Patent Application
20140322340
The present invention relates to the field of food technology and delivery of biologically active compounds via beverages and food. In particular the present invention provides protein (or peptide)-polysaccharide (or oligosaccharide) conjugates, and use thereof for encapsulation, stabilization and protection of active compounds, in particular for clear beverage applications.
One of the most important aims of contemporary food engineering is the enrichment of foods with health enhancing components. There is a growing public awareness for healthy nourishment that includes daily amounts of required micronutrients such as vitamins, essential fatty acids and antioxidants. Along with the tendency to enrich foods and drinks with healthy compounds there is a trend to exclude potentially harmful compounds.
A sub-category of healthy food is nutraceuticals-enriched food, in which a health-promoting bioactive molecule is added to the food or beverage. Many of the nutraceuticals which are desired for enrichment of food and beverages are hydrophobic (hydrophobic nutraceuticals or HN) and thus poorly water soluble, or water-insoluble. Some examples are vitamin A, vitamin D, vitamin E, carotenoids, and ω-3 fatty acids. Many of these HN are also sensitive to oxidation, and other degradation mechanisms.
Enrichment of food and beverages with sensitive HN is a challenge for several reasons: (a) the poor solubility of HN in water, which necessitates the use of a surface active agent (surfactant); (b) the surfactant-HN nanovehicle, or nanocapsule, must be colloidally stable in the target product environment (e.g., temp, pH, ionic strength) during the production and shelf life of the product; (c) HN-loaded nanoparticles must be as small as possible in order to minimize the effect on turbidity (visible light scattering); (d) if the HN is sensitive to oxidation, the vehicle should confer protection to retard HN degradation during shelf life. Oxidation reactions can be retarded by either antioxidant activity of substances in the environment or by encapsulating the sensitive material preventing heavy metals and oxygen from nearing the sensitive HN, and reducing their mobility and reactivity; and (e) all materials composing the vehicle and procedures of its formation must be defined as “generally recognized as safe” (GRAS). Natural food materials and common procedures in food processing are thus preferred.
Several methods have been introduced for enrichment of HN in aqueous solutions, mainly (a) emulsions stabilized by synthetic surfactants such as polysorbate (Tween); (b) emulsions stabilized by low MW natural surfactants such as phospholipids or monoglycerides; and (c) proteins as emulsifiers and nanocapsules (e.g., casein). WO 2007/122613 to the inventor of the present invention describes a system based on re-assembled casein micelles for the delivery of hydrophobic biologically active compounds in food and beverages. US patent application No. US 2011/038987 to the inventor of the present invention teaches the use of beta casein assemblies for enrichment of food and beverages, however the stability of beta casein around pH 4.5-5.5 is very poor, and the protection it provides to sensitive HN is limited. US patent application No. US 2011/0038942 to the inventor of the present invention teaches the formation of beta-lactoglobulin-polysaccharide nanocomplexes for hydrophobic bioactive compounds, for clear drink enrichment with HN, however the non-covalent nature of these complexes limits their application ranges in terms of pH and ionic strength.
Covalently bonded protein-polysaccharides conjugates (PPC) can also act as good emulsifier and for stabilizing agents of HN. One of the dispersant materials frequently utilized is Gum Arabic (gum acacia) which is a natural PPC composed of the polysaccharide arabinogalactan and about 2% protein. The protein regions of the gum Arabic apparently adsorb to hydrophobic droplets in solution. Gum Arabic is exudated as resin from stems and branches of acacia trees, and exhibits wide diversity in structure and properties, depending on period of year harvested, the tree's age and species. Its main disadvantages are its high price and highly variable composition and quality. Therefore, many efforts are aimed at finding good and inexpensive substitutes.1,2
There have been several recent attempts to produce alternative PPC systems under controllable conditions. Possible methods for preparing protein polysaccharide linkage are by enzymatic,3 chemical4 or by electrosynthesis5,6 reactions. A particularly attractive way to form PPC is via the Maillard reaction7 achieved only by heating, which is typical of cooking and food processing. This is most desirable for food applications as it enables the label-friendly statement: “All natural ingredients”. In the Maillard reaction the amino groups originating from the ε-lysine or the amino terminal of the protein are conjugated to an aldehyde group of the saccharide. There are several studies reported regarding glyco-conjugation of milk proteins—whey proteins8-10 or caseins.11-13 Additional substrates used for the Maillard reaction are soy proteins.14-16 Improvement of functional properties via Maillard conjugation of hydrolyzed soy17 and gluten18 proteins was also described.
As a result of the Maillard conjugation a “block-copolymer” with greatly improved functional properties can be obtained. Among the reported improvements are enhanced protein thermal stability, reduction of aggregate sizes,19 reduction of antigenicity,20 improved solubility and antioxidant activity.21,22 Special attention was paid to increased emulsifying capabilities of the Maillard products. Stability tests of oil in water emulsion formation and stabilization demonstrated superiority of the conjugates over non-conjugated proteins.
Only a few examples of the use of Maillard PPCs as encapsulation materials are known in the art. For example, the micellization properties of casein-dextran grafts were studied23,24 and micelles of around 80 nm were produced around the pI of casein. In addition, beta-carotene encapsulation by casein-dextran grafts resulting in 200 nm core shell particles was shown.25 Submicron particles of whey protein-MD conjugates were prepared for conjugated linoleic acid (CLA) encapsulation by dry heating.26 The particle size range disclosed in the art is too high if transparent food solutions (e.g. clear beverages) are to be enriched, and the creation of smaller particles is desirable.
Li27 used Bovine Serum Albumin (BSA) conjugated with dextran to encapsulate ibuprofen, reporting an average size of less than 100 nm, but this system was not proposed for HN delivery in transparent beverage systems, nor were any absorbance or visual results reported to support such applications. Wooster and Augustin28 used maltose or MD of several sizes, conjugated to beta-Lactoglobulin as shell material for encapsulation. They used latex particles as hydrophobic core material. The conjugate-latex particles were up to about 100 nm in diameter. Hiller and Lorenzen29 examined the hydrophobicity of several carbohydrates (dextran, glucose, lactose, pectin) conjugated with several proteins (casein, whey proteins, and combinations of isolated milk proteins), and have shown a decrease in surface hydrophobicity as a function of heating time even after 4 hrs of heating.
Serfert et al.30 used several carbohydrates (glucose, glucose syrup, dextran) conjugated to caseins for microencapsulation of fish oil. They showed an increase in redox index after conjugation with all sugars, meaning the Maillard reaction increased the potential of the protein to act as antioxidant. O'Regan and Mulvihill31 used casein-MD (CN-MD) conjugates and their hydrolyzates as emulsifiers. They showed that conjugation of MD to casein increases the casein's solubility at its pI (pH=4.6).
PPCs have been studied as potential nano capsules but up to date, no method for nano encapsulation of HN was reported to form particles small enough such that, when mixed with a liquid, a clear and transparent solution is obtained.
The present invention provides covalently bonded protein-polysaccharide conjugates (PPC) (including conjugates comprising oligosaccharides and/or peptides) as vehicles for nanoencapsulation of biologically active compounds, particularly nutraceuticals. The PPCs efficiently entrap and protect both hydrophobic (i.e., water insoluble or poorly water-soluble) and certain hydrophilic (i.e., water soluble) nutraceuticals, to provide a composition which, when added to a beverage, disperses so as to provide a clear solution. Advantageously, the conjugates of the present invention protect the nutraceuticals from degradation over a wide range of pH values, both during shelf life and upon gastric digestion. In one embodiment, the PPCs are formed by a Maillard reaction. The PPCs may comprise a Schiff base or Amadori rearrangement products, or keto-enol tautomers. In other embodiments the PPC comprises any other covalent link between the protein (or peptide) and the polysaccharide (or oligosaccharide).
The present invention departs from the known functions of PPCs (e.g., Maillard reaction-based PPCs) as a vehicle for encapsulating nutraceuticals in that it provides for nanoencapsulation of nutraceuticals for clear drink applications at high encapsulation efficiency (possibly >90%), good solubilization, stabilization and protection conferred to sensitive bioactive compounds against degradation. In contrast to known Maillard reaction-based PPC encapsulation products, which have a particle size range that is too large for formation of clear liquid solution, the compositions of the present invention can be added to beverage solution while maintaining transparency and avoiding the formation of turbid solutions or precipitation products. The nano-capsules disclosed by the invention can be incorporated into almost any beverage product without adversely modifying its properties. Advantageously, the compositions of the invention comprises only natural, generally regarded as safe (GRAS), non-toxic ingredients. As such, the compositions of the invention offer significant advantages over the prior art.
A major unique aspect of this invention is the harnessing of covalently linked conjugates of a protein (or peptide) and a polysaccharide (or oligosaccharide) for the stabilization, delivery and protection of insoluble/hydrophobic or soluble/hydrophilic biologically active compounds, particularly nutraceuticals, while maintaining the particle size of the compositions sufficiently small such that, when added to a beverage, a clear solution is formed. The encapsulated compositions not only are the ideal vehicles for stabilizing and delivering biologically active compounds, but their properties enable their incorporation into beverage products (e.g.; water, enriched and/or flavored water, sports drinks, sodas, milk, juice etc.) without compromising the properties of the solution. Furthermore, the encapsulated compositions protect the nutraceutical from degradation over a wide range of pH values (e.g., a pH range of 2.0 to 10.0), both chemically (e.g., during shelf life), or in acidic conditions such as during gastric digestion.
