Polymerized whey protein encapsulated antioxidant compounds and methods of making the same

Abstract

This application relates to antioxidant compounds encapsulated with polylactrin and methods for preparing the same. A method is provided for encapsulating glutathione (GSH), 3,3'-diindolemethane (DIM), coenzyme Q10 (CoQ10), and other hydrophobic antioxidant compounds using whey protein that can be polymerized in a specific manner. Furthermore, compositions comprising glutathione encapsulated with polylactrin (PWP), CoQ10 encapsulated with polylactrin 5 (PWP), and DIM encapsulated with polylactrin (PWP) are provided.

Description

[0001] This application is a divisional application of the application filed on August 17, 2021, with application number 202180066551.6 and invention title "Antioxidant Compound Encapsulated with Polymeric Whey Protein and Preparation Method thereof". Technical Field

[0002] This invention relates to a method of encapsulating glutathione (GSH), 3,3'-diindolemethane (DIM), coenzyme Q10 (CoQ10), and other hydrophobic antioxidant compounds using whey protein that can be polymerized in a specific manner. Compositions comprising glutathione, DIM, coenzyme Q10, etc., encapsulated in polymerized whey protein are described herein. background

[0003] Glutathione (GSH) is an antioxidant found in plants, animals, fungi, and some bacteria. Glutathione is the most abundant thiol in animal cells, ranging from 0.5 to 10 mM. It is present in cytosol and organelles (Guoyao Wu, Yun-Zhong Fang, Sheng Yang, Joanne R. Lupton, Nancy D. Turner. "Glutathione Metabolism and its Implications for Health," Journal of Nutrition (2004) 134(3):489–92).

[0004] Glutathione exists in reduced (GSH) and oxidized (GSSG) states. The ratio of intracellular reduced glutathione to oxidized glutathione is a measure of cellular oxidative stress, with an increased GSSG to GSH ratio indicating greater oxidative stress (A. Pastore et al., "Determination of blood total, reduced, and oxidized glutathione in pediatric subjects". Clinical Chemistry (August 2001) 47(8):1467–9; SC Lu. "Glutathione synthesis". Biochimica et Biophysica Acta (BBA)-General Subjects (May 2013) 1830(5):3143–53). In healthy cells and tissues, more than 90% of the total glutathione library is in reduced form (GSH), and the remainder is in disulfide form (GSSG); (KMHalprin, A. Ohkawara "The measurement of glutathione in human epidermis using glutathione reductase". The Journal of Investigative Dermatology (1967) 48(2): 149–52).

[0005] GSH protects cells by neutralizing (i.e., reducing) reactive oxygen species. The reduction of peroxides illustrates this transformation:

[0006] 2 GSH + R₂O₂ → GSSG + 2 ROH (R = H, alkyl)

[0007] Furthermore, free radicals can be quenched in the body:

[0008] GSH+R → 0.5 GSSG+RH

[0009] Furthermore, glutathione plays a crucial role in cellular regulation and metabolism. For these key metabolic and cellular functions, it would be advantageous to provide glutathione in stable and bioavailable compositions.

[0010] Glutathione lacks oral bioavailability in the digestive tract. Previous encapsulation technologies have been developed, primarily using lipid encapsulation (such as lecithin) combined with synthetic chemicals (such as polysorbate 80) to increase the absorption of poorly absorbed compounds. However, until now, whey proteins or polywhey proteins have not been used in this way. Therefore, a safe, chemical-free alternative is needed to improve bioavailability.

[0011] Finding a way to improve the stability of glutathione in compositions intended for delivery to mammals, particularly human subjects, and reduce its degradation would be a useful contribution to the field. Furthermore, finding a way to optimize the absorption of glutathione and its utilization by the body, such as by improving bioavailability, would be a further useful contribution to the field.

[0012] Regarding the encapsulation of nutrients, certain film-forming compounds and surfactants are useful, such as polyvinylpyrrolidone, polyoxyethylene stearate, sodium cholate, deoxycholate and taurocholate phosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, methylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, hydroxypropyl ethylcellulose, one or more of these can be blended with lecithin.

[0013] 3,3'-Diindolemethane (“DIM”) is an active metabolite of indole-3-methanol derived from cruciferous vegetables and exhibits broad-spectrum anticancer properties. The stability of DIM is a significant challenge in the pharmaceutical industry. Furthermore, DIM suffers from poor oral bioavailability due to its low solubility and high lipophilicity. Encapsulation with whey protein is known to develop nanoparticles with controllable size and properties, a method that can provide protection, preservation, and delivery of sensitive compounds such as aromatic compounds or health supplements.

