments of the intestinal tract and is largely accumulated in the pancreas, pituitary glands and livers of mammals. Animals with selenium deficiency may develop symptoms, such as white muscle disease (WMD) and anemia in cattle and sheep; liver failure, WMD and mulberry heart in swine. Poultry may develop exudative diathesis, reduced growth rate and even death due to lack of selenium in their diets. Selenium also plays vital role in enhancing the immune system of animals and human. For sows fed with selenium-enriched diet, the litter size can be increased. Selenium supplementation during lactation increased the level of total selenium present in milk. In poultry, selenium can improve feather development, increase egg production and improve hatchability. The increased level of selenium could decrease drip loss and improve meat quality of broilers. However, acute toxicity of selenium can also occur at the concentration of between 5 and 25 mg/kg in the animal diets. Chronic selenium toxicity in animals lead to “blind stagger” and “alkali” disease.
The current-Recommended Daily Allowances (RDA) for selenium, established by the Food and Nutrition Board of the National Academy of Sciences (NAS), is 55 μg/day for both male and female adults. Selenium has also been regarded as a high nutritional value and act as antioxidant. The biochemistry of selenium is a complex system that enhances the immune system involves in inflammatory mechanisms that may be linked to diseases such as heart failure, cancer and rheumatoid arthritis in humans (1). Selenium deficiency in human was first reported as the Keshan disease, a type of heart disease called cardiomyopathy that affects young women and children in China. The disease has also been reported in New Zealand and Finland in areas where selenium in the soil is low (2). Although excessive intake of selenium can cause adverse health effects, these are generally observed at doses more than 5 times greater than the RDA.
Schwarz and Foltz found three agents that prevented liver necrosis in rats: vitamin E, cysteine and selenium. The enzyme glutathione peroxidase (GPx) was discovered as a selenium-dependent enzyme (3) and selenocysteine (SeCys) was found to be incorporated into peptide backbone of GPx as an essential component for the catalytic activities of selenium-dependent enzymes (4, 5). The main function of GPx is to protect hemoglobin against oxidative damage by hydrogen peroxide (5-10). The first type is GPx1, of 22 kDa and can be found in many tissues. GPx2 is mainly found in gastrointestinal tract and liver. Plasma GPx (GPx3) can be detected in kidney, heart and lung of mammals, while GPx4 is a phospholipid hydroperoxide found in many tissues. GPx is reported to protect mice from viral-induced myocarditis (11) and benign coxsackievirus in heart muscle in host mice (12). The other types of selenoproteins containing SeCys are iodothyronine-5′-deiodinase (type I and II), which is found in liver and kidney, thioredoxin reductase, Sel-W and Se-P. The selenoprotein Sel-W is a component of WMD that protects animal from the disease. Selenoprotein P, although with unknown function, is an extracellular glycoprotein that contains most of the selenium in plasma. The immune system in a host also has an effect on the association between viral diseases and nutrition. Dietary selenium plays a critical role and could also reduce oxidative stress that lead to pathogenesis of several viral infections, such as hepatitis and influenza (13).
Selenoproteins can be produced via the metabolism of selenium by various organisms. Organic and inorganic selenium may be concentrated in the biomass of plants, mushrooms, yeasts, fungus, Escherichia coli, and Lactobacillus spp. grown in media containing selenite or selenate. Many studies have also reported that selenium-containing compounds may readily enter the pathway of sulfur metabolism, and that O-acetylserine sulphydrylase is likely to be the entry site in Escherichia coli. Selenite metabolism has also been observed in the reduction of selenite-to elemental selenium and the incorporation of selenium into organic molecular structures.