The advantages of the present invention:
1) Smaller size achieved by the entrapment technique, and use of oligomers (peptides and maltodextrin) enabling transparent solutions. Solubility at the protein pI.
2) The protection conferred against degradation by the encapsulation, and by the antioxidant properties of the proteins, and the Maillard reaction products. This advantage is particularly significant compared to low molecular weight emulsifiers.
3) Kosher Parve (in some of the combinations proposed) where vegetable proteins are used.
4) Low allergenicity when using hydrolyzates of proteins not considered allergenic (e.g. rice protein). These advantages are significant compared to milk protein-based systems).
5) Potentially masking of undesired flavors.
6) Potential for improved bioavailability.
According to one aspect, the present invention provides a composition for enrichment of beverages, comprising a nutraceutical encapsulated or entrapped or protected by a conjugate, the conjugate comprising a protein (or peptide) covalently bonded to a polysaccharide (or oligosaccharide) (collectively designated herein “PPC”), wherein the particle size of said composition is sufficiently small such that, when added to a beverage, a clear solution is formed. In one embodiment, the PPC comprises a Schiff base, or Amadori rearrangement products, or keto-enol tautomers based linkage between the peptide and the saccharide. Such a structure may be formed by a Maillard reaction or a Maillard-type reaction.
According to another aspect, the present invention provides a composition for enrichment of beverages, comprising a nutraceutical which is encapsulated or entrapped or protected by a conjugate, the conjugate comprising a protein or polypeptide which is covalently linked to a polysaccharide or oligosaccharide (PPC), wherein the PPC is formed by a Maillard reaction or a Maillard-type reaction, and wherein the particle size of said composition is sufficiently small such that, when added to a beverage, a clear solution is formed.
In general, the compositions of the present invention can have any average particle size as long as they result in a transparent solution when mixed with a liquid. In one embodiment, the average particle diameter of said composition is between about 50 and 100 nm. Preferably, the average particle diameter of said composition is less than about 50 nm and even more preferably less than about 20 nm. Non-limiting examples of particle diameters include less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm or less than about 10 nm. Each possibility represents a separate embodiment of the present invention. The compositions can be homogenized in order to reach the desired particle size.
The solutions of the present invention typically will have an absorbance at 600 nm of below about 0.1, preferably below about 0.075, preferably below about 0.05, more preferably below about 0.02 and even more preferably below about 0.01, with each possibility representing a separate embodiment of the present invention. In one embodiment, the clear or transparent solution has an absorbance at 600 nm of less than about 0.1.
Without wishing to be bound by any particular mechanism or theory, it is contemplated that PPC encapsulation of nutraceuticals leads to the formation of smaller particle size than simple protein encapsulation. This is because conjugation of the oligosaccharide may add steric hindrance which could lead to a small packing parameter, higher curvature, and consequently inhibition of protein aggregation and formation of smaller nanoparticles. The uniqueness of the present invention is based in part on the choice of raw materials, in particular the use of oligosaccharides (e.g. maltodextrin), along with an amphiphilic peptide or protein (e.g. casein, beta-conglycinin), which when covalently bonded under controlled conditions form conjugates (e.g., Maillard reaction conjugates), whose self-assembly, and co-assembly with the nutraceuticals, result in high particle-surface curvature, and hence small nanoparticles—thus enabling the formation of clear solutions.
The molar ratio of carbohydrate to protein used to prepare the PPCs can vary, but in general ranges from about 1:1 to about 1:50 (protein to carbohydrate). Some preferred but non-limiting rations include about 1:1, 1:5, 1:10, 1:20, 1:40 or 1:80 (protein to carbohydrate). Each possibility represents a separate embodiment of the present invention.
In addition, the molar ratio of PPC to nutraceutical can vary, but in general ranges from about 1:1 to about 1:10 (in terms of protein to nutraceutical). Some preferred but non-limiting rations include about 1:1, 1:2, 1:4, 1:6, 1:8 or 1:10 (protein to nutraceutical). Each possibility represents a separate embodiment of the present invention.
In one embodiment the nutraceutical is a hydrophobic nutraceutical (HN), i.e., it generally is poorly soluble or insoluble in water. In another embodiment, however, the nutraceutical may be a hydrophilic nutraceutical, i.e., it is moderately to highly water soluble. Each possibility represents a separate embodiment of the present invention.
In some embodiments the HN is a fat-soluble vitamin. Suitable fat-soluble vitamins include, but are not limited to vitamin D (D2, D3 and their derivatives), vitamin E (α, β, γ, δ-tocopherols, or α, β, γ, δ-tocotrienols), vitamin A (retinol, retinal, retinoic acid), and vitamin K (K1, K2, K3 and their derivatives). Each possibility represents a separate embodiment of the present invention. In specific embodiments the vitamin is vitamin D.
In other embodiments, the HN is an unsaturated fatty acid, including but not limited to linoleic acid, conjugated linoleic acid (CLA), omega-3 fatty acids such as alpha linolenic acid, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and their glycerol-esters or other esters. Each possibility represents a separate embodiment of the present invention. In specific embodiments the unsaturated fatty acid is CLA.
In other embodiments the HN is a sterol, cholesterol or its derivatives. In other embodiments, the HN is a carotenoid including α-, β-, or γ-carotene, lycopene, lutein, zeaxanthin, astaxanthin and others. In some embodiments the HN is selected from phytochemicals, phytoestrogens including phytosterols (e.g. β-sitosterol, campesterol, stigmasterol etc.), isoflavones (genistein, daidzein), stilbenes (e.g. resveratrol, trans-resveratrol), lignans (e.g. Matairesinol) and coumestans (e.g. coumestrol), curcumin, and others. In another embodiment the HN is coenzyme-Q10 (co-Q10). Each possibility represents a separate embodiment of the present invention. In some embodiments, the nutraceutical is water soluble. The nutraceutical may be selected from a polyphenol (e.g., punicalagin), a tannin, a catechin, a flavonoid, an isoflavonoid or a neoflavonoid. Non-limiting examples are epigallocatechin gallate (EGCG), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin (EGC). Each possibility represents a separate embodiment of the present invention. In specific embodiments the nutraceutical is epigallocatechin gallate (EGCG).
In some embodiments, the nutraceutical is an amphiphilic nutraceutical, i.e., it is a chemical compound comprising both hydrophobic and hydrophilic moieties. Any type of protein or peptide, and reducing polysaccharide or oligosaccharide may be used to form the protein-polysaccharide conjugates of the present invention.
The protein in the conjugate may be a vegetable protein, an animal protein, a milk protein, an egg protein, a fungi protein, a microbial protein, an algae protein or any hydrolyzate, peptide or combinations thereof.
In some embodiments, the protein is a vegetable-derived protein, such as but not limited to rice protein, soy protein, pea protein, lupin protein, Zein (corn protein), wheat protein, gluten, and their hydrolyzates. Non-limiting examples of soy proteins are beta-conglycinin and glycinin. In one specific embodiment, the vegetable protein is rich protein hydrolyzate (RPH). In another specific embodiment, the vegetable protein is beta-conglycinin (β-cong).
In other embodiments, the protein is an animal-derived protein. In other embodiments, the protein is a dairy (i.e., milk)-derived protein, such as but not limited to casein, whey protein concentrate (WPC), and whey protein isolate (WPI). In one specific embodiment, the milk protein is casein (which may be in the form of sodium caseinate or an isolated casein such as but not limited to alpha s1, alpha s2, beta or kappa casein, or any combination thereof). In some embodiments the source of casein is sodium caseinate. In other embodiments the source of casein is milk, or milk powder, or any soluble caseinate or casein preparation, or isolated alpha, beta, and/or kappa casein or mixtures of such caseins. In other embodiments, the fungi protein is a hydrophobin. Each possibility represents a separate embodiment of the present invention.
In other embodiments, the protein is any combination of the above vegetable, animal or dairy (milk) proteins, or their hydrolyzates.
The novel nanoencapsulated compositions of the present invention can be introduced into any beverage product to provide a clear solution. Non-limiting examples of beverages include water, soft drinks, juice, milk, tea and coffee.
A certain possible characteristic of the Maillard-based conjugates is their brown color, which may in some embodiments be utilized as a natural pigment for certain beverages.
In other embodiments, the present invention provides methods for the enrichment of beverages with at least one nutraceutical, comprising the step of adding to a beverage a nutraceutical encapsulated by a covalently bonded protein (or peptide)-polysaccharide (or oligosaccharide) conjugate (PPC), wherein the particle size of said composition is sufficiently small such that, when added to said beverage, a clear solution is formed. In one embodiment, the PPC is formed by a Maillard reaction. In another embodiment, the clear solution has an absorbance of less than about 0.1 at 600 nm.
In yet another aspect the present invention provides a method for the preparation of a composition comprising a nutraceutical encapsulated by a covalently bonded protein (or peptide)-polysaccharide (or oligosaccharide) conjugate (PPC) as described herein. The method comprises the following steps:
PPCs can be formed as described in, e.g., Nursten et al, the contents of which are incorporated by reference herein.31 Preferably, the mixing step (iii) occurs comprises slowly adding the nutraceutical solution to the PPC solution while stirring. In some embodiments the method further comprises the step of drying the encapsulated composition. In other embodiments, the method further comprises the step of homogenizing the dried composition so as to reduce the particle size to the desired range.
The solvent used to prepare the nutraceutical or PPC solution can be any food grade solvent. When the nutraceutical is a HN, a water miscible organic solvent which evaporates during the drying of the conjugates is preferably used. Natural or synthetic solvents known in the art can be used according to the teachings of the present invention. In some embodiments the solvent is ethanol. When the nutraceutical is a hydrophilic compound, water also may be used as a solvent.
Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, and are thus included within the scope of this invention.
The present invention provides covalently bonded protein-polysaccharide conjugates (PPC) (including conjugates comprising peptides and oligosaccharides) as vehicles for nanoencapsulation of biologically active compounds, particularly nutraceuticals. The PPCs efficiently encapsulate both hydrophobic (i.e., water insoluble or poorly water-soluble) and hydrophilic (i.e., water-soluble) nutraceuticals, to provide a composition which, when added to a beverage, disperses so as to provide a clear solution. Preferably, the clear solution has an absorbance at 600 nm of less than about 0.1. Advantageously, the conjugates of the present invention enhance the dispersibility of hydrophobic nutraceuticals and protect nutraceuticals from degradation, over a wide range of pH values, both during shelf life and upon gastric digestion. In one embodiment, the PPCs are formed by a Maillard reaction.
The present invention now discloses that co-assembly of biologically active compounds, for example nutraceuticals (which are preferably hydrophobic but can also be hydrophilic or amphiphilic), with PPCs, stabilizes the nutraceuticals and protect them from degradation, even in acidic media such as that found during gastric digestion. These PPC may also protect hydrophilic nutraceuticals against degradation, mainly by oxidation. Such PPC-nutraceutical system facilitates the enrichment of beverage products, while minimizing the effect of the compound incorporation on the beverage properties, and still maintaining its transparency. In one embodiment, the PPCs are Maillard-reaction formed.
Encapsulation of biologically active compounds within PPCs is advantageous over hitherto known encapsulation methods as the compositions comprise only natural components, and their particle size is sufficiently small so as not to form turbid solutions or precipitates when mixed with the beverage of choice. In addition, when the active compound possesses undesirable attributes, the encapsulation in the PPCs diminishes such unwanted features (e.g. in the case of omega 3 fatty acids). Another important potential benefit is the improved bioavailability of the enclosed compound due to its distribution, at a molecular level, over a very large surface area of the PPC-based nanocapsules, and in the case of casein-based PPC, the fact that caseins are evolutionally optimized for ease of digestion and absorption.
Specific embodiments include a method for incorporation of hydrophobic nutraceuticals (HN) such as vitamin D and Conjugated Linoleic Acid (CLA) into rice protein hydrolyzate (RPH)-maltodextrin (RPH-MD) conjugates. Other embodiments include a method for protection of the water-soluble nutraceutical epigallocatechin gallate (EGCG), as well as the hydrophobic nutraceutical vitamin D (VD) using Soy beta-conglycinin-MD Maillard conjugates. Other embodiments include a method for the incorporation of hydrophobic nutraceuticals (HN) such as vitamin D and casein-maltodextrin (CN-MD) conjugates. The methods of the present invention further included the evaluation of the encapsulation processes as well as the protection conferred to the nutraceuticals by the encapsulation process and or by the conjugates themselves.
For example and as disclosed herein for the first time, vegetable proteins (e.g., Rice, Soy or their hydrolyzates), or milk proteins (e.g., casein (CN)), were conjugated to an oligosaccharide (e.g., Maltodextrin (MD)) or polysaccharide using the Maillard reaction or Maillard-type reaction by dry heating (60° C., 79.9% RH for several hours to days). The formation of conjugates was verified by SDS-PAGE, decrease of free amino groups by the o-Phthalaldehyde (OPA) assay, visual color test, DLS, and spectrophotometric absorbance showing increase of peaks at the wavelength region typical for the Maillard products. The conjugation products showed an increase in molecular weights as a function of time with similar reaction rate for pH 4.5, 7.5, 8.8 and significantly higher initial rate for pH 10.5, where most conjugation occurred on the 1st day of heating.
In one embodiment, the co-assembly of rice protein hydrolyzate (RPH)-maltodextrin (RPH-MD) conjugates with vitamin D (VD) or with conjugated linoleic acid (CLA) was examined by mean of DLS particle size distribution and turbidity measurements, and a significant diameter decrease and turbidity reduction were observed in the presence of RPH-MD, indicating the interaction and solubilization effect exerted by RPH-MD conjugates. The degradation of CLA and VD during shelf life at both 4° C. and room temperature, at both neutral and acidic conditions was significantly slower due to nanoencapsulation with RPH-MD, suggesting Maillard conjugates of RPH-MD can serve as nano-vehicles for delivery of HN such as CLA or VD in transparent aqueous systems providing protection against degradation.
In other embodiments, soy beta-conglycinin-MD Maillard conjugates showed better solubility than the mixture of their components. The conjugates, with and without the nutraceutical EGCG gave smaller particle sizes than solutions of MD with and without EGCG, forming clear solutions. The protection provided by the conjugate-based nanoparticles to EGCG was more significant than the protection provided by the simple beta-conglycinin-MD mixture or control sample. These results emphasize the potential of soy beta-conglycinin-MD Maillard conjugates as protective material for clear drink applications.
In another example, for enrichment of clear beverages with a hydrophobic nutraceutical (e.g. vitamin D), CN-MD Maillard conjugate based nanovehicles having diameters of less than 100 nm, were formed. At high VD concentrations (simulating soft drink concentrates), the complexes of VD-conjugate were less turbid than the ones formed by VD and a CN-MD mixture (where each biopolymer was heated separately, then mixed) and much less turbid than VD dispersed in buffer only. Completely clear solutions were obtained with nanoencapsulated VD at doses typical for the final drinks). An industrially feasible fractionation process was developed based on isoelectric precipitation, for enrichment of clear beverages even at pH close to 4.6, the pI of the native casein, where casein nanocapsules would precipitate. Conjugation significantly improved the protection against oxidation conferred to both VD and EGCG. Nanoencapsulation of VD in CN-MD Maillard conjugates conferred significant protection against low pH induced degradation, important for acid drinks, and for survival through gastric digestion. This attribute may be utilized for developing targeted vehicles for enteric delivery of bioactives and drugs.
Overall the study showed the very good potential of Maillard conjugates of proteins and oligosaccharides for nanoencapsulation of nutraceuticals for clear drink applications at high encapsulation efficiency (possibly as high as ˜90%), good solubilization, stabilization and protection conferred to the sensitive bioactive compound against degradation during shelf life, and gastric digestion.
The concentration of the nutraceutical in the PPC can vary depending on the nature of the nutraceutical and its function. Typical concentrations can vary between 0.01 to 100 mg/ml, for example 0.01 to 10 mg/ml, 0.01 to 5 mg/ml or 0.01 to 1 mg/ml.
All references cited herein are hereby incorporated by references in their entirety as if fully set forth herein.
For convenience and clarity certain terms employed in the specification, examples and claims are described herein.
The terms “transparent” as used herein means having the property of transmitting rays of light through its substance so that bodies situated beyond or behind can be distinctly seen. A “clear solution” as used herein means a transparent solution. The term “turbidity” or “turbid” as used herein is the cloudiness or haziness of a fluid caused by scattering of visible light by particles (suspended solids or liquids) that are individually generally invisible to the naked eye. Turbidity can be measured by measuring absorbance at an appropriate wavelength (usually 600 nm is used). Absorbance at 600 nm below 0.1 is generally typical of transparent systems, below 0.05 is typically considered good transparency, and below 0.02 is typically considered excellent transparency. Thus, the solutions of the present invention typically will have an absorbance at 600 nm of below about 0.1, preferably below about 0.075, preferably below about 0.05, more preferably below about 0.02 and even more preferably below about 0.01.
The terms “poorly water-soluble” or “hydrophobic” refer to water solubility of less than about 30 mg/ml, less than about 10 mg/mL, or less than about 1 mg/mL at ambient temperature and pressure and at about pH 7. This corresponds to nutraceuticals which are to be characterized by the commonly used terms “sparingly soluble”. “slightly soluble”, “very slightly soluble”, “practically insoluble” and “insoluble”, all of which are used herein interchangeably.
A “nutraceutical”, also known as a functional food (or its component), is generally any one of a class of food ingredients or dietary supplements including vitamins, minerals, herbs, healing or disease-preventative foods or food components that have medical or pharmaceutical effects on the body. Examples of non-polar or hydrophobic nutraceuticals include, but are not limited to fatty acids (e.g., omega-3 fatty acids, DHA and EPA or their esters); fruit and vegetable extracts; vitamins A, D, E and K; phospholipids, e.g. phosphatidyl-serine; certain proteoglycans such as chondroitin; certain amino acids (e.g., iso-leucine, leucine, methionine, phenylalanine, tryptophan, and valine); various food additives, various phytonutrients, for example lycopene, lutein and zeaxanthin; certain antioxidants; plant oils; fish and marine animal oils and algae oils. It is to be understood that certain nutraceuticals can also be referred to as therapeutics as well as cosmetic compounds.
Some non-limiting examples of hydrophobic nutraceuticals include, but are not limited to:
(a) Fat-soluble vitamins including vitamin D (D2, D3 and their derivatives), vitamin E (α, β, γ, δ-tocopherols, or α, β, γ, δ-tocotrienols), vitamin A (retinol, retinal, retinoic acid), vitamin K (K1, K2, K3 and their derivatives).
(b) Unsaturated fatty acid, including but not limited to linoleic acid, conjugated linoleic acid (CLA), omega-3 fatty acids including alpha linolenic acid, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and their esters, including their glycerol esters.
(c) A sterol, cholesterol or its derivatives.
(d) Carotenoids including α-, β-, or γ-carotene, lycopene, lutein, zeaxanthin, astaxanthin and others.