[0014] Certain methods for encapsulating 3,3'-diindolylmethane (“DIM”) and similar substances using ultrasonication are known. See, A. Khan, et al., “Physicochemical and Microstructural Properties of Polymerized Whey Protein Encapsulated 3,3'-Diindolylmethane Nanoparticles,” Molecules (2019) 24:702.

[0015] If a way can be found to improve encapsulation methods to provide DIM encapsulated with polymeric whey protein, for example, with better stability, solubility, and other improved properties (such as oral bioavailability), it would contribute to the chemical and formulation industry. Invention Overview

[0016] A composition is described, the composition comprising polymeric whey protein encapsulating glutathione.

[0017] A method for preparing microencapsulated glutathione of polymeric whey protein is described, the method comprising the steps of: dissolving whey protein concentrate powder in water at about 8%-12% w / v to provide an aqueous solution; heating the whey protein concentrate solution to at least 80°C for about 15-25 minutes to provide a polymeric whey protein solution; adding an antioxidant compound to the polymeric whey protein solution during a cooling process; stirring with a homogenizer or shear mixer to provide a clear, homogeneous solution; and separating the microencapsulated antioxidant compound of polymeric whey protein to provide a powder, for example by freeze-drying or spray drying.

[0018] Furthermore, a method for preparing polymeric whey protein microencapsulated DIM (PWP-DIM) is described, the method comprising the steps of: (a) dissolving whey protein concentrate powder in water at 10% w / v to provide an aqueous solution; (b) heating the whey protein concentrate solution to at least about 70°C-80°C to provide a polymeric whey protein solution; (c) adding DIM to the polymeric whey protein solution; (d) adjusting the pH to a range of about 6.5 to about 9.0; (e) cooling the polymeric whey protein solution; (f) stirring during cooling from about 80°C to about 45°C to provide a clear, homogeneous solution; and (g) separating the polymeric whey protein microencapsulated DIM.

[0019] One objective is to prepare PWP-DIM, which has significantly better encapsulation to better protect it as it passes through the digestive tract (i.e., the gastrointestinal tract).

[0020] Another objective is to prepare PWP-DIM, which has improved absorption in the digestive tract.

[0021] Furthermore, a method for preparing a microencapsulated antioxidant compound of polywhey protein is described, the method comprising the steps of: (a) dissolving whey protein concentrate powder in water at 10% w / v to provide an aqueous solution; (b) heating the whey protein concentrate solution to approximately 70°C-80°C for approximately 15 minutes to provide a polywhey protein solution; (c) adding glutathione to the polywhey protein solution; (d) stirring to provide a clear, homogeneous solution; and (e) separating the polywhey protein microencapsulated glutathione, for example by freeze-drying, to provide a powder.

[0022] In one embodiment, the method for preparing microencapsulated antioxidant compounds of polymeric whey protein is used to prepare microencapsulated glutathione of polymeric whey protein (PWP-GSH).

[0023] In another embodiment, the method for preparing microencapsulated antioxidant compounds of polywhey protein is used to prepare microencapsulated coenzyme Q10 of polywhey protein (PWP-CoQ10). Attached Figure Description

[0024] Figure 1 A standard curve for determining reduced glutathione (GSH) using HPLC was plotted. The standard curve was constructed as a function of concentration versus peak area.

[0025] Figure 2 A standard curve was plotted to determine reduced glutathione (GSH) using the assay kit. The standard solutions were plotted as absolute OD values ​​compared to GSH concentrations (μM).

[0026] Figure 3 The plasma concentration (μmol / L) of reduced glutathione (GSH) in mice as a function of time was described after oral administration of the test sample.

[0027] Figure 4 A standard curve was plotted to determine the antioxidant activity of plasma. The standard curve was plotted by determining the OD values ​​of different standard samples at 0.1, 0.2, 0.4, 0.8, and 1.0 mM.

[0028] Figure 5 The in vivo antioxidant activity of mouse plasma after oral administration of free GSH, WPC / PWPC-based GSH, and WPC / PWPC, as measured by an assay kit (ABTS method), is depicted in the examples. The vertical bars at individual time points are arranged from left to right as follows: control, WPC, PWP, GSH, WPC-GSH, and PWP-GSH.

[0029] Figures 6A-6F In another embodiment, the concentrations (μM) of GSH in mouse tissues measured after oral administration of free GSH, WPC / PWPC-based GSH, and WPC / PWPC are depicted. The vertical bars at individual time points are arranged from left to right as follows: control, WPC, PWP, GSH, WPC-GSH, and PWP-GSH. Figures 6A-6F The measurements are shown for the brain (A), heart (B), lungs (C), kidneys (D), liver (E), and intestines (F).

[0030] Figure 7 In another embodiment, the weight curves of rats in grams were depicted in a 28-day feeding test in which PWP-GSH was incorporated into the feed.

