COMPOSITIONS AND METHODS FOR THE MAINTENANCE OF ADEQUATE IRON INTAKE IN A MAMMAL
20170258830 · 2017-09-14
Assignee
Inventors
Cpc classification
A61K9/0095
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A23P10/30
HUMAN NECESSITIES
International classification
A61K9/50
HUMAN NECESSITIES
A23P10/30
HUMAN NECESSITIES
Abstract
A preparation of microspheres comprises a multiplicity of discrete microspheres, in which the microspheres comprise iron entrapped within a protein matrix, and in which the protein in the protein matrix is at least partially denatured. The protein of the protein matrix comprises denatured, de-calcified, whey protein. Methods for making the preparation of microspheres are also described.
Claims
1. A preparation of microspheres comprising a multiplicity of discrete microspheres, in which the microspheres comprise iron entrapped within a protein matrix, and in which the protein in the protein matrix is at least partially denatured.
2. A preparation of microspheres according to claim 1 in which the protein in the protein matrix is depleted in divalent metal ions.
3. A preparation of microspheres according claim 2 in which the protein in the protein matrix is decalcified.
4. A preparation of microspheres according to any preceding claim in which the protein matrix comprises whey protein, another milk protein, or pea protein.
5. A preparation of microspheres according to any preceding claim in which the protein matrix comprises denatured, decalcified, whey protein.
6. A preparation of microspheres according to any preceding claim in which the microspheres comprise 0.1% to 5% iron (dry weight %).
7. A preparation of microspheres according to any preceding claim in which the microspheres comprise 0.1% to 2.0% iron (dry weight %).
8. A preparation of microspheres according to any preceding claim in which the microspheres comprise 0.1% to 1.0% iron (dry weight %).
9. A preparation of microspheres according to any preceding claim in which the microspheres comprise 0.25% to 0.75% iron (dry weight %).
10. A preparation of microspheres according to any preceding claim in which the iron is ferrous (II) iron, optionally selected from iron sulphate, iron chloride, iron ascorbate, and an iron carbohydrate complex.
11. A preparation of microspheres according to any of claims 1 to 9 in which the iron is a ferric (III) iron, optionally selected from iron (III) maltose or iron (III) maltol.
12. A preparation of microspheres according to any preceding claim in which the microspheres comprise a coating of a citrate, phosphate, or ascorbate salt.
13. A preparation of microspheres according to any preceding claim in which the microspheres have a mean particle size of 60-250 μm as determined using laser diffraction.
14. A preparation of microspheres according to any preceding claim in which the microspheres have a mean particle size of 60-150 μm as determined using laser diffraction.
15. A preparation of microspheres according to any preceding claim in which the microspheres have a mean particle size of 60-120 μm as determined using laser diffraction.
16. A preparation of microspheres according to any preceding claim in which the protein matrix of the microspheres comprises a gelled filamentous protein network.
17. A preparation of microspheres according to any preceding claim in which the microsphere comprises 75-95% at least partially denatured protein that is optionally depleted in divalent metal ions.
18. A preparation of microspheres according to any preceding claim in which the microsphere comprises 85-95% at least partially denatured protein that is optionally depleted in divalent metal ions.
19. A preparation of microspheres according to any preceding claim in which the microsphere comprises (as a dry weight %): 75-95% denatured, optionally de-calcified, whey protein; and 0.1-5.0% iron.
20. A preparation of microspheres according to any preceding claim in which the microsphere comprises (as a dry weight %): 85-95% denatured, optionally de-calcified, whey protein; and 0.1-5.0% iron.
21. A preparation of microspheres according to any preceding claim in which the microsphere comprises (as a dry weight %): 75-95% denatured, optionally de-calcified, whey protein isolate; and 0.1-5.0% iron.
22. A preparation of microspheres according to any preceding claim in which the microsphere comprises (as a dry weight %): 85-95% denatured, optionally de-calcified, whey protein isolate; and 0.1-5.0% iron.
23. A preparation of microspheres according to claim 1 comprising 75-95% at least partially denatured, de-calcified, whey protein (% dry weight) and 0.1 to 5.0% ferrous or ferric iron (% dry weight), in which the microspheres have an average dimension of 60-250 μm as determined using laser diffraction.
24. A preparation of microspheres according to any preceding claim suspended in a liquid.
25. A preparation of microspheres according to any preceding claim in a dried format.
26. A dietary supplement comprising a preparation of microspheres according to any preceding claim.
27. A dietary supplement according to claim 26 and provided as a unit dose product.
28. A dietary supplement according to claim 27 in which each unit dose product comprises 10-40 mg iron.
29. A pharmaceutical composition comprising a preparation of microspheres according to any preceding claim in combination with a suitable pharmaceutical excipient.
30. A preparation of microspheres according to any of claims 1 to 25, for use in a method of treating or preventing iron deficiency in a mammal.
31. A preparation of microspheres according to any of claims 1 to 25, for use in a method of maintaining adequate iron intake in a mammal.
32. A composition comprising a preparation of microspheres according to any of claims 1 to 25, in which the composition is selected from a food or beverage product, an animal feed, or a health supplement for humans or animals.
33. A method of making a preparation of microspheres according to the invention, comprising the steps of: preparing a suspension of at least partially denatured protein; extruding the suspension through a vibrating nozzle to form a laminar jet in which break-up of the extruded laminar jet into microdroplets is induced by applying a sinusoidal frequency with defined amplitude to the nozzle; and curing the microdroplets in an acidification bath, wherein a separate iron-containing solution is simultaneously extruded through the vibrating nozzle; or wherein the acidification bath comprises iron; or wherein a separate iron-containing solution is simultaneously extruded through the vibrating nozzle and the acidification bath comprises iron.
34. A method according to claim 33, comprising the steps of: preparing the iron-containing solution; separately preparing the suspension of at least partially denatured protein; delivering the solution and suspension to the nozzle; simultaneously extruding the solution and suspension through the nozzle in a laminar jet in a manner that produces the microdroplets; and curing the microdroplets in the acidification bath such as an acetate bath to generate the microspheres comprising the iron entrapped within a crosslinked protein matrix.