(e) Phytosterols (e.g. β-sitosterol, campesterol, stigmasterol etc.), isoflavones (e.g., genistein, daidzein), stilbenes (e.g. trans-resveratrol), lignans (e.g. Matairesinol) and coumestans (e.g. coumestrol), and others.
(f) Polyphenols (e.g., punicalagin), tannins.
In yet other embodiments the nutraceutical is selected from a bioactive peptide, such as casein-phosphopeptide (CPP) and other calcium-binding peptides.
Nutraceuticals for use in the compositions of the present invention may also be hydrophilic (i.e., water soluble). In such embodiments, the nutraceutical is selected from a catechin a flavonoid, an isoflavonoid or a neoflavonoid. Non-limiting examples are epigallocatechin gallate (EGCG), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin (EGC).
(i) Vitamin D (VD):
VD is an oil-soluble vitamin which is photochemically synthesized in the skin during exposure to ultraviolet radiation (UVB) of mid-day sunlight. It is crucial for multi-system function: calcium and bone metabolism, muscle function, insulin reactivity, cell differentiation, immune system function and more. Adequate VD status was linked to reduced risks for fractures, hypertension, diabetes, cancer and more. VD status is far below optimal in many countries all over the world, mainly due to avoidance of sun exposure to prevent melanoma, and the use of sunscreen which blocks VD synthesis. Also, the nutritional sources of VD are scarce, and cannot provide sufficient amounts when sun exposure is lacking. Therefore, it is imperative to enrich staple foods and drinks with VD to raise its consumption by large populations.
VD tends to oxidize readily in aqueous solution and especially under acidic conditions. The structures of the two most prominent forms of vitamin D, vitamin D2 and vitamin D3, are shown below:
(ii) Conjugated Linoleic Acid (CLA)
CLA is mainly found in meat and milk products from ruminant animals. CLA has been attributed with diverse health benefits which include immune response enhancement, atherosclerosis reduction, growth enhancement, anti-diabetic, anti-atherogenic and antiadipogenic properties. In addition, it has been reported that CLA can inhibit the proliferation of various cancer cell lines and act as an inhibitor of chemically induced carcinogenesis.
Both VD and CLA are hydrophobic compounds that readily dissolve in oil or organic solvents but have very poor water solubility. When added to water, VD and CLA provide unstable turbid suspensions. Moreover, both of the compounds are subjected to oxidative processes, which lead to loss of its bioactivity and to decreased nutritional quality when it is used as a food additive. Vitamin D was found to be very unstable in aqueous solutions and even more so in acidic conditions. Because of the presence of conjugated double bonds in the molecular structure of CLA, its oxidative stability was shown to be extremely low. As demonstrated herein, the Maillard protein-polysaccharide conjugates used as nanovehicle formers according to the principles of the present invention helped solubilize these and other HN in stable transparent solutions, while protecting them from various degradation reactions.
(iii) Epigallocatechin-3-Gallate EGCG:
EGCG is the major catechin found in green tea, comprising 50%-60% of the total catechin mass. EGCG is a water soluble compound, readily oxidized at neutral and alkaline pH, and degraded to yellow products absorbing visible light at wavelength of 425 nm. Animal studies indicated that the consumption of green tea and green tea products with high levels of EGCG and other catechins may have a significant effect toward the prevention of tumors, cardiovascular disease, neurodegenerative disease, obesity and other adverse medical conditions. The chemical structure of EGCG is shown below:
As used herein, the term “casein” refers to the predominant protein in milk, comprising the subgroups αs1, αs2, β and κ.
Casein is organized in micelles. Casein micelles (CM) are designed by nature to efficiently concentrate, stabilize and transport essential nutrients, mainly calcium and protein, for the neonate (1). All mammals' milk contains casein micelles. Cows' milk contains 30-35 g of protein per liter, of which about 80% is casein.
Casein micelles are spherical colloids, 50-500 nm in diameter (average of 150 nm) (2), made of the main four caseins: αs1-casein (αs1-CN), αs2-CN, β-CN, and κ-CN (molar ratio ˜4:1:4:1 respectively). The caseins are held together in the micelle by hydrophobic interactions and by bridging of calcium-phosphate nanoclusters bound to serine-phosphate residues of the casein molecules. The structure of the casein micelles is important for their biological activity in the mammary gland as well as for their stability during processing of milk into various products, as well as for the good digestibility of the nutrients comprising the micelles. The micelles are very stable to processing, retaining their basic structural characteristics through most of these processes.
The choice of casein for use as the protein part for the conjugates of the present invention stems from the excellent amphiphilicity of caseins, their low price, and their large number of side-amine residues. An average casein molecule contains 13.6 lysine (Lys) residues, which may theoretically serve as Maillard-conjugation sites.
Maltodextrin (MD) is an oligosaccharide formed by hydrolysis of starch. Hydrolyzates of starch are characterized by the “dextrose equivalent” (DE) value. DE is defined as the percentage of the total solids that have been converted to reducing sugars following starch hydrolysis. MD is defined as hydrolyzed starch having DE of 3-20. The higher the DE is, the lower the average molecular weight is and thus the MD is more easily dissolved in water. Starch consists of D-glucose monomers linked with an α(1-4) glycosidic linkage. There are two types of starch: amylose—a linear form consisting mainly of α(1-4) linked glucose units, and amylopectin—a branched form of starch, wherein the side chains are linked to the backbone via an α(1-6) linkage.
Due to the branching, the density of amylopectin is lower than the density of amylose. MD derived from corn starch was used in some embodiments of the present invention since corn starch contains about 70% amylopectin—a branched form of starch, to obtain a larger hydrophilic part of the conjugate. This is advantageous for steric repulsion, and for increasing the curvature of the nanoparticles formed—so that their radii would be smaller. A MD molecule has only one reducing end, which is a desired feature, so that the conjugation would not lead to gelation, but only to low molecular weight copolymers.
A scheme of the initial stages of the Maillard reaction is shown in Scheme 1:31
See, Reference 31 for a description of the Maillard reaction. The initial stage includes two reactions: 1. Amine-sugar condensation in which a covalent bond is formed between an amine of a protein and a reducing end of a saccharide, a water molecule is released, and a Schiff base is formed. 2. Amadory rearrangement in which a series of isomerizations form an Amadory compound.
During the initial stage, the products remain as whole saccharide-protein copolymers. In most cases lysine is the most reactive residue in proteins, while Tryptophan and Arginine are less reactive. The condensation reaction is affected by the water activity (aw). The reaction rate is accelerated at aw between 0.5 and 0.8, while at lower aw values the reactants lose their mobility, and at higher aw they are diluted. [19] The reaction rate also increases with increasing pH for two reasons: the reactive form of the amine group is the unprotonated form, and the reactive form of the sugar is the open chain form, which is more prevalent at higher pH conditions. [38] The formation of Schiff base was found to be rate limiting. The formation of Amadory compounds was found to be the most temperature sensitive reaction of the initial stage. The Amadory reaction rate rises sharply at temperatures above 70° C. and at pH above 8. [19] To gain mostly products of the initial stage with minimum degradation and\or polymerization (typical of later stages), reaction conditions should be kept below 70° C. and pH<8.
A scheme of the intermediate and progressive stages of the Maillard reaction is shown in Scheme 2:31
The intermediate stage includes three reactions: 1. An exit of a water molecule from the saccharide. 2. Breakdown of the saccharide. 3. Strecker degradation—a degradation of the amino acid. During this stage the saccharide-protein copolymer breaks, free radicals form and react. Because free radical formation is involved the reactions are highly non-specific.
In the finals stage fragments are polymerized to form melanoidins—a high molecular weight polymer compounds. The routes of degradation and polymerization are greatly affected by the pH.
It has been reported that during the Maillard reaction, antioxidants are formed, which is important for the functionality of Maillard reaction conjugates as protective nanoencapsulators for oxidation sensitive nutraceuticals.
The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.
aw—water activity
BSA—Bovine serum albumin
CN—Casein
Conjugated linoleic acid (CLA)
DE—dextrose equivalent
DHA—Docosahexaenoic acid
DLS—Dynamic light scattering
EGCG—Epigallocatechin-3-gallate
Em—Emission
EPA—Eicosapentaenoic acid
Ex—Excitation
GRAS—Generally recognized as safe
HN—Hydrophobic nutraceutical
Lys—Lysine
M—Molar
MD—Maltodextrin
MW—molecular weight
NR—Nile red
O.D.—Optical density
OPA—Ortho-phthaldialdehyde
PAGE—Polyacrylamide gel electrophoresis
PPC—Protein polysaccharide conjugate
RH—Relative humidity
RP-HPLC—reversed phase high performance liquid chromatography
SDS—Sodium dodecyl sulfate
UV—Ultraviolet
VD—Vitamin D
VIS—Visible
The objective of this experiment was to form Maillard conjugates of Rice Protein Hydrolyzates (RPH) and maltodextrin (MD), characterize them and evaluate their potential for nanoencapsulation of hydrophobic nutraceuticals, preferably for clear liquid systems.
Materials
Maltodextrin (MD) of dextrose equivalent (DE) 19 with an average molecular weight of approximately 10 KDa, was donated by Productos de maiz S.A. Corn Products international (Munro, Argentina). RPH was donated by Cognis Ltd. (Dusseldorf, Germany). It was produced by specific proteolysis resulting in peptides of 2-3 KDa, and obtained in a form of a 30% solution, preserved with a sodium benzoate, at pH 4.5.