[0031] Figure 8 In another embodiment, a feed consumption curve for rats in grams per rat per day was depicted during a 28-day feeding test in which PWP-GSH was incorporated into the feed.

[0032] Figure 9 In another embodiment, a food efficiency curve for rats in a 28-day feeding test incorporating PWP-GSH into their feed is depicted.

[0033] Figure 10A Microscopic images of stained sections of several organ tissues from male rats in a 28-day feeding test in which 4% by weight of PWP-GSH was incorporated into the diet compared to a control were depicted.

[0034] Figure 10B Microscopic images of stained sections of several organ tissues from female rats in a 28-day feeding test in which 4% by weight of PWP-GSH was incorporated into the feed compared to a control were depicted.

[0035] Figure 11 A standard curve was plotted to determine the content of reduced GSH in solid samples using the reduced glutathione (GSH) assay kit (A006-2-1).

[0036] Figure 12 TEM images of PWPC and PWPC-GSH, determined using standard techniques, are depicted. The scale at the bottom is 1.0 micrometer.

[0037] Figure 13A The particle size distribution of whey protein-encapsulated glutathione nanoparticles (e.g., PWPC-GSH) based on whey protein was depicted.

[0038] Figure 13B The particle size distribution of whey protein-encapsulated glutathione nanoparticles (e.g., PWPI-GSH) based on WPI was depicted.

[0039] Figure 14 The polydispersity index (PDI) of whey protein-encapsulated glutathione nanoparticles (e.g., PWPI-GSH) based on WPC and WPI starting materials was characterized. The letter ac indicates significant (P≤0.05).

[0040] Figure 15 The zeta potential (mV) of whey protein-encapsulated glutathione nanoparticles (e.g., PWPI-GSH) based on WPC and WPI starting materials was depicted. The letter ad indicates significant (P≤0.05).

[0041] Figure 16A The apparent viscosity (mPa-sec) of whey protein-encapsulated glutathione nanoparticles (e.g., PWPC-GSH) based on WPC was described as a function of the shear rate (1 / s).

[0042] Figure 16B The apparent viscosity (mPa-sec) of whey protein-encapsulated glutathione nanoparticles (e.g., PWPI-GSH) based on WPI was described as a function of the shear rate (1 / s).

[0043] Figure 17AThe circular dichroism x 10⁻¹⁰ of whey protein-encapsulated glutathione nanoparticles (e.g., PWPC-GSH) based on whey protein was depicted. 3 deg cm 2 dmol -1 Compared to wavelength (nm).

[0044] Figure 17B The circular dichroism x 10⁻¹⁰ of whey protein-encapsulated glutathione nanoparticles (e.g., PWPI-GSH) based on WPI was depicted. 3 deg cm 2 dmol -1 Compared to wavelength (nm).

[0045] Figure 18A The FT-IR spectra of whey protein-encapsulated glutathione nanoparticles (e.g., PWPC-GSH) based on WPC were depicted using standard techniques.

[0046] Figure 18B The FT-IR spectra of glutathione nanoparticles (e.g., PWPI-GSH) encapsulated with whey protein based on WPI were depicted using standard techniques.

[0047] Figure 19 The average particle size (nm) of PWPI standards and PWPI-CoQ10 samples, measured by standard methods at ratios of 100:1, 80:1, 60:1, 40:1 and 20:1 (PWPI:CoQ10), is depicted.

[0048] Figure 20 The polydispersity index (PDI) of PWPI standards and PWPI-CoQ10 samples, measured by standard methods at ratios of 100:1, 80:1, 60:1, 40:1, and 20:1 (PWPI:CoQ10), is described.

[0049] Figure 21 The zeta potential (mV) of PWPI standards and PWPI-CoQ10 samples, measured by standard methods at ratios of 100:1, 80:1, 60:1, 40:1 and 20:1 (PWPI:CoQ10), is depicted. Detailed description

[0050] In one aspect, the present invention relates to the microencapsulation of both fats and water-soluble antioxidants using polymerized whey protein, with the explicit aim of protecting nutrients from degradation and optimizing their absorption and utilization in vivo. For example, the inventors of this invention have contributed to and described the method for preparing polymerized whey protein-encapsulated glutathione (PWP-GSH) according to the present invention in: S. Zhang, et al., “Polymerized Whey Protein Concentrate-Based Glutathione Delivery System: Physicochemical Characterization, Bioavailability and Sub-Chronic Toxicity Evaluation,” Molecules (2021) 26:1824, which is hereby incorporated herein by reference in its entirety.

[0051] In another aspect, the present invention relates to a novel method for manipulating whey proteins by adjusting temperature and pH to allow the proteins to be encapsulated around nutrients (particularly antioxidants) for protection and optimal absorption and utilization. Antioxidants that have been successfully encapsulated using the method of the present invention include glutathione, diindolemethane, and coenzyme Q10. Current research is underway to apply this technology to dietary supplements (e.g., tableting, encapsulation, and incorporation into ready-to-drink powders) and to allow for the successful incorporation of antioxidants into a variety of foods and beverages to enhance their health benefits.