35. A method according to claim 33, comprising the steps of: extruding the suspension of at least partially denatured protein through the vibrating nozzle; and curing the microdroplets in the iron-containing acidification bath to generate the microspheres comprising the iron entrapped within a crosslinked protein matrix.
36. A method according to any of claims 33 to 35 in which the acidification bath comprises an acetate buffer having a pH of 3.5 to 4.5, a concentration of 0.15M to 0.25M, and optionally a temperature of 40-50° C.
37. A method according to any of claims 33 to 36 in which the microspheres are immersed in a buffer comprising a citrate, ascorbate or phosphate salt for a period of time sufficient to allow coating of the microspheres with a citrate, ascorbate or phosphate salt.
38. A method according to any of claims 33 to 37 in which the microspheres are dried.
39. A method according to any of claims 33 to 38 in which the protein suspension or solution comprises 5-15% de-calcified denatured whey protein (w/v).
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0073] In this specification, the term “microsphere” should be understood to mean a particle, typically a generally spherical particle, having a mean particle spherical diameter of 60-250 μm, 60-150 μm, 60-120 μm, and preferably 60-100 μm as determined using the laser diffraction technique and comprising a gelled filamentous network formulated from at least partially denatured protein material for the space-filling effect of iron encapsulation. The term “discrete” means that the microspheres are separate from each other and not-interconnected. The term “de-calcified” as applied to microspheres should be understood to mean that the microspheres comprise less than 1.0%, preferably less than 0.1%, and ideally only trace amounts of calcium (dry wt %) as determined by standard HPLC methodologies.
[0074] Particle size analysis was performed by dispersing the microencapsulated iron in MilliQ water after which size distribution was measured at 25° C. in triplicate for each manufactured batch by dynamic light scatter (Mastersizer 2000, Malvern, Worcestershire, UK). Microsphere diameter and size distribution were determined as a function of polymerization time, iron concentration and pH of whey protein suspension. In this way, the optimum conditions or reproducible microsphere size was determined. Encapsulated iron particles had a spherical shape with a specified size ranging between 0.2-2000 μm. For the purpose of this invention, the particle size distribution was measured by dispersity (D, formerly polydispersity index) which is a measure of the distribution of particle size in a fluid; the higher the value of dispersity the lower the uniformity in particle size. The range for different iron formulations within this invention was 0.25-0.5 (Mastersizer 2000).
[0075] Production of mono-dispersed microspheres with a very narrow size distribution typically involves the generation of a steady flow of the whey protein polymer through the nozzle at controllable rates, which will provide optimum breakup of the extruded liquid jet into droplets optimally suitable for ferrous encapsulation (
[0076] Microspheres size can be varied within a certain range by regulating the frequency of vibration and/or the flow rate of the protein with higher frequencies and lower jet velocities enabling the generation of smaller whey protein-iron droplets. Nonetheless, the main factor governing iron microsphere size using the present invention is the nozzle diameter, either single or concentric whereby the final microsphere diameter is approximately 1.25× the size of the chosen nozzle, using this specific technique.
[0077] Optimal breakup of the jet results in the formation of a single bead chain, which can be monitored using a stroboscopic lamp (
[0078] Suitably, to avoid coalescence in droplets in the air, the protein suspension is adjusted to a pH above the isoelectric point (pI) most suitable to induce a strong negative charge on the denatured de-calcified whey protein droplets. This generates droplet repulsion and dispersal of the chain into a cone-like shape (
[0079] Suitably, when the protein is whey protein, the whey protein suspension has a pH value above 4.8, typically above 5.2, and ideally above 5.4. Having a strong negative charge on the denatured de-calcified whey protein droplets has been shown to provide for improved microsphere formation. Thus, the pH of the whey protein “feed” solution/suspension to the nozzle is important to help generate optimum microsphere morphology at a reproducible size.
[0080] Suitably, the microspheres are capable of binding to the intestine, especially the lumen of the duodenum or upper jejunum, and releasing the iron at the lumen. In one embodiment, the microspheres have a porous surface, as determined using AFM techniques.
[0081] Atomic force microscopy (AFM) images were also obtained using Asylum Research MFP-3D-AFM (Asylum Research UK Ltd., Oxford, UK) to investigate the frequency of pores on the surface of the microspheres. The microcapsules are poured onto microscope glass slides, incubated for slide adsorption for approx. 20 minute, washed with ultraclean water and dried in a nitrogen flow leading to a linear surface coverage of microspheres on the slide. Suitable microsphere samples could then be chosen in transmitted light DIC contrast prior to AFM scanning. The AFM head is then placed onto a Zeiss Axiovert 200 inverted optical microscope. AFM analysis was relatively slow since microspheres were delicate and easily damaged mechanically if the force exerted by the AFM tip was too high. Microscope images were obtained in intermittent contact mode with standard silicon non-contact cantilevers (NCH) provided by Asylum Research, UK. Resonance frequency was approx. 300 kHz and spring constant of 40 N/m. Digital resolution was 512×512 pixels. The tip radius of cantilever was less than 10 nm and the opening angle between 40° and 50°. The applied force between the cantilever tip and ferrous capsules was minimised in order to alleviate the influence of the AFM tip on the capsule surface topography during pore scanning Measurements were started by scanning a random area of 1 m, with subsequent zoom to 20 nm. These images were used to evaluate the morphology of microsphere pores. Scan size was decreased gradually until pores could be viewed clearly. Iron-whey protein microspheres were analysed and scanned using typical working distance of 2-8 nm for visualization of the pores.
[0082] Ideally, the microspheres have a coating of a promotor capable of opening tight junctions in epithelial cells. Preferably, the promotor is a citrate salt, ideally tri-sodium citrate, or a phosphate salt, ideally di-sodium phosphate, or another salt such as an ascorbate salt. This additional coating was required to enhance iron bioavailability.
[0083] Typically, the microsphere of the invention comprises (as a dry weight %): [0084] 75-95% at least partially denatured protein that is optionally depleted in divalent metal ions; and [0085] 0.1-5.0% iron.
[0086] Typically, the microsphere of the invention comprises (as a dry weight %): [0087] 75-95% denatured, optionally de-calcified, protein (optionally whey protein); and [0088] 0.1-5.0% iron.