A previous study by Turner et al. has shown that selenate and selenite may have entered the. cells of E. coli through the sulfate permease system (cysA, cysU, cysW) (14). However, only the synthesis of selenate was inhibited when the system is repressed suggesting the uptake of selenite by bacterial cells could be via an undefined carrier. They also reported that selenate exhibits a lower affinity for the sulfate permease pathway and its uptake is repressed by the presence of cysteine. Two organisms, Selenomonas ruminantium and Cldstridium pasteurianum, were discovered to have difficulty in transporting and metabolizing sulfate or selenate (15). Non-specific uptake rates for selenite and selenate by the sulfate permease in different bacterial species may be due to the differences in the kinetics of uptake between species (15). Selenite was believed to be bound to a protein through the vicinal thiol groups and released in the form of metallic selenium after accepting four electrons (16). Nickerson and Falcone suggested that the thiol content of protein could have determined the amount of selenite bound. The studies suggested that arsenite inhibition of the selenite reduction is not reversed by a monothiol substance and that selenite rapidly binds to SH-group in a divalent manner to form —S—Se—S linkages (16). Tomei et al. demonstrated that the transformation of selenate and selenite to elemental Se by Desulfovibrio desulfuricans occurred intracellularly when grown in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor (17).
Size exclusion and cation or anion exchange chromatography are common techniques used for the separation of various selenoamino acids (18-21). Inductively coupled plasma-mass spectrometry (ICP-MS) is a reliable technique that can detect sensitively most elements, inclusive of trace metals (18, 22-26). The ICP-MS technique has a detection limit of parts per trillion (ppt) or less with a 98-percent confidence level, established by several analytical laboratories (23). Various modular configurations involving the use of ICP-MS include quadrupole mass spectrometer, vacuum system or detector with liquid chromatography, high performance liquid chromatography, gas chromatography and/or mass spectrometry (LC-/HPLC-/GC-MS) have been used to study Se-enriched yeasts (18, 23-27).
Inorganic selenium (selenates and selenites) and selenium-enriched yeasts are commercially available as feed supplements in animal diets. Numerous types of selenium-yeast products consisting of varying quantities of organic and inorganic forms of selenium have been marketed in the feed industry (28). Studies have shown that the concentration of SeMet in selenium-yeast was found to range from 23% to 63%. However, the detection of SeMet in commercially available selenium-enriched yeast, Sel-Plex® (Alltech, Inc., USA), using HPLC was reported to involve the use of cyanogen bromide, which generates hydrogen cyanide.
Lactic acid bacteria (LAB) are naturally found in the gastrointestinal tract of human and animals. It is widely believed that they prevent the growth of putrefactive microorganisms in the gut system. Lactic acid bacteria is a group of Gram positive, non-pathogenic bacteria that can ferment carbohydrates to produce lactic acid. These bacteria can also possess proteolytic, lipolytic and β-galactosidase activities. Genera of LAB include Lactococcus, Lactobacillus, Streptococcus and Pediococcus. Lactobacillus acidophilus has been mixed with spray-dried selenium-enriched yeast products as supplements for the equine (29). Studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique have shown that selenium can become incorporated into bacterial proteins of L. delbrueckii subsp. bulgaricus (30, 31). However, no study to date has demonstrated the ability of Pediococcus spp. to take up and convert inorganic selenium into organic forms.
The invention consists of a novel strain of bacteria identified herein as SP80, a non-spore forming and Gram-positive coccus, which is identified with API biochemical and sugar fermentation tests (97.3-99.9% confidence), ribotyping (83% confidence) and 16S rRNA sequencing (99.52%) as Pediococcus pentosaceus. Bacteria of the strain SP80 grow in a. sulfur-limiting medium that has been supplemented up to at least 1000 ppm with inorganic selenium compounds, such as sodium selenite. Analysis of the cell-free extracts of the bacteria showed an uptake of selenium and the presence of both inorganic and organic selenium compounds.
The selenium-enriched bacteria are fed to animals as a source of selenium. In a preferred embodiment, mice fed control diets including a selenium-depleted diet, a diet enriched with inorganic selenium and a diet supplemented with selenium-enriched yeast, were compared with mice fed a diet including the selenium-enriched bacteria. The mice fed the selenium-enriched bacteria showed an improved feed conversion rate and enhanced Glutathione Peroxidase (GPx) activity in heart, kidney and liver tissues, indicating a higher level of absorption and retention of selenium in these tissues.