[0052] The advantage of the current method is that it achieves a technically simplified and more natural approach, without using certain chemical reagents that natural food or dietary supplement companies typically do not want.

[0053] In the primary embodiments, the encapsulation of antioxidant compounds and other derivatives is more uniform and consistent, which allows for better protection of the active ingredients in the digestive tract. Furthermore, these method embodiments provide better encapsulation when observed under a microscope, for example, using SEM or TEM.

[0054] Compared to free GSH, the PWP-GSH compositions and formulations described herein exhibit improved in vivo absorption, better pharmacokinetics, better bioavailability, and higher antioxidant capacity. Compared to comparable GSH products, the PWP-GSH compositions and formulations described herein also exhibit improved in vivo absorption, better pharmacokinetics, and better bioavailability.

[0055] Microencapsulation

[0056] Microencapsulation is a technique for protecting a variety of biomolecules. See, Whey Protein Production, Chemistry, Functionality, and Applications, edited by Mingruo Guo (one of the inventors of this invention), Chapter 7, “Whey Protein Functional Properties and Applications in Food Formulation,” pp. 157–204 (and references cited therein), (Wiley: Hoboken, NJ, 2019), which is incorporated herein by reference.

[0057] Formulations can be prepared in any product form suitable for human consumption, including reconfigurable powders, ready-to-eat liquids, parenteral (or intravenous) formulations, and dilutable liquid concentrates, all of which are well known in the field of nutritional formulation. As used herein, the amount of a component present in a formulation or composition refers to the amount when the formulation or composition is prepared for consumption by a human individual.

[0058] The formulation or composition may optionally be sterilized and subsequently used on an ready-to-eat basis, or it may be stored as a concentrate. The concentrate can be prepared by spray drying the liquid formulation as described above, and the formulation can be reconstituted by rehydrating the concentrate. The formulation concentrate is a stable liquid with a suitable shelf life.

[0059] For powder examples of formulations or compositions containing GSH, whey protein-encapsulated GSH (WP-GSH), or polymeric whey protein-encapsulated GSH (PWP-GSH) used in the methods of the present invention, the powder can be reconstituted with a suitable aqueous liquid, preferably water. Reconstituteable powders are typically in the form of flowable or substantially flowable particulate compositions, or at least specific compositions that can be easily scooped and measured with a spoon or similar other device, wherein these compositions can be easily reconstituted by the intended user with a suitable aqueous fluid (typically water) to form a liquid formulation or composition. In this context, “immediate” use generally means within about 48 hours, most typically within about 24 hours, preferably immediately after reconstitution. These powder examples include spray-dried, agglomerated, dry-mixed, or other known or other effective particulate forms. The amount of nutritional powder required to produce a volume suitable for one serving can vary.

[0060] Nutritional formulations used in the methods of this invention can be packaged and sealed in single-use or multi-use containers and then stored under ambient conditions for up to about 36 months or longer, more typically from about 12 to about 24 months. For multi-use containers, these packages can be opened and then covered by the end user for reuse, provided that the covered package is then stored under ambient conditions (e.g., avoiding extreme temperatures) and the contents are used within about one month.

[0061] Compositions for delivering oral formulations of dietary supplements (containing GSH, whey protein-encapsulated GSH (WP-GSH), or polymeric whey protein-encapsulated GSH (PWP-GSH)) can be administered orally, for example, with an inert diluent or with an absorbable edible carrier, or can be encapsulated in hard or soft-shell gelatin or hydroxypropyl methylcellulose (i.e., hydroxypropyl methylcellulose) capsules, or can be compressed into tablets, or can be directly incorporated into food. For oral administration, dietary compositions containing GSH, whey protein-encapsulated GSH (WP-GSH), or polymeric whey protein-encapsulated GSH (PWP-GSH) can be incorporated with excipients and used in the form of ingestible tablets, lozenges, tablets, capsules, elixirs, suspensions, syrups, sheets, etc. Tablets, lozenges, pills, capsules, etc., may also contain the following: binders, such as astragalus gum, gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate, microcrystalline cellulose, etc.; disintegrants, such as potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and may contain sweeteners (such as sucrose, lactose, or saccharin) or flavorings (such as peppermint, wintergreen oil, or cherry flavoring). When the dosage unit is in capsule form, it may contain a liquid carrier in addition to the materials of the types mentioned above. Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrups or elixirs may contain active compounds, sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavorings. Oil-in-water emulsions may be better for oral administration to infants because they are water-miscible, thus masking their oiliness. Such emulsions are well-known in pharmaceutical science.