[0089] Preferably, the microsphere of the invention comprises (as a dry weight %): [0090] 85-95% denatured, optionally de-calcified, protein (optionally whey protein); and [0091] 0.1-5.0% iron.
[0092] Suitably, the microsphere of the invention comprises (as a dry weight %): [0093] 75-95% denatured, optionally de-calcified, whey protein isolate; and [0094] 0.1-5.0% iron.
[0095] Ideally, the microcapsule of the invention comprises (as a dry weight %): [0096] 85-95% denatured, optionally de-calcified, whey protein isolate; and [0097] 0.1-5.0% iron.
[0098] The term “as a dry weight %” means as a weight % of the microcapsule in a dry format (i.e. having less than 1% water), for example a microcapsule in a spray dried format.
[0099] In this specification, the term “at least partially denatured” should be understood to mean a protein preparation that comprises at least 10%, 20%, 30%, 40% or 50% denatured protein as determined by Ellman's technique (detailed below).
[0100] In this specification, the term “denatured whey protein” should be understood to mean a whey protein preparation that comprises greater than 96%, 97%, 98% or 99% denatured whey protein as determined by Ellman's technique (detailed below), and which is substantially free of non-denatured whey protein and ideally also substantially free of hydrolysed or partially hydrolysed whey protein. Hence, the use of fully denatured protein is the optimized encapsulation material to be used for the delivery of ferrous iron in microspheres. The denatured whey protein is generally a denatured whey protein concentrate or denatured whey protein isolate. Methods for denaturing whey protein will be known to those skilled in the art, and include heat denaturation and pressure-induced denaturation. In a preferred embodiment of the invention, the whey protein is heat denatured at a temperature of 70° C. to 140° C., preferably 78° C. to 121° C. Suitably, when the whey protein has a pH within the 5-9 range and a concentration of greater than 7%, it is heated at a temperature of greater than 70° C. for more than 15 minutes. Typically, when the whey protein has a pH within the 5.5-7.5 range and a concentration of greater than 9%, it is heated at a temperature of greater than 70° C. for more than 25 minutes is heat denatured for a time of between 30 and 50 minutes. Usually, the whey protein is agitated during denaturation. Normally, the β-lactoglobulin has a degree of denaturation of at least 60%, 70%, 80%, 90% or 95%, preferably greater than 80%, as determined using HPLC technique.
[0101] Ellman's reagent (2-nitro-5-mercaptobenzoic acid, DTNB) was utilised to determine the number of free thiol groups available after protein denaturation. The presented assay was performed in the absence of any reagents such as urea or SDS, because under these conditions, only the thiol groups of interest, those of the surface of the protein aggregate, were determined for encapsulation purpose. The number of thiol groups free for ferrous encapsulation was calculated using a molar extinction coefficient for DTNB of 13,600 M.sup.−1 cm.sup.−1.
[0102] Generally, denatured protein is first “activated” by heat to fully denature and unfold its globular structure. This creates a protein network, driven by physical interactions for iron encapsulation. Hence, the polymerisation conditions and residence time are highly important for the rigidity and strength of denatured de protein microspheres for successful iron delivery to the duodenum and upper jejunum of mammals.
[0103] Typically, the protein employed in the process of the invention has at least 90%, 94% or 98% protein content (on a moisture and fat free basis).
[0104] Suitably, the protein is depleted in divalent metal cations. Ideally, the protein is de-calcified. In this specification, the term “depleted in divalent metal cations” or “de-calcified” as applied to the protein should be understood to mean that the protein has most of its divalent metal cations (or calcium) replaced with monovalent cations, as determined using a technique described in http://en.wikipedia.org/wiki/Cation-exchange_capacity. Preferably, the protein, ideally denatured protein, comprises less than 1% divalent metal cations (or calcium), more preferably less than 0.1% divalent metal cations (calcium), and ideally only trace amounts of divalent metal cations (or calcium) (w/v). Many methods of divalent metal cation depletion (or de-calcification) of protein will be apparent to those skilled in the art, including (a) cation exchange, (b) acidification to pH 4.6-7 with subsequent dialysis and/or ultrafiltration and/or diafiltration, and or (c) through use of a divalent metal cation (calcium) chelating or sequestering agent.
[0105] Preferred denatured whey protein (i.e. WPI) for encapsulation purposes have calcium removed by a cation exchange method for efficient encapsulation of ferrous compounds for improved bioavailability in the blood serum. Preferably, the cation exchange has been performed on a resin bearing strongly acidic groups, preferably sulfonate groups. The functional groups are sulphonic acid groups that can be obtained in the Na.sup.+ form or alternatively converted to the K.sup.+ form. The use of the Na.sup.+ or K.sup.+ form is preferred.
[0106] Typically, the whey protein (i.e. WPI) applied to the cation exchanger preferably has the pH in the range of 5.0-8.0, more preferably 5.5-6.5. Once whey protein has passed through the column its pH increases. If it increases above 7.0, it will generally be adjusted to about 6.5-7.0 to make it more possible to mask the taste of ferrous compounds during proposed encapsulation procedures.
[0107] The treatment of whey protein (i.e. WPI) for use as an encapsulation agent for iron, typically ferrous iron, firstly requires the removal of calcium by acidification, followed by dialysis and ultrafiltration, the pH is adjusted to be in the range 4-8, preferably 4.4-7.0, more preferably 4.6-6.8. The preferred ultrafiltration membrane requires a nominal molecular weight cut off of 10,000 Daltons or less. For encapsulation purposes, it is preferred to neutralise (pH 5-7) the solution and filter the solution (through 0.45 micron) to remove any denatured material generated during the powder production. A chelating agent such as citric acid, citrate or typical food acidulants can also be added in order to remove the calcium. In general, chelating agents may be used before, during or following ultrafiltration or independently of an ultrafiltration or diafiltration. However, for this application, it is preferable to remove calcium using a chelating agent such as citrate before the ultrafiltration process.