Inorganic selenium compounds, as used in this disclosure, include salts of selenium, and preferably alkali-metal salts of selenite and selenate, and more preferably sodium selenite.
Organic selenium compounds include seleno-amino acid compounds and complexes, and preferably selenomethionine, selenocystine, selenocysteine, Se-methylselenocysteine, gamma-glutamyl-Se-methylselenocysteine, gamma-glutamyl-selenomethionine, selenocystathionine and Se-adenosyl selenohomocysteine.
Lactic acid bacteria is a group of Gram positive, non-pathogenic bacteria that can ferment carbohydrates to produce lactic acid. Genera of LAB include Lactococcus, Lactobacillus, Streptococcus and Pediococcus.
Materials and Methods
Gastrointestinal tracts from three healthy chickens were obtained from a local market certified by the Agri-Food & Veterinary Authority of Singapore. Sections comprising the duodenum, jejunum and ileum of each intestinal tract were macerated and their contents were inoculated into de Man Rogosa Sharpe broth pH 6.3 (MRS) (Becton, Dickinson, USA) and incubated in an incubator set at 30° C. under 5% CO2 for 24 h. Overnight cultures were streaked for isolation of pure colonies on MRS agar, pH 6.3 before sub-culturing in MRS broth using the same conditions and kept in 40% glycerol at −80° C. for long-term storage. Gram staining and biochemical tests (API® 50 CH test kit; bioMerieux, USA) were performed to identify the strains of lactic acid bacteria isolated from the gastrointestinal tract of healthy chickens.
Results
A total of 120 strains of microaerophilic and anaerobic bacteria were isolated from the duodenum, jejunum and ileum portions of the intestinal tract of healthy chicken using the MRS media, pH 6.5. Using the Gram-staining technique and biochemical tests, the morphologies and identities of these bacteria were identified. It was found that 97 strains of the bacteria isolated were Gram-positive bacteria with the majority showing a rod-shape morphology (Table 1).
aA total of 120 strains of bacteria were isolated from the gastrointestinal tract of healthy chicken. Out of these, 23 strains were Gram-negative bacteria.
Materials and Methods
A strain of LAB, identified herein as SP80 was selected out of the 97 strains previously isolated and screened for growth in media containing sodium selenite (25, 50, 100 and 200 ppm). The bacterium was selected based on the growth rate and ability of the cells to convert sodium selenite to amorphous selenium, indicated by the formation of red-pigmented colonies.
SP80 was grown in MRS broth at 30° C. under 5% CO2 for 24 h. Five-ml portions of the culture were centrifuged at 5700×g for 10 min and the supernatant discarded. The pellet was then resuspended in CHL media (bioMérieux, USA) before dispensing into the cupules of the API® 50 CH test strips. The strips were incubated at 37° C. for 48 h. After 48 h, the results of the biochemical tests were analyzed using the automated identification software, API® LAB Plus™ (bioMérieux, USA). This strain was also found to be heat resistant at 60° C. for 30 min (data not shown). Results of the characterization study indicated that the bacterium has 97.3-99.9 percent identity with Pediococcus pentosaceus (Table 2).
The strain of Pediococcus pentosaceus SP80 isolated in our laboratory fermented nine different types of sugars, including amydalin, arbutin, maltose and trehalose. Consistent with the report by Facklam et al., (1989) P. pentosaceus was shown to react with salicin, maltose and trehalose (32). Our study indicated that P. pentosaceus SP80 is a Gram-positive non-spore forming coccus that grows well under microaerophilic conditions at 30° C. Samples of P. pentosaceus SP80 have been deposited with the American Type Culture Collection (ATCC) under accession number PTA 6736.