[0062] GSH was determined using HPLC.

[0063] Optimal conditions for HPLC analysis:

[0064] Column: symmetry C18

[0065] Mobile phase: water: acetonitrile = 90:10

[0066] Flow rate: 0.5 ml / min

[0067] Wavelength: 218 nm (UV detector)

[0068] Injection volume: 0.5 μL

[0069] GSH powder was dissolved in ultrapure water to prepare a 10 mg / ml stock solution. A series of standard solutions (0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml) were obtained by diluting the stock solution with ultrapure water. GSH was identified using HPLC. Figure 1 As shown, the standard curve is plotted as a function of concentration and peak area.

[0070] The compositions and methods described in the above embodiments can be further understood by referring to the following examples. Additionally, the following non-limiting examples are provided to illustrate the invention. However, those skilled in the art will understand that it may be necessary to modify the procedures used in any given embodiment of the invention, for example, changing the order or steps of the method.

[0071] Example 1

[0072] Whey protein-GSH mixture (WP-GSH)

[0073] A whey protein concentrate (WPC) solution (10% protein, w / v) was prepared by dissolving whey protein concentrate powder in deionized water at room temperature and then stirring (700 rpm) for 2 h. The stock solution was stored overnight at 4 °C for complete hydration. The whey protein solution was warmed to ambient temperature and then mixed with reduced GSH at weight ratios of WPC (powder):GSH of 1:1, 1:1.5, and 1:2. The mixture was stirred for 20 min to achieve complete dissolution.

[0074] Polymeric whey protein-encapsulated GSH (PWP-GSH)

[0075] A whey protein concentrate (WPC) solution (10% protein, w / v) was prepared by dissolving whey protein concentrate powder in deionized water at room temperature and then stirring (700 rpm) for 2 h. The solution was stored overnight at 4 °C for complete hydration. The whey protein solution was then allowed to return to ambient temperature, and the pH was adjusted to 7 or 8, followed by heating at 80 °C for 15 min. Polymeric whey protein (PWPC) solution was obtained by rapidly cooling the heated whey protein solution to room temperature (25 °C ± 1 °C) in a water-ice mixture. The PWPC solution was then mixed with GSH powder at weight ratios of PWPC:GSH of 1:1, 1:1.5, and 1:2. The mixture was stirred for 20 min to achieve complete dissolution.

[0076] Stability test

[0077] The stability of all whey protein-based GSH solutions was observed at room temperature for 20 h. Table 1 below lists the stability of whey protein-based glutathione solutions (including those from the polymerization examples).

[0078] Table 1

[0079]

[0080]

[0081]

[0082] As shown in Table 1, the PWP-GSH samples stabilized for at least 4 hours.

[0083] Example 2

[0084] Pharmacokinetics Studies

[0085] ICR mice (male, SPF, 3 weeks old, weighing 18 to 22 g) were provided by Beijing HFK Bioscience Co., Ltd. (Beijing, China). Reduced glutathione (GSH) assay kit (A006-2-1) and total antioxidant capacity assay kit (ABTS method) (A015-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

[0086] All mice were kept in plastic laboratory animal cages in a ventilated room. The room was maintained at 20℃±2℃ and 60%±10% relative humidity, using a 12-hour light / dark cycle. Water and commercial laboratory-complete food for the mice were freely available. They were allowed to acclimatize to this environment for 7 days before treatment. All animal experiments were approved by the Animal Welfare and Research Ethics Committee of Jilin University (Approval No.: SY201905018).

[0087] Blood collection. Before blood collection, add 30 μL of heparin solution to a 1.5 mL centrifuge tube and vortex. Collect a blood sample (approximately 0.5 mL) by removing one eye of the mouse. Centrifuge the blood at 6000 rpm for 2 min at room temperature. Transfer the supernatant plasma to a new centrifuge tube. Analyze plasma GSH concentration using a GSH assay kit.

[0088] The GSH stock solution (1 mmol / L) was diluted to prepare a series of standard solutions with concentrations of 0 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 100 μmol / L. The standard solutions were plotted as absolute OD values ​​relative to GSH concentrations (…). Figure 2 ).

[0089] A. Blood samples were collected from six mice in each group at several time points (0, 15 min, 30 min, 1 h, 2 h, and 4 h) following oral administration of free GSH, whey protein-based GSH, whey protein, GSH based on poly-lactamase, and poly-lactamase via gavage. The dose of whey protein-based GSH was adjusted to have an equivalent of 100 mg / kg GSH. The GSH concentration in the blood samples was determined using an assay kit.

[0090] Table 2 shows the test groups and the gavage volumes.

[0091] Table 2

[0092]

[0093] like Figure 3 As shown, after oral administration, the PWP-GSH test material provided an excellent and significant increase in plasma GSH concentration.