[0108] Suitably, the concentration of the at least partially denatured protein solution/suspension is from 4 to 30%, preferably from 7 to 30%, and ideally from 9 to 16% (w/v). Typically, the protein is whey protein, ideally denatured de-calcified protein, optionally in the form of a dispersion, in which partial gelation of the β-lactoglobulin produces a protein agglomerates. Typically, the suspension is subject to a filtration process prior to being delivered to the nozzle. In one preferred embodiment, the suspension is passed through a plurality of filters having a gradually decreasing pore size. Ideally, the final filter has a sub-micron pore size, for example 0.1 to 0.9 microns. Ideally, the second liquid stream has a pH of 4.8-6.8, the pH ideally suited for the generation of a negative charge on the denatured whey protein droplets during extrusion.
[0109] Ideally, the at least partially denatured (optionally de-calcified) whey protein employed in the process of the invention has a β-lactoglobulin content of at least 30%, 40%, 50%, 60%, 70% or 80% (w/w).
[0110] In this specification, the term “ferrous iron” should be understood to mean iron in a form having a +2 oxidation state, for example an iron II compound. Examples of ferrous iron include iron II salts such as ferrous sulfate, ferrous chloride, ferrous nitrate or iron oxides, or heme iron. Preferably, the ferrous iron is an iron II salt. For the purpose of this invention, the details for the spectrophotometric technique are provided for the determination ferrous iron concentration in microspheres (via calorimetric analysis). Microspheres are reacted with specialized reagent to form a colored microsphere with complexed ions. The intensity of the colored species is measured using a Cary spectrophotometer. A calibration curve (absorbance versus concentration) is constructed for iron +II and the concentration of the unknown iron in microspheres samples. This methodology is based on the change in the intensity of the colour of a solution with variations in concentration. Colorimetric methods represent the simplest form of ferrous iron analysis for this present invention.
[0111] It is possible to determine iron concentrations at the ppm level. 2,2′-Bipyridyl (bipy), Mw=156.20, forms an intensely red complex with iron (II) which may be exploited to determine iron concentrations in the ppm range. The reaction is:
3bipy+Fe.sup.2+<===>Fe(bipy).sub.3.sup.2+
[0112] The complex formed with encapsulated ferrous iron conforms to an octahedral geometry with coordinate covalent bonds being formed between the adjacent sp.sup.3d.sup.2 hybrid orbital's of the Fe.sup.2+. The complex is chiral; there are left-handed and right-handed non-superimposable optically active forms. The molar absorptivity of the iron-bipyridyl complex is 8650 L/mol/cm at the wavelength of maximum absorbance. The complex forms rapidly, is stable over the pH range 3 to 9, efficiently measures iron(II) concentrations in the range of encapsulation utilized in the present invention, and also lowers levels such as 0.5 to 8 ppm.
[0113] Suitably, the ferrous iron solution has a pH of less than 5, and ideally of less than 4.5.
[0114] In this specification, the term “citrate, ascorbate or phosphate salt” should be understood to mean sodium citrate, ideally tri-sodium citrate or alternatively ascorbic acid, and phosphate salt should be understood to mean di-sodium phosphate. The citrate, ascorbate and phosphate salts have been found to significantly improve the absorption of ferrous iron in the mammalian gastrointestinal tract. Without being bound by theory, it is believed that this is due to the citrate, ascorbate or phosphate acting as an absorption promotor, opening tight junctions in epithelial cells lining the lumen of the GI tract, and thereby facilitating delivery of the ferrous iron compounds. Citrate also binds calcium, a non-competitive inhibitor of the divalent metal transporter DMT1. Suitably, the citrate or phosphate bath has a salt concentration of 0.2 to 0.4M, typically 0.25 to 0.35M, and ideally about 0.4M. Typically, the citrate or phosphate bath has a pH of 4-6, suitably about 4.5 to 5.5. Generally, the citrate or phosphate bath has a temperature of 15-25° C., typically 18-22° C.
[0115] In this specification, the term “acidification bath” refers to a bath composed of an organic acid, for example acetic acid, that has a pH sufficiently low to cure the microdroplets to form solid microspheres. The bath is typically free of calcium ions. Suitably, the bath has an organic acid concentration of 0.1 to 0.3M, typically 0.15 to 0.25M, and ideally about 0.2M. Typically, the bath has a pH of 3 to 4.5, suitably about 4. Generally, the bath has a temperature of 40-50° C., typically about 45° C. Typically, the acidic curing solution comprises a surfactant to prevent or inhibit agglomeration of the formed microspheres. Suitably, the surfactant is a polysorbate surfactant, ideally Tween 20.
[0116] Suitably, the formed microspheres are subject to an extended curing period in the acidification bath, for a period of at least 15 minutes (after gelation), and preferably for a period of at least 20 minutes. In a preferred embodiment of the invention, the formed microspheres are cured for a period of time from 20 to 180, 20 to 120, or 20 to 60 minutes. Ideally, the acidic curing solution is agitated during the curing process.
[0117] The microspheres of the invention are typically capable of surviving intact during passage through the mammalian stomach and capable of releasing the ferrous iron in the gastrointestinal tract distally of the stomach, for example in the small intestine. The term “surviving intact in the stomach” means that the microspheres are resistant to gastric and peptic break-down and release of iron in the gastric conditions of the mammalian stomach during gastrointestinal transit.
[0118] A preferred method of producing the microdroplets is a vibrating nozzle technique, in which the denatured de-calcified protein and iron salt are prepared separately and not mixed until just prior to or during extrusion through a nozzle and laminar break-up of the extruded laminar jet is induced by applying a sinusoidal frequency with defined amplitude to the spray nozzle. Examples of vibrating nozzle machines are the ENCAPSULATOR (Inotech, Switzerland) and a machine produced by Nisco Engineering AG, or equivalent scale-up version such as those produced by BRACE GmbH and the like.
[0119] Typically, the nozzle has an aperture of between 60 and 250 microns, preferably between 100 and 200 microns, suitably 140 and 160 microns, and ideally about 150 microns.