O-phthaldialdehyde (OPA), Trizma base, SDS, vitamin D3 (VD), conjugated linoleic acid (CLA), mercaptoethanol, acrylamide-bisacrylamide mixture, and ammonium persulphate were obtained from Sigma-Aldrich (Rehovot, Israel). Methanol and Acetonitrile, both of HPLC grade, were obtained from LabScan (Dublin, Ireland). NaOH was obtained from Merck (Darmstadt, Germany), absolute ethanol from BioLab (Jerusalem, Israel), and sodium tetraborate from Laba Chemie (Mumbai, India). SDS-PAGE size markers and coomassie brilliant blue 250-R stain were obtained from Bio-Rad. Bromophenol blue was obtained from Fluka.
The pH 7 PBS buffer comprised 30 mM NaH2PO4/Na2HPO4. The pH 3 citrate buffer comprised 30 mM sodium citrate. 0.02% (w/w) sodium azide was added to the buffers as a preservative.
Methods
A 50 mg/ml solution of RPH and MD at 1:1 w/w ratio was prepared in doubly distilled water (ddw). The pH of the obtained solution was 4.5; pH values of 7.5, 8.8 and 10.5 were obtained by adding 1M NaOH. The control solutions of the RPH without MD were prepared at the same conditions. The solutions were then freeze-dried, and placed in an oven at 60° C. A constant relative humidity of 79.9% was achieved by placing the samples in a desiccator containing saturated KBr solution. Samples were removed from the oven every 24 hr during 0-5 days, freeze-dried again and stored for further analysis.
Entrapment of Hydrophobic Nutraceuticals (HN) within the RPH-MD Micellar Nanoparticles.
The co-assembly of the HN with the RPH-MD was achieved by slowly adding the VD or CLA, dissolved in ethanol, into the RPH-MD solution during stirring. The HN ethanol solutions were prepared at different concentrations while the final ethanol concentration was kept constant and did not exceed 4%.
The Maillard reaction products were analyzed by electrophoresis method based on Tricine-SDS-PAGE, for low Mw peptides resolution. Three-layer gel was prepared as follows: the lower separating gel was composed of 16.5% of acryl amide:bisacrylamide (19:1), the intermediate gel—10% acryl amide:bisacrylamide (29:1) and the upper stacking gel—4% acryl amide:bisacrylamide (29:1). The sample buffer contained 24% (v/v) glycerol, 1% SDS, 0.6 Tris and ˜2 mg/ml bromophenol blue. The samples in a form of lyophilized powder were dissolved in the sample buffer. Then mercaptoethanol was added to a final concentration of 5% (v/v) and the samples were incubated for 5 min at 100° C. while vigorously stirring. Final sample concentration of 50 mg/ml on a protein basis was obtained. A volume of 20 μl was loaded into the gel wells. The voltage was adjusted to 30 V during the first hour of electrophoresis and to 150 V during the next two hours. After the electrophoresis the gels were immersed in a fixation solution (30% methanol, 10% acetic acid) for 0.5 hr, then stained in a coomassie brilliant blue R-250 for 1 hr and washed by 10% acetic acid.
The RPH-MD graft samples were analyzed by the OPA assay to determine the degree of conjugation. The OPA reagent was prepared as described in the literature.33,34 The following compounds were diluted with water to 100 ml: 80 mg OPA (dissolved in 2 ml 95% ethanol); 50 ml 0.1 M sodium tetraborate, 5 ml 20% SDS; 0.2 ml of 2-mercaptoethanol. The OPA reagent was prepared immediately before use. The RPH-MD samples at a concentration of about 0.1 mg/ml on protein base (taking the RPH Mw as 2.5 KDa, this concentration corresponds to 4E-5 M) were prepared in DDW with 0.02% azide. 0.05 ml of the sample was added to 2 ml of OPA reagent. This solution was briefly stirred and the absorption at 340 nm was measured after 2-min incubation at room temperature (RT). A standard curve was obtained by using L-leucine as a reference compound. Reference samples with a concentration ranging from 1.5E-4 to 1.5E-3 M were prepared in DDW with 0.02% sodium azide and the L-leucine determination was performed as described above.
The particles size distribution analysis was performed by dynamic light scattering (DLS) analyzer (NICOMP—380, Particle Sizing Systems Inc., Santa Barbara, Calif., USA) as described in previous publications.35
The conjugate samples were characterized as follows. Two mg/ml (on a protein base) solutions were prepared from the dry heated samples of RPH with and without MD and compared to the unheated control. The RPH-MD powder was dissolved in a pH 7 PBS and stirred overnight for complete hydration. The samples were filtered through 0.45 μm syringe filters (polyvinylidene fluoride, Durapore filter (Millipore, Carrigtwohill, Co. Cork, Ireland).
Additionally, the HN RPH-MD co-assembled particles were analyzed at different concentrations of the VD or CLA.
The HN samples turbidity was estimated by absorbance measurements at 600 nm using an Ultrospec 3000 spectrophotometer (GE Healthcare, Waukesha, Wis., USA) or the Synergy HT Multi-Detection Microplate Reader.
The protection conferred by the RPH-MD against HN deterioration with time was evaluated. Different shelf life conditions of temperature 4° C. or 23° C. and pH, neutral or acidic, were tested. Neutral pH conditions were obtained by a pH7 PBS. Acidic conditions for CLA were obtained by a pH 3 citric buffer. Acidic conditions (pH 2.5) for VD were obtained by addition of HCl.
Following the incubation of HN co-assembled samples under the above shelf life conditions, HN extraction was carried out. Then the HN concentration in the samples was quantified using reversed phase HPLC (RP-HPLC), 4.6×100 mm C18-C2 column, on an Akta basic HPLC system equipped with a 3 simultaneous wavelengths UV detector (GE Healthcare, Waukesha, Wis., USA). The volume of the injection loop was 100 and the operation temperature was 24° C.
The concentration of the vitamin D was 0.02 mg/ml, a high dosage intended for simulating beverage concentrates, which are later diluted during bottling, but must be colloidally and chemically stable. The CLA concentration was 0.1 mg/ml which is the highest concentration that could be achieved in visually transparent samples with the help of the conjugates. A molar ratio of HN to RPH of about 1:1 was chosen.
Vitamin D extraction and HPLC quantification was based on Kazmi et al57. Samples (1 ml) were placed into 12 ml test tubes, followed by the addition of 3.75 ml of a methanol:chloroform mixture (2:1). The tubes were vortexed and 1.25 mL of chloroform were added to each tube, which was again vortexed for 1 min. Samples were centrifuged for 10 min at 1500×g and 4° C. The clear chloroform layer at the bottom of each test tube was collected using a glass syringe and transferred to an evaporation vial. The chloroform extract was dried under a flow of nitrogen gas, reconstituted in 2 mL of the mobile phase [see composition below], and left undisturbed for at least 15 min. Operating conditions were: ambient temperature (24±1° C.); mobile phase was methanol: acetonitrile: water (49.5:49.5:1 v/v); flow rate was 0.3 ml/min; and the absorbance was measured at 254 nm and 228 nm.
To extract CLA, isopropanol (0.5 ml) was added to 0.5 ml sample. After vortexing for 30 s, hexane (1 ml) was added, and the tube was vortexed again for 15 min. Then, the samples were centrifuged (1,900×g for 5 min), and the upper hexane layer was collected. Then the hexane was evaporated by nitrogen (99.997% purity) and the concentrate was re-dissolved in 2 ml of acetonitrile/0.14% of acetic acid (vol/vol). Aliquots of the latter were injected into the HPLC system. The separation of CLA was performed with a mobile phase of acetonitrile/water/acetic acid (70/30/0.12, v/v/v) at a flow rate of 1.5 ml/min and CLA was detected at 234 nm, which was found optimal in a spectrum scan (not shown).
The dispersion of VD with and without RPH-MD was homogenized by Micro DeBee (Bee International Inc. South Easton Mass., USA) ultra-high pressure homogenizer: process pressure: 20-25 kpsi, Orifice diameter: 0.1 mm. The sample composition was 0.1% protein, 0.5% ethanol, VD concentration 0.02 mg/ml, buffer PBS pH 7. The sample volume subjected to homogenization was 25 ml. The influence of the homogenization process on particles size was evaluated by DLS measurements performed 2 hr after the homogenization.
SDS-PAGE analysis was used to verify the conjugation between the RPH and MD, and study the effect of pH on this process. This is a method of choice, as it separates by molecular size, thus enables to visualize higher molecular weight block-copolymer conjugates formed during the Maillard reaction of the saccharides and protein molecules.
Additional pH conditions of 4.5, 8.8 and 10.5 are shown in
The gel image was analyzed by the ImageJ, Java image processing software and the protein-to-conjugate conversion yield was evaluated according to the following procedure. The initial band area is spread between 2 and 5 kDa. On the final day of the heat treatment the band staining was distributed quite evenly between 2 and 15.7, 16.3, 19, 24 kDa for pH 4.5, 7.5, 8.8, 10.5, respectively. The conjugation yield was evaluated from the band area growth. The resulting yields were 65, 66, 71 and 77% for pH 4.5, 7.5, 8.8 and 10.5, respectively.
The reaction of RPH with MD resulted in browning which intensified with heating time (
The degree of RPH-MD conjugation was evaluated by quantifying the reduction of free amino groups as was quantified by the OPA assay. The results were summarized in
A typical absorbance change with heating time is shown in
To summarize the above results it can be determined that among the pH values studied, the Maillard reaction is quickest and most efficient at pH 10.5. However, for food applications, processing at extreme alkali conditions is not recommended.60 Moreover, pH 10.5 is less preferable in Maillard process due to high extent of melanoidins formation. For samples at initial pH of 4.5, 7.5 or 8.8 after 4 days of heating the conjugation similarly progressed to a significant extent. Based on the above considerations, subsequent experiments focused on the RPH-MD samples obtained after 4 days of heating at initial pH of 7.5.