[0094] B. In vivo antioxidant activity

[0095] Trolox stock solution (10 mM) was diluted to 0.1, 0.2, 0.4, 0.8, and 1.0 mM. A standard curve was plotted by determining the OD values ​​of different standard samples. Figure 4 The antioxidant activity of plasma is expressed as a multiple of the capacity of Trolox, where Trolox's TAOC (Total Antioxidant Capacity) is 1.

[0096] The total antioxidant capacity of all samples was also measured using an assay kit, and the results are as follows: Figure 5 As shown. The in vivo antioxidant activity of plasma in mice after oral administration of free GSH, WPC / PWPC-based GSH, and WPC / PWPC was measured using an assay kit (ABTS method).

[0097] like Figure 5 As shown, the PWP-GSH sample exhibited the most robust antioxidant activity over time.

[0098] C.GSH's organizational distribution

[0099] To evaluate the organ delivery efficiency of GSH, tissue samples from the brain, heart, kidney, liver, lung, and intestine were collected at 0, 1, 2, 4, and 6 h after oral administration to six mice in each group. The tissues were homogenized, proteins were precipitated, and centrifuged. GSH content was then analyzed using an assay kit. Figure 6A –6F).

[0100] like Figures 6A-6F As shown, the distribution of free GSH in various tissues was significantly greater than that in the control. Furthermore, the distribution of GSH in the PWP-GSH group was significantly greater than that in the control in various tissues.

[0101] D. Toxicity study of GSH based on polymerized whey protein (PWP-GSH)

[0102] Whey protein concentrate was supplied by Fonterra Co-operative Group (Auckland, New Zealand). Sodium pentobarbital, formalin, and anhydrous ethanol were supplied by Beijing Works (Beijing, China). 3-week-old SD rats were supplied by Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China).

[0103] Food preparation products

[0104] GSH based on polymeric whey protein concentrate (PWP-GSH) was prepared according to Example 1, wherein the concentration of polymeric whey protein was 10% and the ratio of whey protein to GSH was 1:1. The prepared polymeric whey protein GSH was then dried using a freeze dryer (Alpha 1-4LDplus, Germany). The powdered polymeric whey protein-based GSH was then incorporated into normal feed at percentages of 0.5%, 1%, and 4% (w / w) (corresponding to 0.25%, 1%, and 2% GSH percentages, respectively). These diets were prepared by Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). Dosage was set based on the daily intake of healthy individuals (100 mg / day). Based on an average weight of 60 kg, the human dose was 1.6 mg / kg. According to the conversion formula, this is equivalent to 10 mg / kg for rats. Setting the dose to 0.5%, 1%, and 4% corresponds to 25, 50, and 200 times the human daily intake, respectively.

[0105] Experimental Design

[0106] Eighty 3-week-old rats (half male and half female) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). All rats were kept in plastic laboratory animal cages in a ventilated room. The room was maintained at 20℃±2℃ and 60%±10% relative humidity, using a 12-hour light / dark cycle. Water was freely available. Before treatment, the rats were acclimatized to the environment for 7 days, during which their general condition did not change significantly. After acclimatization, the rats were randomly assigned to four groups (10 rats / sex / group) based on their mean body weight. At randomization, the individual body weight in each group was within ±20% of the overall mean. Compared with the control group, the low, medium, and high dose groups received 0.5%, 1%, and 4% whey protein-based GSH in their diet, respectively. All animal experiments were approved by the Animal Welfare and Research Ethics Committee of Jilin University (Approval No.: SY201905018).

[0107] Clinical observation

[0108] Throughout the study, changes in hair condition, skin, mucous membranes, secretions and excretions, autonomic nervous system activity, gait, and posture were observed in each rat.

[0109] Weight and food intake

[0110] Individual body weights were measured and recorded at 4-day intervals for a total period of 28 days. Final body weights were recorded prior to the planned necropsy (after fasting). Feed intake was also measured and expressed as average food consumption (g / rat / day) calculated for the corresponding time intervals.

[0111] Blood collection

[0112] At the end of the experiment, all animals were fasted for 12 hours before blood collection, but were allowed to drink water. Rats were injected with a 2% sodium pentobarbital solution at a level of 0.2 ml / 100 g. Two separate blood samples were then collected via the heart for hematology and serology. For hematological analysis, blood samples were collected through EDTA-2K coated tubes, and the following parameters were determined using an Exigo animal hematology analyzer: white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), platelet count (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution (RDW), mean platelet volume (MPV), lymphocytes (LYM), neutrophils (GRAN), monocytes (MONO), lymphocyte percentage (LYM%), granulocytes (GRA), and monocyte percentage (MON%).