[0120] Suitably, the frequency of operation of the vibrating nozzle is from 900 to 3000 Hz. Generally, the electrostatic potential between nozzle and acidification bath is 0.15 to 0.3 V. Suitably, the amplitude is from 4.7 kV to 7 kV. Typically, the falling distance (from the nozzle to the acidification bath) is less than 50 cm, preferably less than 40 cm, suitably between 20 and 40 cm, preferably between 25 and 35 cm, and ideally about 30 cm. The flow rate of suspension (passing through the nozzle) is typically from 3.0 to 10 ml/min; an ideal flow rate is dependent upon the nozzle size utilized within the process.
[0121] In one embodiment, the process involves a step of detecting the size of the initial microspheres generated, comparing the detected size of the microspheres with a predetermined desired size, and wherein the detected size differs significantly from the predetermined desired size, the microspheres are diverted away from the acidification bath until the detected size correlates with the predetermined desired size.
[0122] In this specification, the term “composition” should be understood to mean a composition of matter that comprises microspheres of the invention. Suitable compositions include comestible products such as food products and beverages, and food supplements in any form, for example unit dose products, powders, and the like.
[0123] Typically food products include health drinks, yoghurts and yoghurt drinks, health bars, and the like.
[0124] The preparation of microspheres of the invention may be provided in a dried form, for example a spray-dried, drum dried, dehydrated, or freeze dried form, or they may be provided as a suspension is a suitable solvent, for example water.
[0125] The present invention also provides pharmaceutical compositions. Such compositions comprise an effective amount of the microspheres, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In this specification, the term “effective amount” should be taken to mean an amount of the microspheres of the invention which results in a clinically significant inhibition, amelioration or reversal of development or occurrence of iron deficiency. This may be enough to prevent the occurrence of iron deficiency, such as maintaining adequate amounts of iron in the body (sub-clinical), or therapeutic use in treatment of clinical iron deficiency.
[0126] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the preparation of microspheres are administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain an effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[0127] Denatured de-calcified whey protein isolate (WPI) is preferable for the generation of robust encapsulation matrices and also contains an ideal protein gelation character in order to achieve controlled release or ferrous (Fe2.sup.+) molecules from gelled protein encapsulation microspheres, with a spherical diameter less than 250 microns. Whey protein concentrate (WPC) is also a possible encapsulation material however, higher concentrations are required to adjust for the low (but significant) levels of fat, cholesterol and carbohydrate, in the form of lactose.
[0128] One aspect of this technology involves the use of denatured de-calcified whey protein isolate/concentrate for the efficient encapsulation of iron supplements, ideally ferrous (Fe2.sup.+) supplements. This represents a novel processing step for commercial encapsulation of iron for targeted bioavailability. Commercial WPI has low (but significant) levels of calcium; hence it is preferably removed in order to generate an optimum encapsulation material for iron encapsulation. Hence, this processing step is preferred for i) efficient iron compound encapsulation and ii) enhanced bioavailability of iron within the blood serum of mammals.
[0129] In general, when iron and milk proteins are declared on the label together, iron absorption will be compromised as a result of calcium ions present in the dairy source. Hence, the efficient removal of calcium ions provides a viable solution for commercial iron encapsulation and targeted delivery to the blood serum. Functional denatured de-calcified whey protein microspheres have shown the ability to stabilise and deliver iron in the blood serum of mammals.
[0130] This initial process step involves WPI treatment by ion exchange to replace calcium with monovalent cations and dried to form a proteinaceous ingredient useful for the preparation of encapsulated ferrous iron supplements. Methods of de-calcification include (i) cation exchange on an ion exchanger charged substantially with a single species of monovalent cation (<90% of the exchangeable ions is a single species such as Hydrogen) (ii) acidification to pH 4.6-7 with subsequent dialysis and/or ultrafiltration and/or diafiltration or (iii) by addition of a chelating agent and/or binding a proportion of calcium ions (ionic or colloidal calcium) with a chelating or sequestering agent. In this context of ferrous encapsulation, it is understood that a small proportion of the cations bound to a cation exchange resin may be resistant to exchange with the desired cation
[0131] An alternative methodology for the calcium-depleted WPC for ferrous iron encapsulation applications is prepared comprising:
[0132] (a) providing a low fat milk solution, for example whey protein concentrate, in liquid form;
[0133] (b) removing of calcium ions therein by a method chosen from at least of (i) cation exchange on an ion exchange in a form bearing a monovalent cation species, or (ii) acidification to pH 4.6-7 optionally with subsequent dialysis; and
[0134] (c) concentrating the solution obtained by ultrafiltration, optionally with diafiltration, to form WPI having at least 90% dry weight of protein.
[0135] The calcium-depleted WPI is suitably dried and then re-dissolved in the optimum composition for ferrous encapsulation purposes. De-calcified whey protein isolate (WPI) used to form the ferrous microspheres, was preferably initially denatured at appropriate environmental conditions (pH, salt, solid concentration) to enable the production of a soluble dispersion of protein aggregates suitable for extrusion and encapsulation in the presence of sodium acetate and ferrous sulfate. This invention can be used to stabilize ferrous compounds in the matrix network whey protein microspheres. This process occurs instantaneously when whey protein comes into contact with ferrous sulfate at defined conditions of salt, pH and temperature.
[0136] The preparation of de-calcified whey protein (i.e. WPI) to form ferrous encapsulation material typically involves:
[0137] 1. Dispersion of de-calcified WPI in water with concentrations in the range of 4-30% (w/w), more preferably between 7-30% (w/w), ideally between 9-16% (w/w). This may be achieved using high shear stirring in a blade mixer or Ultra-Turrax in the presence of a surfactant in the range of 0.01-0.1% (w/w) ideally in the range 0.04-0.09% w/w) with a pH in the range of 5.0-9.0, ideally in the pH range 6.0-7.0
[0138] 2. Application of filtration to remove any denatured material with filtration pore size between 5 micron and 0.2 micron, more preferably between 0.45 microns and 1.2 micron. This may be achieved by sterile vacuum filtration.
[0139] 3. Application of heat treatment to expose the specific hydrophobic sites in the protein material to enable controlled ferrous encapsulation. Protein denaturation is suitably performed between 60-140 degrees Celsius, preferably between 70-121 degrees Celsius at pH in the range of 5.0-8.5, preferably in the range of 6.0-8.2. This process is entitled “protein activation” since it exposes protein regions to enable iron encapsulation and disulfide bond formation for protein polymerization in denatured structures.