Interaction of RPH-MD with CLA or VD
Next, the functionality of the conjugates for nanoencapsulation of model hydrophobic nutraceuticals (HN), conjugated linoleic acid (CLA), and vitamin D (VD) was investigated by DLS and turbidity measurements. It was hypothesized that, the co-assembly of HN and the amphiphilic conjugates should result in the formation of smaller colloidal particles, compared to aggregates of the HN alone dispersed in water, consequently leading to formation of more transparent solutions. Moreover, it was hypothesized that the encapsulation of the HN by a “shell” of the conjugates would provide some protection against degradation. The RPH itself might serve as such amphiphile and further improvement of its amphiphilic and protective properties could be achieved by the Maillard based block-copolymers.
This phenomenon is further depicted in
As mentioned above, VD was another model HN studied. VD particle sizes with and without RPH and MD mix or conjugate, at a concentration range of 10 to 300 μg/ml are shown in
The turbidity test shown in
To further improve clarity and homogeneity, an ultra-high pressure homogenization process was employed on the samples of VD with and without the conjugate. In
After the interaction between RPH-MD and HN was evidenced by DLS and turbidity measurements, the protective capability of the conjugate nanovehicles was evaluated. The CLA and VD are known to be unstable if exposed to oxygen and/or acidic conditions. The entrapment within the RPH-MD nanocapsules was hypothesized to provide some protection to CLA and VD against degradation processes during product shelf life. Without wishing to be bound by any particular mechanism or theory, it is hypothesized that the protection capabilities of the proposed system are not only due to immobilization and physical shielding of the HN by the conjugates, but also due to antioxidant features of the conjugates and their building blocks and Maillard reaction by-products. It was recently shown that the hydrolyzed proteins possess antioxidant features. Maillard products were also shown to serve as antioxidants.
The protection of VD against a 2 hr acidic pH treatment is shown in
Next, the shelf life of VD was examined at RT and at 4° C. in blank, mix and conjugate samples and the results were summarized in
The Maillard reaction of RPH and MD was examined by SDS-PAGE analysis as a function of time at different pH conditions. For pH 4.5, 7.5 and 8.8, similar progression was observed showing gradual Mw increase of the oligo-peptides species due to covalent bonding with the MD molecules. For pH 10.5, after one day of heating a significant Mw increase was obtained i.e. the reaction rate was the highest at this pH. These results were confirmed by color development studies. The absorbance measurement showed an increase as a function of reaction time at spectral region typical to Millard reaction products. The free amino residues concentration decreased with the Maillard reaction progression as was shown by the OPA analysis. The highest decrease was obtained for pH 10.5 samples in agreement with other methods of analysis. The amino residues content decreased mainly on the first day of heating and then stayed approximately unchanged during the rest of the time.
The size distribution of the obtained RPH-MD conjugates was measured by DLS and an enlargement of particle size compared to the control samples was shown which served an additional proof for the conjugation process, and a possible clue for some self-assembly.
The interaction of the RPH-MD with two model HN: CLA and VD, was studied by DLS and turbidity analyses. Significant reduction of the HN particle size in the presence of RPH-MD was achieved, which also resulted in a decrease in turbidity of the mixture (i.e., increased transparency). These effects were attributed to the solubilization capability of the RPH-MD due to co-assembly with the HN. RPH-MD conjugates were found to be more effective dispersants than the RPH-MD mixture, i.e. the conjugation has improved the RPH amphiphilic properties.
The protection of VD and CLA during the shelf life by the RPH-MD mix and conjugate was evaluated. It was found that both model HN compounds underwent significant degradation under acidic conditions but significant improvement was achieved in presence of RPH-MD with considerable advantage of the conjugate over the mix. Examination of CLA shelf life at neutral pH and RT showed significant improvement of the residual percentage of CLA in the presence of the RPH-MD conjugates. After 14 days it was 0, 70 and 75% for blank, mix or conjugate samples, respectively. For VD after 22 days of the simulated shelf life at neutral pH and 4° C., 28, 37 and 52% of residual VD were obtained for blank, mix or conjugate samples, respectively. These results demonstrate the good potential of the HRP-MD Maillard conjugates (and Maillard conjugates in general) as natural nanoencapsulating materials for HN for application in clear drinks.
The objective of this part of the study was to form Maillard conjugates of soy proteins and maltodextrin, characterize them and evaluate their potential for nanoencapsulation of nutraceuticals, preferably for clear liquid systems.
Soy protein beta-conglycinin was kindly donated by Solbar (Solbar Plant Extracts Ltd, Ashdod, Israel). Beta conglycinin was chosen as it is more amphiphilic than the other major soy protein, glycinin. It was dialyzed against distilled water with Spectra/Por molecular porous membrane tubing (M.W.C.O 12-14 kDa). However, it is apparent to a person of skill in the art that other soy proteins including but not limited to glycinin can also be used to form the Maillard conjugates of the present invention.
(−)-Epigallocatechin-3-gallate (EGCG) (CAS registry number 989-51-5) (EG-090, purity>90% by HPLC) EGCG was chosen as the model hydrophilic nutraceutical compound for this study, because of its highly important health benefits, and the challenge of protecting water-soluble nutraceuticals. Additionally, it is a good model for nutraceutical study, as it changes its color upon oxidative degradation. It was purchased from Shanghai Angoal Chemical Co. (Shanghai, China).
Freeze dried solutions of beta-conglycinin (β-cong) and Maltodextrin (MD) DE=19 at molar ratios of 1:1, 1:2, 1:4, 1:8 were heated (60° C. at 79% RH) for 6 hrs to form conjugates by Maillard reaction. After heating, the samples were freeze dried. The Maillard reaction products were dissolved in phosphate buffer solution (PBS) pH 6.87, 30 mM.
Conjugate solution (β-cong: MD molar ratio 1:8), mixture solution (β-cong: MD molar ratio 1:8) and β-cong solution were centrifuged at 15700′ g for 1 min. B-cong concentration was 0.25% w/v in all three solutions. The pellet was dried overnight in an oven at 100° C. and then weighted by an analytical balance. The percentage of soluble material was calculated as:
C=the conjugate/mixture/β-cong concentration
V=the solution volume
Wp=the pellet weight
Particle size distribution was determined by a dynamic light scattering (DLS) analyzer (NICOMP™ 380, Agilent Technologies, Inc., Santa Barbara, Calif., USA) equipped with an Avalanche Photo Diode (APD) detector, used at a fixed angle θ=90°. The 90 mW laser wavelength was 658 nm. Mono- bi- or tri-modal distributions were calculated from the scattered light intensity fluctuations, by Nicomp™ cumulants analysis of the autocorrelation function. Measurements were made in duplicate at 23° C.
Conjugate solution (β-cong: MD molar ratio 1:8), mix solution (β-cong: MD molar ratio 1:8) and β-cong solution were centrifuged at 15700′ g for 1 min, and the supernatant was collected. EGCG solution (0.125% w/v 30 mM PBS, pH 2.5) was added to the supernatant. The final EGCG concentration was 0.0125% w/v. The final con/mix/β-cong concentration was 0.092% w/v and the pH of the final solution was 6.69. After adding the EGCG solution, the samples were vortexed for 20 seconds.
After preparation, the samples were placed in 1 cm path length spectrophotometer cuvettes and covered with parafilm. Absorbance at 425 nm was recorded with time at room temperature for 300 hrs.
The Maillard reaction products at different molar ratios (β-cong: MD) were dissolved in PBS pH 6.87, 30 mM, and particle size distribution was measured by DLS. The results are shown in
To separate the two populations, the samples were centrifuged at 15700′ g (13000 rpm) for 1 min. After centrifugation, the pellets were dried overnight in an oven at 100° C., and then weighted by analytical scales. The percentage of soluble material was calculated as described in the Methods section; results are shown in
Mix solution was obtained by adding MD solution to β-cong solution, at the same b-cong:MD molar ratio as that of the conjugate. MD and β-cong were subjected to the same heat treatment as the conjugates (60° C. at 79% RH for 6 hrs).
As seen from
(−)-epigallocatechin-3-gallate (EGCG) was chosen as an oxidation-sensitive bioactive. EGCG is one of the main effective constituents of green tea; it is a water soluble polyphenol which is highly unstable in neutral and alkaline solutions.
The co-assembly of the conjugates with EGCG was studied by measuring particle sizes. The changes in size distribution of the system were also measured after 48 hrs. Results are shown in
EGCG degrades irreversibly from a colorless clear solution of the fresh compound, to a yellow solution of the deterioration products, mainly due to oxidation and the formation of dimers. To estimate the protection against oxidation provided by the conjugate, the absorbance of EGCG with the conjugate was measured at 425 nm. Protection provided by control sample of the mixture solution of β-cong and MD was also monitored. Results are shown in
These results of this study emphasize the potential of soy β-cong-MD Maillard conjugates as nanoencapsulation material for clear drink applications. The conjugates showed better solubility than the mixture of their components. The conjugates, with and without EGCG gave smaller particle sizes than solutions of MD with and without EGCG. The conjugation apparently facilitated formation of smaller-more soluble entities, with particle sizes that remained below 20 nm after at least 48 hrs from preparation. The protection provided by the conjugate-based nanoparticles to EGCG was more significant than the protection provided by the mixture control sample.