[0113] For serum chemistry analysis, blood samples were centrifuged at 10,000 rpm for 3 min at room temperature. The supernatant serum was transferred to a new centrifuge tube, and the following parameters were determined using an SMT-120V automated biochemical analyzer: albumin (ALB), calcium (Ca), creatinine (Crea), total bilirubin (TB), total protein (TP), inorganic phosphorus (PHOS), urea (UREA), amylase (AMY), triglycerides (TG), glucose (GLU), BUN / CR ratio (U / C), creatine kinase (CK), globulin (GLOB), and aspartate aminotransferase (AST).

[0114] Organ weight, gross autopsy and histopathology

[0115] At the end of the procedure, all rats were anesthetized with sodium pentobarbital and bled out by transverse incision of the thoracic cavity. A complete gross pathological examination was then performed visually during necropsy. The brain, heart, lungs, liver, spleen, kidneys, bladder, ovaries, uterus, testes, epididymis, and seminal vesicles of all animals were removed and weighed. The relative weight of each organ (or paired organs) was calculated based on the final individual weight measured on the end of the day. Except for the testes, which were fixed with Bouin solution, tissue sections of these organs were fixed in 10% formaldehyde-buffered, embedded in paraffin, sectioned at 2–5 μm, mounted on glass microscope slides, stained with standard hematoxylin-eosin, and examined using an optical microscope. All histopathological procedures were performed at the College of Animal Science and Veterinary Medicine, Jilin University.

[0116] result

[0117] During the experiment, no adverse effects were observed in the experimental group compared to the control group.

[0118] No adverse effects on the clinical appearance of the treatment-related animals were observed during the experiment. Body weight gradually increased as the treatment period progressed. Figure 7 There were no statistically significant differences in body weight among the female groups. For the male groups, starting at day 16, 1% of the males were significantly different from those in the control group. Body weight changes were observed only in the male groups, and there was no dose-dependent effect.

[0119] Figure 8 and Figure 9The results of food consumption and food efficiency in rats over 28 days are shown. No PWPC-GSH-related toxicity was observed in the experimental groups, although there were some significant differences between the experimental and control groups at some time points. On day 8, the food efficiency of the 0.5% and 4% female groups showed significant differences compared to the control (p<0.05).

[0120] Table 3 shows the serum biochemistry of male rats in the 28-day toxicity study.

[0121] Table 3

[0122]

[0123]

[0124] Note: * indicates a significance level of 0.05 compared to the control group, ** indicates a significance level of 0.01 compared to the control group.

[0125] Serum biochemical results of male rats are shown in Table 3 above. Albumin levels in the 4% male group were 33.01 ± 1.34 g / L, significantly lower than the control group (p < 0.05). The low serum albumin level may be due to a synthetic defect. This change could have two causes. Hepatic absorption of albumin may be reduced due to hepatitis. Another cause may be renal excretion dysfunction due to low nephrogenicity, which could lead to the excretion of large amounts of albumin in the urine. Globulin levels in the 4% male group were 28.1 ± 2.77 g / L, significantly lower than the control group (p < 0.05). However, this value was within the normal range (15-28 g / L). Aspartate aminotransferase levels in the 4% male group were 91.17 ± 8.13 U / L, significantly lower than the control group (p < 0.05). However, this value was also within the normal range (39-111 U / L). The alanine aminotransferase level in 4% of males was 39 ± 7.73 U / L, significantly lower than that in the control group (p < 0.05). However, this value was within the normal range (20–61 U / L). Aspartate aminotransferase and alanine aminotransferase are indicators of liver function. An increase in these two parameters may indicate some pathological changes in the liver, while a decrease may not be clinically significant. Compared with the control, amylase levels in all experimental groups were significantly lower (p < 0.05), which is a benefit. Creatine kinase levels in the experimental groups were significantly lower than those in the control group (p < 0.05), which is a benefit. Glucose levels in 4% of males were significantly lower than those in the control group and were within the normal range (2.78–7.50 μmol / L). Calcium levels in the 1% and 4% male groups were significantly lower than those in the control group (p < 0.05). The decrease in calcium levels may be due to (1) parathyroid hormone deficiency; (2) vitamin D deficiency or metabolic abnormalities; (3) chronic kidney disease.

[0126] In summary, significant changes in serum biochemistry were mainly observed in the 4% male group, and these changes were primarily related to liver or kidney function.

[0127] Table 4 shows the serum biochemistry of female rats in the 28-day toxicity study (n=10).

[0128] Table 4

[0129]

[0130]

[0131] Note: * indicates a significance level of 0.05 compared to the control group, ** indicates a significance level of 0.01 compared to the control group.