[0140] Protein denaturation will unfold the globular whey protein molecule to reveal free thiol groups that are required for protein polymerization and gelation. For this embodiment of the invention, Ellman's reagent (2-nitro-5-mercaptobenzoic acid, DTNB) was utilised to determine the number of free thiol groups available after protein denaturation. The presented assay was performed in the absence of any reagents such as urea or SDS, because under these conditions, only the thiol groups of interest, those of the surface of the protein aggregate, were determined for encapsulation purpose. The number of thiol groups free for ferrous encapsulation was calculated using a molar extinction coefficient for DTNB of 13,600 M.sup.−1 cm.sup.−1.
[0141] The turbidity of the denatured de-calcified whey protein suspension was also an important test utilised to verify the optimum heat required to fully denature whey protein solutions. Turbidity measurements were performed at 25° C. on a Cary
[0142] Spectrophotometer (Varian, Netherlands) equipped with a temperature control unit. The turbidity was measured at 500 nm in quartz cuvettes with a path length of 2 mm.
[0143] The denatured protein suspension is generally aseptically extruded through a nozzle into a tempered (45° C.) sodium acetate bath (pH 4.0, 0.2M) with 25 mg ferrous sulfate. The acetate-ferrous gelling bath is typically continuously agitated to avoid coalescence or flocculation of microspheres during protein polymerization i.e. sphere formation. Pliable microspheres were generally polymerized at room temperature, recovered and suitably dispersed in tri-sodium citrate (0.4M) and subsequently washed with sterile water. This method represents protein polymerization as a result of ionic gelation of de-calcified denatured whey protein i.e. denatured protein gelation in the presence of ferrous sulfate in acetate solution.
[0144] Alternatively, the de-calcified denatured protein suspension can be extruded through a concentric nozzle with 25 mg (±0.334 mg) of ferrous sulfate (no flocculation) into an sodium acetate gelling bath (pH 4.0, 0.2M) with continuously agitation to avoid coalescence or flocculation of microspheres during polymerization i.e. sphere formation. Pliable microspheres were polymerized at room temperature, recovered and dispersed in tri-sodium citrate (0.4M) and subsequently washed with sterile water. This method represents protein polymerization as a result of isoelectric focusing i.e. protein gelation at pH conditions close the isoelectric point of denatured de-calcified whey protein.
[0145] The above parameters provide the optimum denatured globular protein rheological property for use of de-calcified denatured WPI as an encapsulation material of this specific ferrous invention. Rheological properties include gel strengths, viscosity and stress-strain characteristics when subject to either static or dynamic deformation.
[0146] Formation of microspheres typically involves use of an encapsulator. Suitably, the iron encapsulator consists of a single orifice (
[0147] Microsphere size can be varied within a certain range by regulating the frequency of vibration and/or the flow rate of the polymer with higher frequencies and lower jet velocities enabling the generation of smaller microspheres of entrapped Ferrous. Nonetheless, the main factor governing microsphere size the nozzle diameter (
[0148] Optimal breakup of the jet results in the formation of a single bead chain, which can be monitored using a stroboscopic lamp (
[0149] Droplet entry into the polymerization solution with ferrous is of paramount importance for microsphere morphology. Upon landing in the agitated bath, droplets are gelled by ionotropic gelation with acetate buffer in the presence of iron. Surface tension issues were overcome by the addition of a surfactant (Tween-20), a common food ingredient. This practical approach significantly reduced surface tension and reduced the incidence of non-spherical microspheres and coalescence by avoiding the delayed departure of Ferrous droplets from the gelling solution surface.
[0150] Monodisperse microspheres were prepared for encapsulation of Ferrous Sulfate using de-calcified denatured whey protein dispersions. In the present invention, the Whey Protein reservoir was delivered to the nozzle via a feed line utilizing nozzles with 40-100 μm diameters. The nozzle was connected via a PTFE membrane to a vibrating device, which was insulated from the surrounding structures by rubber mounts to avoid the generation of resonance frequencies in the system. The flow of the Protein dispersion to the nozzle was accomplished using a peristalic pump with maximum extrusion volume of 1 Litre. The production of <1 litre of microspheres was sufficient to meet the requirements of this preliminary study; hence the encapsulator resembled a batch-reactor on this occasion only. Sterile protein suspensions were equilibrated to room temperature. The denatured Whey Protein suspension was aseptically extruded through a nozzle into a tempered (45° C.) sodium acetate bath, pH 4.0, 0.2M, with 25 mg ferrous sulfate. Protein polymerization at this specific temperature will induce the production of physical interactions such as hydrophobic or Van der Waals interactions. The gelling bath was continuously agitated to avoid coalescence or flocculation of microspheres during polymerization i.e. sphere formation. Pliable microspheres were polymerized at room temperature, recovered and dispersed in tri-sodium citrate and subsequently washed with sterile water. The acetate buffer conditions (0.2M; 45° C.; pH 4.0) are favorable for iron encapsulation since the i) pH is optimum for ferrous stability; ii) the temperature is favorable for iron solubility; and iii) buffer concentration is optimised to enable the entrapment and stability of divalent cations, such as ferrous sulfate. A subsequent curing step in a tri-sodium citrate buffer (0.3M; 20° C.; pH 5.0) further enhances iron bioavailability and bioaccessability in the blood serum.
EXPERIMENTAL
[0151] Generation of Microspheres
(a) De-Calcification of Whey Protein
[0152] This initial process step in the presented invention involves WPI treatment by ion exchange resins to replace calcium with monovalent cations for the preparation of whey protein for encapsulation purposes. The current invention utilized WPI treated with a single (<90% of the exchangeable ions is a single species such as Hydrogen) or mixed resins (where large proportion i.e. approx. 60% of the resin is charged with hydrogen ions and the significantly lower proportion i.e. 40% is treated with sodium ions to produce a de-calcified WPI. In the present invention, approx. 90% resin is charged with strongly acidic groups, preferably sulfonate groups. The functional groups are sulphonic acid groups that can be obtained in the Na.sup.+ form or alternatively converted to the K.sup.+ form to produce a de-calcified WPI with optimum encapsulation properties. This invention utilised a cation exchange on an ion exchanger charged substantially with a single species of monovalent cation (<90% of the exchangeable ions is a single species such as Hydrogen). The WPI applied to the cation exchanger preferably has the pH in the range of 5.0-7.0. When the void volume is eliminated and the column is running the WPI solution, the pH requires regular adjustment to pH 6.5-7.0 using 0.2M HCl. The following step involves acidification using citrate (0.2M) to pH 4.6-7.0 and filtration (through 0.45 micron) to remove any denatured material generated during ion exchange. The next step involves ultrafiltration using a membrane with 10,000 Daltons or less Molecular weight cut-off (MWCO).