The objective of this part of the study was to form Maillard conjugates of casein and maltodextrin (MD), characterize them and evaluate their potential for nanoencapsulation of hydrophobic nutraceuticals, preferably for clear liquid systems. The behavior of the nanocapsules during simulated gastric digestion was also studied.
Maltodextrin (MD) of dextrose equivalent 19 which corresponds to approximately 10 KDa was donated by Productos de maiz S.A. Corn Products international (Munro, Argentina). Caseinate was donated by Strauss-group, and was manufactured by Molkerei Meggle Wasserburg GmbH and co. (Casinella QN lot number 901155). O-phthaldialdehyde (OPA), Trizma Base, SDS, Vitamin D3 (VD3), Vitamin D2 (VD2), mercaptoethanol, Acrylamide/bis-Acrylamide, Ammonium persulphate, pepsin from porcine gastric mucosa (3200-4500 units/mg), Nile red, were obtained from Sigma-Aldrich (Rehovot, Israel). Methanol and Acetonitrile both of HPLC grade were obtained from LabScan (Dublin, Ireland). NaOH was obtained from Merck (Darmstadt, Germany). Ethanol absolute—BioLab (Jerusalem, Israel), Sodium Tetraborate-Laba Chemie (Mumbai, India). SDS-PAGE size markers and Coomassie Blue 250-R stain were obtained from Bio-Rad. Bromophenol blue was obtained from Fluka.
Epigallocatechin-3-gallate (EGCG) (CAS registry number 989-51-5) (EG-090, purity>90% by HPLC) was purchased from Shanghai Angoal Chemical Co., Ltd. (Shanghai, China).
Acetone was purchased from Frutarom, Israel.
Caseinate powder was dissolved in Doubly Deionized water with 0.02% (w/w) sodium azide over-night. Later it was dialyzed for 48 hrs, frozen and freeze-dried. Freeze dried solutions of Caseinate and MD at different molar ratios were heated (60° C. at 79% RH) for 4, 6, and 8 hrs. (Similar ranges of conditions were used28, 64-67 to form conjugates without significant progression of the Maillard reaction to degradation and/or polymerization). After heating the conjugates were freeze-dried again.
All characterization procedures were made on both CN-MD conjugates (conjugates) and CN with MD mixture (mixture). Mixture control-samples of casein (CN) and MD were heated separately, than mixed.
The Maillard reaction was tracked by electrophoresis method using PHAST system (Pharmacia LKB Biotechnology, GE Healthcare), PhastGel gradient 8-25, and PhastGel SDS Buffer Strips, both manufactured by GE Healthcare were used. The samples in a form of lyophilized powder were dissolved in a sample buffer (50 mM Tris, 1% SDS, 2.5% mercaptoethanol, 10% glycerol, 1 mM EDTA, 0.025% bromophenol blue). The samples were incubated for 5 min at 95° C. with vigorous stirring. A final sample concentration of 5 mg/ml on protein basis was obtained. A volume of 1 μl was loaded on the gel. After the electrophoresis the gels were immersed in a fixation solution (30% methanol, 10% acetic acid) for 0.5 hr, then stained in a Coomassie Brilliant Blue R-250 for 1 hr and washed by 10% acetic acid solution.
The RPH-MD graft samples were analyzed by the OPA assay to determine the degree of conjugation. The OPA reagent was prepared as described above in Example 1. The following compounds were diluted with water to 100 ml: 80 mg OPA (dissolved in 2 ml 95% ethanol); 50 ml 0.1 M sodium tetraborate, 5 ml 20% SDS; 0.2 ml of 2-mercaptoethanol in ethanol. The OPA reagent was prepared immediately before use. The CN-MD conjugates and mixture samples at concentration of 0.5 mg/ml on casein base were prepared in DDW with 0.02% azide. 0.05 ml of the sample was added to 2 ml of OPA reagent. This solution was briefly stirred and absorption at 340 nm was measured after a 2-min equilibration at room temperature. A standard curve was obtained by using L-leucine as a reference compound. Reference samples with a concentration ranging from 1.52E-5 to 7.62E-3 M were prepared in DDW 0.02% sodium azide and the L-leucine determination was performed as described above.
The samples at concentration of 1 mg/ml were dissolved in ddw, and acidified to pH=4.6 (pI of casein), followed by separation of the precipitate by centrifugation at 1000 g for 10 minutes. Then, the supernatant was transferred to a new tube and pH was adjusted back to 7 with NaOH 5 M. The protein content of the pre-separated solution and supernatant were measured by absorbance at 278 nm. The yield of separation was defined as follows:
Absorbance was measured by Ultrospec 3000 UV/Visible Spectrophotometer, GE healthcare.
The particle size evaluation was performed by dynamic light scattering (DLS) analyzer (NICOMP—380, Particle Sizing Systems Inc., Santa Barbara, Calif., USA). The detector angle was set to 90 degrees. Samples of mixture, conjugates, and supernatant with and without HN were analyzed. ND (“neutral density”) filter (light intensity adjustment, which is an indication of the amount of scattered light from the sample) was kept in the range of 70-120, by adjusting sample concentration, to avoid multiple scattering.
The co-assembly of the HN with the conjugates was achieved by addition of the VD or Nile red (NR) dissolved in ethanol into the CN-MD solution during stirring. The HN ethanol solutions were prepared at different concentrations while the final ethanol concentration was kept constant 0.25% (vol/vol). All solutions containing VD were flushed with argon gas to prevent oxidation.
A. Extraction of VD using phase separation in a separatory funnel of diethyl ether: petroleum-ether was accomplished as was previously described.36 While this procedure was good enough for extracting the VD from a mixture solution, it was insufficient for extracting it from the conjugates, as they stabilized an emulsion and no phase separation occurred, preventing extraction (an indication of the superior encapsulation capacity of the conjugates compared to that of the mix).
B. Extraction of VD using a method based on Kazmi et al:37 1 ml of sample solution was put in a glass centrifuge tube, 3.5 ml of methanol:chloroform (2:1 vol/vol) were added, then the tube was vortexed for 20 seconds, followed by addition of 1.5 ml chloroform and vortexing for 60 seconds. Argon gas was added to the headspace and the tubes were capped and centrifuged for 10 minutes at 1500 g at 4° C. Two ml of the clear chloroform layer at the bottom of each test tube were transferred to an evaporation vial using a glass syringe. The chloroform extract was dried under a flow of nitrogen gas, reconstituted in 1 mL of the high performance liquid chromatography (HPLC) mobile phase [methanol:acetonitrile:water (49.5:49.5:1 v/v)], the tube headspace was filled with argon gas. The tubes were left undisturbed for 15 min, after which the samples were put on ice until injection to HPLC. Operating conditions were: ambient temperature (˜24° C.); mobile phase was methanol: acetonitrile: water (49.5:49.5:1, by vol); flow rate was 0.3 ml/min; and the absorbance was measured at 265, 254 and 228 nm.
C. VD degradation at pH=2.5: VD2 and VD3 in buffer solutions were made by addition of VD stock solution in ethanol into phosphoric acid buffer at pH=2.5, to a final concentration of 0.05 mg/ml. Samples were incubated at room temperature for 2 hrs and then analyzed for VD. Peak areas were compared to calibration curves.
The protection of VD2 by the conjugates as a function of time, compared to controls of mixture and buffer was evaluated. Simulated shelf life conditions of temperature 4° C. and pH 7 were tested. For pH 7 a NaH2PO4/Na2HPO4 buffer was used. Samples were flushed with argon, heated for 2 minutes at 80° C., and kept at 4° C. for 15 days. VD was analyzed, before heating, after heating and after 1, 3, 5, 9, 13, and 15 days. Extraction was carried out, then the VD content was quantified using reversed phase HPLC (RP-HPLC), equipped with 4.6×100 mm C18-C2 Pharmacia column and a triple wavelength UV detector. The volume of the injection loop was 100 μl. The operation temperature was 24° C. The initial concentration of VD2 was 0.05 mg/ml (simulating a concentrate of an enriched soft drink). The molar ratio of VD to CN was 1:1.
EGCG was chosen as a model for a sensitive water soluble nutraceutical. It was dissolved in phosphoric acid buffer (20 mM) pH=2.5, and added to solutions of the conjugate, and the controls: CN: MD mixture, CN, MD, PBS (pH=7) and phosphoric acid buffer (pH=2.5). In mixture and conjugate solutions casein concentration was 5 mg/ml, MD: CN molar ratio was 4. In casein and MD solutions each substance concentration was the same as in the mixture and the conjugate. EGCG concentration was 0.9 mg/ml. Samples were kept at room temperature.
EGCG oxidation was measured by determining absorbance at 425 nm, based on the observation that EGCG oxidation products absorb at 425 nm.
Nile read (NR) was chosen as a model for a hydrophobic nutraceutical, as it is similar in structure and properties (Table 1) to such hydrophobic nutraceuticals like VD, and it can be easily determined by spectrophotometry, and spectrofluorometry. Moreover, it “reports” of its binding or hydrophobic entrapment, by changing its fluorescence. Nile red is known as a probe for hydrophobic domains and as a probe for protein hydrophobic surfaces. When in water it adsorbs to the glass and does not fluoresce.
The chemical structures of a—Nile red, b—Vitamin D2, c—Vitamin D3 are set forth below:
NR stock solution in ethanol was prepared at a concentration of 0.16 mg/ml=502.6 μM. NR was added during vortexing to the conjugate, mixture or buffer solution, at a concentration of 1.3 μM, to minimize inner filtering effect in fluorescence measurements.