Materials and Methods
Culture of Pediococcus pentosaceus SP80 on media containing sodium selenate and selenite. Pediococcus pentosaceus SP80 obtained from −80° C. freezer was resuscitated and grown in MRS broth at 30° C. under 5% CO2 for 24 h. The cells were then inoculated into sulfur-limiting media (SLM; pH 6.0) containing 40 g/L of buffered peptone water (BPW; pH 7.4), 32 g/L Lab-Lemco powder, 16 g/L yeast extract (Oxoid, UK), 80 g/L glucose, 20 g/L sodium acetate trihydrant, 8 g/L di-potassium hydrogen phosphate anhydrous, 0.8 g/L magnesium chloride, 0.2 g/L manganese (II) chloride (Merck, Germany), 8 g/L citric acid and 4 g/L Tween® 80. The medium was then supplemented with sodium selenate (0, 250 ppm) or selenite (0, 1, 10, 50, 100, 250, 500 and 1000 ppm) (Sigma Chemicals, USA). The cultures were incubated at 30° C. under 5 % CO2 for 24 h. Serial dilutions and plate count were performed using BPW and MRS agar, respectively.
Separation of organic and inorganic selenium by anion exchange chromatography. Overnight cultures containing Pediococcus pentosaceus SP80 were centrifuged at 20,000×g for 15 min and washed with phosphate buffered saline (PBS-1×; pH 7.4). The cell pellet containing SP80 was then resuspended in PBS-1× before disruption using a cell-sonicator (Misonix, USA) set at 6 W with 30-sec intervals for 15 cycles. Cell debris was treated with 1.5 M nitric acid (Merck, Germany) at 80° C. for 20 min. Cell free extracts were then obtained after being spun at 1000×g for 15 min. The pH of cell free extract was adjusted to 5.0-5.5 using 1 M sodium hydroxide (Merck, Germany). One-ml portion of the cell free extract was dispensed into a 1-cm (diameter)×20 cm (height) anion exchanger column (BIO-RAD, USA) packed with Dowex® 1-8 X (Sigma Chemicals, St Louis), with particle size of 100-200 mesh, to a height of 10 cm. Elution was performed with 0.01 M of sodium chloride and hydrochloric acid at 0.2 and 0.5 M, respectively. Five-ml fractions of the eluant were collected for Se analysis using ICP-MS.
Detection of total selenium by ICP-MS. An Elan 6100 ICP-MS (Perkin Elmer, USA) at PSB Corporation, Singapore was stabilized for one hour prior to injection of samples. A 3-point standard calibration was performed using the atomic spectroscopy standard at 10, 20 and 50 ppb, respectively. The atomic spectroscopy standard contains 10 ppb each of manganese, copper, rhodium, cadmium, indium, barium, lead and uranium. The accepted correction coefficient was set at less than 0.995. Five-ml portions of samples were then subjected to the Elan 6100 for ICP-MS analysis.
Results and Discussion
Putative strains of LAB were grown on MRS agar plates containing various concentrations of selenate and selenite. Strains that grew on MRS agar plates containing sodium selenate (25, 50, 100 and 200 ppm) formed white colonies, similar to those grown on control medium. In contrast, red-pigmented colonies were observed on agar media containing sodium selenite of up to 200 ppm. Generally, the color intensity of the colonies ranged from pink to dark red, corresponding with the increase in concentrations of sodium selenite used in the media. The coloration of colonies observed in the LAB cells grown on media containing sodium selenite may be due to the presence of amorphous selenium deposited by the bacteria. Gharieb (1995) has demonstrated that the reduction of selenite to elemental selenium on Czapek-Dox agar has resulted in the red-pigmented fungi colonies (33). Candida albicans was shown to rapidly reduce selenite but this reduction was inhibited by methionine, formate, fluoride, dinitrophenol and certain sulfhydryl poisons present in the medium (34).