(b) Encapsulation of Ferrous Iron—Example 1
[0153] Calcium-depleted WPI is dried and then re-dissolved in the optimum composition for ferrous encapsulation purposes. The ferrous iron encapsulation system was prepared using the calcium-depleted WPI preferably contains (per 100 gram) from 125 mg—elemental iron and 95 gram protein, in absence of calcium. Microspheres composed of a ferrous sulfate gel core and a hydrogel membrane were prepared using the co-extrusion jet-break-up technique. A stock solution of whey protein was prepared in phosphate buffer (pH 7; 2M) in a blade mixer or Ultra-Turrax in the presence of a surfactant in the range of 0.01-0.1% (w/w) at pH range 6.0-7.0. The solution is filtered through a 0.45-0.2 microns under a pressure of 4-65 bar prior to ensure the removal of denatured protein material generated during powder processing. Whey protein isolate (WPI) was subsequently heat-denatured at appropriate environmental conditions (pH 7.0, >78° C.; 4-9% w/w protein content). Heat treatment is performed ideally between 70-140° C. at pH in the range of 5.0-8.5. Heat denaturation is performed under agitation (150-200 rpm) to enable the production of a soluble suspension of protein aggregates suitable for ferrous encapsulation. Heat denaturation is performed for a min. 30 minutes/max. 90 minutes to allow full denaturation and exposure of specific hydrophobic sites in the whey protein molecule. It is noteworthy that the protein requires full denaturation; this process fully denatures whey protein to permit the exposure and “unfolding” of hydrophobic sites on the globular protein structure for optimum ferrous encapsulation. This step is entitled the “Protein Activation” due to the fact that it “activates” whey protein for an inventive ferrous encapsulation process via denaturation processes.
[0154] After Protein Activation (i.e. heat denaturation), the solution of aggregates ware rapidly cooled to 20° C. using ice. The solutions are then diluted to approx. 5-9% (w/w) protein concentrations (using MilliQ water) before modification and stored at 4° C. for 6-8 hours with no agitation.
[0155] After heat denaturation, dilution and specified storage, the protein suspension was adjusted to optimum pH value to generate strong negative charge on the protein particles (i.e. >pH 4.8, ideally greater than pH5.1, the isoelectric point of β-Lactoglobulin (β-Lg). Following protein equilibration, the suspension of negatively charged protein aggregates are aseptically extruded through a single nozzle into a tempered sodium acetate bath (45° C.; pH 4.0, 0.2M), containing 25 mg ferrous sulfate/200 mL. Spherical microspheres were obtained by the application of a vibrational frequency with defined amplitude to the co-extruded jet and collected in a acetate gelling placed 18 cm below the nozzle and agitated by a magnetic stirrer (length 4 cm) in dish with diameter 35 cm. Polymer flow rate and vibration frequency were empirically determined for the specific viscosity & concentration of denatured whey protein in the “activated”, negatively charged form. The appearance of a protein gelation upon contact with gelling bath, occurred at a minimal de-calcified denatured protein concentration of 1.75% (9% diluted to 1.75%) induced by protein aggregation and non-covalent chemical interactions. For the purpose of optimum ferrous encapsulation in homogenous, spherical microspheres, (for delivery to the duodenum and upper jejunum) the protein concentrate must be greater than 3% (w/w) de-calcified whey protein at pH greater than 5.1 to promote physical interactions such as hydrophobic and van der Waals interactions within the ferrous protein network. The acetate-ferrous gelling bath is continuously agitated to avoid coalescence or flocculation of microspheres during protein polymerization i.e. bead formation and ferrous encapsulation. Pliable microspheres were agitated (50 rpm) at room temperature in the acetate bath for minimum 30 minutes. These microspheres are then recovered and dispersed in tri-sodium citrate (0.4M) and subsequently washed with sterile water. This step sequesters calcium ions from the local environment in order to avoid ferrous contact with calcium ions upon microsphere digestion and ferrous release. After extrusion and chemical cross-linking of the protein polymer membrane in acetate and citrate, the microspheres were used immediately autoclaving at 121° C. for 15 minutes for sterilisation. This method represents whey protein polymerization as a result of ionic gelation i.e. protein gelation in the presence of ferrous sulfate in acetate buffer solution.
(b) Encapsulation of Ferrous Iron—Example 2
[0156] Similar to the aforementioned method i.e. protein de-calcification, heat denaturation and preparation, the suspension of negatively charged protein aggregates were aseptically extruded through a concentric nozzle into a tempered (45° C.) sodium acetate bath (pH 4.0, 0.2M). For this reason, the encapsulator was fitted with concentric nozzle with an internal diameter of 80 μm and an external diameter of 100 μm. Two syringe pumps (200 series, KD Scientific, Boston, USA) were connected to the encapsulator to supply ferrous sulfate through the central nozzle and de-calcified denatured protein solution through the external nozzle. The inner nozzle contained de-calcified denatured whey protein suspension with a slightly higher protein concentration for gelation. Spherical microspheres were obtained by the application of a vibrational frequency with defined amplitude to the co-extruded jet and collected in a acetate gelling bath (which may alternatively contain the ferrous sulfate placed 18 cm below the nozzle and agitated by a magnetic stirrer (length 4 cm) in a dish with diameter 40 cm. Polymer flow rate, ferrous flow rate and vibration frequency were empirically determined for the specific viscosity & concentration of whey protein (in the denatured, negatively charged form) and ferrous sulfate at bioavailable pH conditions (<4.5). The appearance of a protein-iron gelation upon contact with acetate gelling bath, occurred at a minimal de-calcified denatured protein concentration of 2.5% (9% diluted to 2.5%) induced by protein aggregation and non-covalent chemical interactions. For the purpose of optimum ferrous encapsulation using a concentric nozzle system, the protein concentration must be greater than 5.5% (w/w) de-calcified whey protein at pH greater than 5.1 to promote the generation of homogenous, spherical microspheres for duodenum and upper jejunum delivery, with suitable physical interactions i.e. hydrophobic and van der Waals interactions within the ferrous protein network (interactions are temperature dependent). Pliable microspheres were polymerized at room temperature, recovered and dispersed in tri-sodium citrate (0.4M) and subsequently washed with sterile water. This step sequesters calcium ions from the local environment in order to avoid ferrous contact with calcium ions upon microsphere digestion and ferrous release. After extrusion and chemical cross-linking of the polymer membrane in acetate and citrate, the capsules were used immediately autoclaving at 121° C. for 15 minutes. This method represents protein polymerization as a result of isoelectric focusing i.e. protein gelation at pH conditions close the isoelectric point of de-calcified denatured whey protein.