NR was added as mentioned above to ethanol, buffer pH=2.5, water, conjugate solution, and mixture solution. Casein concentration was 5 mg/ml, MD: CN Molar ratio was 4. NR final concentration was 1.3 uM. Absorbance was measured in a quartz cuvette (path length=10 mm) using Ultrospec 3000 UV/Visible Spectrophotometer, (GE healthcare).
All fluorescence measurements were done using a Fluorolog 3-22, (Horiba Jobin Yvon, Edison, N.J. USA). Emission spectra was measured at excitation wavelength (ex) of 570 nm, slit width was 5 nm for both excitation and emission. NR final concentration was 1.3 μM emission spectrum was measured in a quartz cuvette sized 10 mm*2 mm at a right angle mode. Blank measurements were also taken and were two orders of magnitude smaller than that of the NR signal.
Evaluating HN Release from Nanocapsules by Examination of NR Adsorption to Glass:
Free NR in aqueous solution adsorbs to the glass walls, which may serve as a convenient way to study its release behavior from nanocapsules. To examine whether NR adsorbs to glass when in water, NR was added to 1 ml of water in a glass vial to a final concentration of 1.3 μM. The vial was left for 2 hrs, then the water was transferred to a new glass vial, and 1 ml of acetone was added to the new vial. At the same time 2 ml of water:acetone (1:1 vol/vol) solution was added to the first vial. After 1 hr both solutions were read for florescence intensity at ex:570, em:645 which is the intensity peak at water:acetone 1:1. Concentration was calculated from the intensity using a NR in water:acetone (1:1) calibration curve.
Since it was validated that all free NR adsorbs to the glass when in water, detection of NR which did not adsorb to the protein (or was released from it after protein digestion) was carried out as follows: NR was added to CN-MD mixture, to conjugate (casein concentration was 3 mg/ml, MD: CN molar ratio was 4) and to buffer (all at pH=2.5 in phosphoric acid buffer 20 mM) to a final concentration of 1.3 μM. Then pepsin was added to some of the samples at a concentration of 0.15 mg/ml, in order to reach pepsin: CN mass ratio of 1:20 according to the method by Mandalari et al.79. The samples were left for 2 hrs at 37° C. while gently stirring to simulate gastric digestion. After two hours, protein aggregates sedimented to the bottom of the vials in which pepsin was present. The solution and the aggregates were taken out using a syringe and put into new vials. Into the old vials we added 1 ml of water: acetone solution. After 1 hr the acetone: water solution with NR which had adsorbed to the glass was read for fluorescence intensity (ex:570, em:645). NR fluorescence was measured with time to validate that no bleaching occurred during the procedure (data not shown).
MD and Casein Conjugation Characterization: Conjugation of Casein with MD DE=6
As seen in
MD DE=6 was not used in later work. Rather, MD with greater solubility was used in order to allow the formation of concentrated homogeneous solutions before conjugation.
Conjugation of Casein with DE=19 MD
An average casein molecule contains about 13.6 lysine residues. The ratio which resulted in largest decrease in free lysines was 1:4 so theoretically more lysines could have reacted with MD. Without wishing to be bound by any particular mechanism or theory, there are two possible explanations why this did not occur are: 1. Steric hindrance: not all lysines are accessible, and even more so, after one or two MD molecules attached to the casein, the access of additional MD molecules to the casein is even more difficult. 2. During freezing, phase separation occurred and good contact between the MD and the casein was not achieved.
A better conjugation ratio may be achieved by quench-freezing of the mixed solution of MD with casein.
At a ratio of 1:4 it is expected that the maximum decrease in free lysines would be 4/13.6=31%, which means residual amines of about 69%. The observed decrease was of 60%, i.e. more than expected. An explanation for this phenomenon may be that progressive Maillard reactions caused further decrease of free lysines below 69%.
The number of lysine per casein molecule was calculated as follows:
Examination of sedimentation at the casein pI
With the aim of fractionating the Maillard products and concentrating the conjugates in a simple, industrially feasible procedure, sedimentation of the unconjugated casein was performed at its pI, and the supernatant containing the conjugates which are still soluble at this pH was collected. The sedimentation at the casein pI, and the yield calculation was performed as described in the Materials and Methods section. The yield calculation was based on spectrophotometric determination of protein concentrations in the supernatant and original solution.
According to
Based on the results presented in
Nanoencapsulation of VD Using pI-Fractionated (Supernatant) Conjugates.
The supernatant was collected; freeze dried and later dissolved in buffers at pH 2.5, 4.6 and 7. VD3, predissolved in ethanol, was added to those solutions during vortexing. The final casein concentration was 1 mg/ml, VD: CN molar ratio was 1:1. The solutions were then measured for size distribution using DLS. Size distribution of particles containing VD with supernatant conjugate at different pH is shown in
Comparing VD Nanocomplexes Made of CN:MD Conjugates Vs. CN:MD Mixture, and Evaluating the Effect of VD:CN Molar Ratio.
The range of ratios described in
Future studies were conducted with VD: CN molar ratios of 1 and 2 because at those concentrations and ratios mostly particles smaller than 100 nm were formed. At VD concentrations of above 0.05 mg/ml large aggregates were formed. In addition, VD without the conjugates tended to aggregate as expected at all concentrations. Shelf life experiments were conducted for VD at the highest concentration that the conjugate can stabilize and prevent its aggregation, i.e. 0.05 mg/ml. Casein concentration was 3 mg/ml in order to reach 1:1 VD:CN molar ratio.
VD degrades at low pH, hence the protection conferred by the nanoencapsulation against its degradation at pH 2.5 was evaluated. The results are presented in
Residual VD during a simulated shelf life study is shown in
Without wishing to be bound by any particular mechanism or theory, several possible reasons for this improved protection are contemplated: 1. The interactions between the protein and VD cause its immobilization, thus reducing its reactivity in various reactions. 2. The conjugate nanocapsules shield the entrapped VD from external degradation factors, both chemical (oxidizing agents) and physical (e.g. UV radiation). 3. It was reported that Maillard products act as antioxidants. It is very likely that a combination of these mechanisms confers the observed protection.
Epigallocatechin gallate (EGCG) is a water soluble nutraceutical, extracted from green tea. It tends to oxidize at neutral pH and degrade into yellow products that absorbs at 425 nm. It was shown in a previous study that the interaction with milk proteins (beta lactoglobulin), particularly after they undergo heat treatment, can reduce the oxidation rate of EGCG significantly.
Studying Entrapment and Release from the Nanocapsules Upon Simulated Gastric Digestion, by Using Nile Red—a Fluorescent Model for a Hydrophobic Nutraceutical.
Interactions of Nile Red (NR) with Proteins and Solvents
Interaction of the conjugate and mixture with a hydrophobic nutraceutical was examined with a model molecule-NR whose fluorescence emission wavelength and quantum yield depend on the polarity of its environment. NR can be used as a model for Vitamin D2 and Vitamin D3 because all these molecules are very similar in Mw and hydrophobicity (as may be described by log P: log of the octanol-water partition coefficient, P). NR absorbance spectra in ethanol, in the conjugate, in the mixture and in a buffer solution are shown in
Subsequent experiments used 570 nm as the excitation for NR fluorescence measurements, as it is a wavelength that NR absorbs both in water and in conjugate/mix solution. Emission spectra of NR in different solvents (ex:570 nm) are shown in
In
Adsorption of NR to the Glass Surface when Added to Water: A Way of Probing the Release of a Model Hydrophobic Nutraceutical from Nanoparticles.
NR was used as a model for HN (e.g. VD), to study its release from the conjugate or the mixture during gastric digestion. NR was added to buffer, mixture and conjugate solutions (3 mg/ml CN) at pH=2.5, then pepsin was added to some of the solutions (1:20 pepsin:CN mass ratio). The solutions were stirred for 2 hrs in a 37° C. water bath. After stirring was stopped, aggregates of digested conjugate and mixture began to sediment in the samples containing pepsin.
Quantitative results were obtained by transferring the solution and sediment to new vials and addition of water:acetone (1:1). One hour later, water: acetone which extracted NR back from the glass was examined for fluorescence emission. Fluorescence intensity of NR adsorbed to glass is shown in
1. CN-MD Maillard conjugate based nanovehicles for enrichment of HN, having diameters of less than 100 nm, were successfully formed.
2. The complexes of VD-conjugate were less turbid than the ones formed by VD-mixture, and much less turbid than VD dispersed in buffer only, at the high concentrations studied, simulating soft drink concentrates (Completely clear solutions were obtained with nanoencapsulated VD at doses typical for the final drinks).
3. The supernatant conjugate can be used for enrichment of clear beverages even at pH close to 4.6 which is the pI of the native casein, where unconjugated casein nanocapsules would precipitate.
3. Conjugation significantly improved the protection against oxidation conferred to both VD and EGCG.
4. Conjugation does not significantly change the ability of caseins to bind HN.
5. Enzymatic hydrolysis by gastric pepsin of the casein was not followed by release of the hydrophobic molecules from the protein. It is likely that it remains bound to shorter peptides.
While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the spirit of the invention.
This application claims the benefit of and priority from U.S. Provisional Application No. 61/447,773, filed on Mar. 1, 2011, the entirety of which is incorporated herein by reference for the teachings therein.
| Number | Date | Country | |
|---|---|---|---|
| 61447773 | Mar 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13410001 | Mar 2012 | US |
| Child | 14269153 | US |