Our screening experiments have demonstrated that some strains of the isolated 97 LAB were not inhibited by sodium selenite of up to 200 ppm. In addition, a number of these strains were found to be fast growers, with their colonies appearing only after 12 h of incubation (Table 3).
apercentage of bacterial cells that grew
b250 ppm of selenate or selenite in media
The strain of Pediococcus pentosaceus identified herein as SP80 also grows well in a sulfur-limiting medium (SLM) containing up to 1000 ppm of selenite and produces a brick red pigmented culture when compared to those grown in selenate-enriched media. We suspected that SP80 may have metabolized sodium selenite and portions of which were converted to elemental Se. It is possible that excess elemental Se may then be deposited to the exterior of the cells and in the media as brick-red pigment. The reduction of selenite or selenate into elemental Se by microorganisms that causes the appearance of brick red pigments in the culture media were also observed by various researchers (14, 30, 31).
In our current study, selenium was detected in the cell free extract of the HNO3-digested SP80 cells grown in SLM supplemented with 250 ppm of sodium selenite. Using ICP-MS technique, 32.20 ppm of total selenium was detected from hydrolyzed cells of SP80. No selenium was detected in the cell free extract of SP80 grown in the control media. When the cell free extract of selenium-enriched SP80 was passed through an anion exchanger, two organic and inorganic selenium fractions were eluted separately and collected. The fractions were subjected to analysis by ICP-MS and the results of this study indicated that the organic and inorganic fractions contained 4.34 and 21.7 ppm of selenium, respectively (
Accordingly, a novel strain of lactic acid bacteria, strain SP80 was able to grow in a sulfur-limiting medium containing high concentrations of selenate and selenite. The strain has been identified using biochemical and sugar fermentation tests as Pediococcus pentosaceus. Pediococcus pentosaceus SP80 when cultured in a sulfur-limiting medium was found to take up selenite, convert or reduce it to elemental Se, forming a brick-red precipitation. The organic and inorganic selenium present in P. pentosaceus SP80 were fractionated using an anion exchange chromatography technique. The selenium content in both organic and inorganic fractions was analyzed and quantified using ICP-MS analysis. The selenium concentrations in the organic and inorganic fractions were found to be 4.34 and 21.7 ppm, respectively.
Materials and Methods
Bacterial Culture and Culture Conditions. The pure culture of Pediococcus pentosaceus SP80 was resuscitated and grown in a sulfur-limiting medium (SLM; pH 6) supplemented with 10 ppm of sodium selenite (Sigma Chemicals, USA). The bacteria were grown at 30° C. under 5% CO2 condition for 24 h.
Scale-Up Fermentation and Freeze Drying of Bacterial Culture. A 2-L B-Braun Biostat® B-DCU Stirred Tank Fermenter (Braun, Germany) was used to produce 10 liters of fermented culture containing approximately 108 CPU per ml of P. pentosaceus SP80. The medium was sterilized in the fermenter by autoclaving at 121° C. for 20 min. After the medium has cooled to room temperature, a 1-percent overnight seed culture of P. pentosaceus SP80 was inoculated into the pre-sterilized sulfur-limiting medium containing 10 ppm of sodium selenite. The fermentation temperature was maintained at 30° C. The culture was allowed to grow in the fermenter for 24 h. The 24-h culture was then freeze-dried at −40° C. under vacuum.
Design of Trial
Preparation of mice feed supplements and diets. The mice feed was obtained from a local feed importer. The composition of the mice feed is set out in Table 4. The mice pellets were ground and passed through a mesh-20 size sieve.
Experimental feed A is the negative control diet containing ground feed alone (Table 5). Experimental feeds B, C, and D are negative control diets supplemented with sodium selenite, freeze-dried SP80 prepared and described as before, and Sel-Plex®, a commercial source of selenium-enriched yeast (Alltech, USA), respectively (Table 5). Except for the negative control diet, the final concentrations of total selenium in experimental feeds B, C and D were adjusted to 0.3 ppm, respectively.