[0157] Characterisation of Microspheres Image analysis was performed using a Leica TCS SP5 confocal scanning laser microscope (CSLM) (Leica Microsystems, Wetzler, Germany) for the purpose of micro-capsule morphology and porosity assessment within the micro-structure. Micro-capsules with encapsulated ferrous sulfate were stained using the Nile Red (BD Biosciences, San Jose, Calif.), by integrating dye concentrations optimized for ferrous detection. Thickness of the optical section was in the order of several micrometers. Changing the plane of focus vertically up and down by 10 μm had no effect on the apparent membrane thickness. Image analysis was performed using ×63 magnification objective with numerical aperture of 1.4. Confocal illumination was provided by an argon laser (488 nm laser excitation) and red-green-blue images (24 bit), 512 by 512 pixels, were acquired using a zoom factor of 2.0, giving a final pixel resolution of 0.2 μm/pixel.
[0158] Atomic force microscopy (AFM) images were also obtained using Asylum Research MFP-3D-AFM (Asylum Research UK Ltd., Oxford, UK) to investigate the morphology i.e. depth, width and frequency of pores on the surface of the microspheres. The microspheres are poured onto microscope glass slides, incubated for slide adsorption for approx. 20 minute, washed with ultraclean water and dried in a nitrogen flow leading to a linear surface coverage of microspheres on the slide. Suitable microsphere samples could then be chosen in transmitted light DIC contrast prior to AFM scanning. The AFM head is then placed onto a Zeiss Axiovert 200 inverted optical microscope. AFM analysis was relatively slow since microspheres ware delicate and easily damaged mechanically if the force exerted by the AFM tip was too high. Microscope images were obtained in intermittent contact mode with standard silicon non-contact cantilevers (NCH) provided by Asylum Research, UK. Resonance frequency was approx. 200 kHz and spring constant of 30 N/m. Digital resolution was 512×512 pixels. The tip radius of cantilever was less than 10 nm and the opening angle between 30° and 40°. The applied force between the cantilever tip and ferrous microspheres was minimised in order to alleviate the influence of the AFM tip on the microspheres surface topography during pore scanning Measurements were started by scanning a random area of 1 m, with subsequent zoom to 20 nm. These images were used to evaluate the morphology of microspheres pores. Scan size was decreased gradually until microsphere pores could be viewed clearly. Iron-whey protein microspheres were analysed and scanned using typical working distance of 2-6 nm for visualization of pores dimensions and general morphology.
[0159] The gel structure of ferrous-protein microspheres exhibited a repeating network of ferrous cavities and protein strands, with diameter ranging 22-58 nm, is shown in
[0160] This AFM technique is optimally calibrated to determine the release location of produced microspheres with added acetate and citrate coatings.
[0161] Atomic Force Microscopy has the ability to screen membrane porosity. This present invention reveals (the first) AFM screening of porosity on the surface of microspheres. This inventive technique provides a tool for qualitative and quantitative analysis demonstrates of pore distribution (i.e. homogenous or sporadic) across the microspheres surface.
[0162] Microscopy analysis demonstrated the presence of deep pore cavities on microspheres surfaces after both acetate and citrate treatments.
[0163] In-Vivo-Data
[0164] B:
[0165] A prospective, randomized, cross-over, single-blind, cross-over study with at least 1 week wash-out was carried out to evaluate the pharmacokinetics (% relative bioavailability, total serum iron AUC.sub.0-4h (μM.Math.h)) of a single oral dose of the invention compared to the same dose of a market leading oral iron formulation. The single oral dose was made up of 15 g of microspheres of the invention (dry weight) made according to Example 2 above, suspended in 200 mls of apple juice, in which the 15 g of microspheres included 25 mg (±0.334 mg) of iron. We excluded subjects taking iron supplements and those with haemoglobin >11.5 g/dL, serum ferritin >150 μg/L, haemochromatosis or other iron storage disorders, gastro-intestinal disorders, inflammatory bowel disease, any medications and any intolerance to any of the components of either formulation.
[0166] Each dose was taken orally in the fasting state (>8 hours) as a drink, after fasting since midnight the night before the study. Blood samples were obtained over a 4 hour period in the fasting state (in all cases baseline, 2, 4 hours post dose). During this time they were not permitted to drink tea or coffee, but are allowed to drink water ad libitum an hour after the initial sample is taken.
[0167] Samples were taken by a trained, qualified nurse and all evaluations were performed under medical supervision. Samples were analysed for serum iron, total iron binding capacity (TIBC) and serum ferritin. Baseline venous blood samples were also drawn to obtain a full blood count and measure and haemoglobin levels prior to inclusion and administration of the test articles. All analyses were carried out in a blinded fashion by an externally validated esoteric laboratory operating under INAB accreditation. The primary endpoint of the study was the AUC0-4 h of total serum iron (μM.Math.h) over 4 hours post-dose. The results (n=3) are presented in
[0168] Participants also completed a questionnaire on the taste and acceptability (including adverse effects) of the supplements (See
[0169] The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.
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