Pre-treatment of study animals. Thirty-two adult mice used in the experiment were divided into 4 treatment groups (Table 5). Each group consisted of 4 male and 4 female mice. Four mice of the same gender were kept in one cage. There was a short acclimatization period of 1 week before feeding them with the various diets. All mice received the control diet for 1 week before the intervention.
Animal management. All mice were kept at 25° C. in an incubator. Feed and water were available ad libitum. The weight of all mice and the feed consumed was monitored on a weekly basis throughout the period of study.
Glutathione Peroxidase Assay
On the sixth week, mice were randomly selected from each treatment group per cage to be sacrificed and their serum, heart, liver and kidney were obtained for analysis. Prior to dissection, tissues were perfused with 0.9% NaCl containing 0.16 mg/ml of heparin to remove red blood cells and clots. One-gram of tissue was then homogenized in 4-8 ml of cold buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 1 mM DTT. The suspension was then centrifuged at 10,000×g for 20 min at 4° C. After 20 min, the supernatant was removed for analysis using the Cellular Calibiochem®'s Glutathione Peroxidase Assay Kit (Cat# 354104) (Merck KGaA, Germany). The bioavailability of selenium was determined using a spectrophotometer at 340 nm to measure the glutathione peroxidase (GPx) activity in the serum, heart, liver and kidney of the mice from each treatment group. The calculation of GPx activity is determined by dividing the sum of the net rate of NADPH and the reduction X sample factor by 0.0062.
Analysis of Data
Results from the treated groups were-compared with the control groups and between treatments using the GraphPad Prism statistical package (GraphPad Software, Inc., USA) to test for significant difference at p<0.05. Body weight gains, feed intakes, feed conversion ratios and GPx activities were also subjected to analyses of variance and the standard errors of difference tested for statistical significance.
Analysis of the Results
In the current study, mice are fed on different diets containing organic and inorganic selenium from various sources. Regardless of their origins, the mineral, selenium has to be ingested, absorbed and retained within the mice in order for it to perform its function. Glutathione Peroxidase (GPx) is a tetrameric protein weighing approximately 85,000 D. GPx has 4 atoms of selenium bound as seleno-cysteine moieties that confer the catalytic activity. Therefore, the Glutathione Peroxidase assay was performed to compare the glutathione peroxidase activities between different treatment groups to reflect the level of Se absorbed and retained, in various tissues of mice fed with selenium from various sources. In our study, the GPx activity in various organs was consistently higher in male mice fed on diets containing Se-enriched bacteria when compared to the negative control diets (P<0.05) (Tables 7-9). Results from the mice trial also consistently showed that the GPx activity in livers, kidneys and hearts was higher in mice fed on diets containing Se-enriched bacteria compared to those that fed on Se-enriched yeast (P<0.05) (Tables 7-9). Our data also indicated that the activities of GPx within the liver and heart of female mice fed on diets containing Se-enriched bacteria were higher compared to the negative control diets (P<0.05) (Tables 8, 9). In terms of feed intake efficiency, it is interesting to note that although a standard 5-gram of the respective feed was given to each mouse per day, mice fed with Se-enriched bacteria showed a significant enhancement in feed conversion rate (FCR) of the male mice compared to those fed on negative control diets (P<0.005) (Table 6).
a,bValues with different superscripts in the same column are significantly different [P < 0.05]; each mean value represents an average of 4 replicates of male or female mice per cage throughout 6 weeks of feeding.
a,bValues with different superscripts in the same column are significantly different [P < 0.05]; each mean value represents an average of 4 replicates of male or female mice per cage after 6 weeks of feeding.
a,bValues with different superscripts in the same column are significantly different [P < 0.05]; each mean value represents an average of 4 replicates of male or female mice per cage after 6 weeks of feeding.
a,bValues with different superscripts in the same column are significantly different [P < 0.05]; each mean value represents an average of 4 replicates of male or female mice per cage after 6 weeks of feeding.
The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.