[0132] Serum biochemical results of female rats are shown in Table 4. The total protein level in the 1% female group was 68.02 ± 4.31 g / L, significantly higher than that in the control group (p < 0.05). However, it was within the normal range (53–69 g / L). The elevated total protein level may be due to chronic liver disease. Amylase and glucose levels in the 1% and 4% female groups were significantly lower than those in the control group (p < 0.05). Inorganic phosphorus levels in the 1% and 4% female groups were significantly higher than those in the control group. However, they were within the normal range (1.87–3.6 μmol / L).

[0133] hematology

[0134] Table 5 shows the hematology of male rats in the 28-day toxicity study (number of animals = 10).

[0135] Table 5

[0136]

[0137]

[0138] Note: * indicates a significance level of 0.05 compared to the control group, ** indicates a significance level of 0.01 compared to the control group.

[0139] PWPC-GSH powder had no treatment-related adverse effects on hematological parameters in male rats. However, some statistically significant differences were observed between the control and treatment groups. PLT and MPV in the 4% group were significantly different from those in the control group (p<0.05). MPV and LYM in the 0.5% group were also significantly different from those in the control group (p<0.05).

[0140] Table 6 shows the hematology of female rats in the 28-day toxicity study (number of animals = 10).

[0141] Table 6

[0142]

[0143]

[0144]

[0145] Note: * indicates a significance level of 0.05 compared to the control group, ** indicates a significance level of 0.01 compared to the control group.

[0146] PWPC-GSH powder had no treatment-related adverse effects on hematological parameters in female rats. However, some statistically significant differences were observed between the control and treatment groups. The RBC count in the 1% group was 6.76 ± 0.56 × 10⁻⁶ compared to the control. 12 The HGB / L value was significantly higher (p<0.05). This change is likely due to dehydration and should not be considered related to the test substance. HGB in the 1% female group (14.46±0.88 g / dL) and the 4% female group (14.10±0.36 g / dL) were significantly higher than the control (p<0.05). However, these values ​​are within the normal range (13.2–16.4 g / dL) and should not be considered an adverse effect.

[0147] relative organ weight

[0148] The results for relative organ weights are shown in Tables 7 and 8. The final body weight of male rats fed with PWPC-GSH powder was significantly lower than that of the control group (p<0.05). Compared with the control, there were no significant differences in the relative weights of organs in all rats fed with PWPC-GSH powder, except for the liver and kidney in the 4% male group.

[0149] Table 7 shows the relative organ weights of male rats in the 28-day toxicity study (number of animals = 10).

[0150] Table 7

[0151]

[0152]

[0153] Note: * indicates a significance level of 0.05 compared to the control group, ** indicates a significance level of 0.01 compared to the control group.

[0154] In female rats, the final body weight of rats in the 4% group was significantly reduced (p<0.05). There was no significant difference in relative organ weight between rats fed with PWPC-GSH powder and the control group.

[0155] Table 8 shows the relative organ weights of female rats in the 28-day toxicity study (number of animals = 10).

[0156] Table 8

[0157]

[0158] Note: * indicates a significance level of 0.05 compared to the control group, ** indicates a significance level of 0.01 compared to the control group.

[0159] Pathology and histopathology

[0160] Figure 10A and 10B Stained sections of several tissues from male and female rats in the 4% feeding group compared to the control group are shown. Tissue samples were prepared and stained using standard methods and observed using standard microscopy.

[0161] in conclusion

[0162] The results of the PK study showed that serum intake of GSH encapsulated with polywhey protein (PWP-GSH) was three times higher than that of Kyowa's Setria GSH. Three to four hours after administration, rats fed with PWP-GSH showed significantly higher levels of GSH in the brain and liver tissues compared to those fed a commercial GSH diet. Compared to controls, the 4% male-fed group showed some changes in body weight, serum biochemistry, and relative organ weight parameters. Therefore, it can be concluded that the no-observed-adverse-effects (NOAEL) level is estimated to be at least 1% for male rats and 4% for female rats, corresponding to approximately 50 and 200 times the human daily intake values, respectively.

[0163] In summary, compared with the standard control, whey protein-encapsulated GSH (WP-GSH, PWP-GSH) has significantly improved bioavailability and is a safe delivery system.

[0164] Example 3

[0165] Preparation of glutathione (PWP-GSH) encapsulated with polymeric whey protein

[0166] Whey protein concentrate (5 kg) was dissolved in distilled water at a concentration of 10% (w / v) and stored overnight at 4°C. The whey protein concentrate solution was then heated at 80°C for 15 minutes. After cooling to room temperature, the PWPC solution was mixed with GSH powder (5 kg) at a weight ratio of PWPC:GSH = 1:1. The mixture was stirred for 20 minutes to ensure complete dissolution. After blending, the mixture was freeze-dried to provide the PWP-GSH powder product.

[0167] Reduced glutathione (GSH) assay kit (A006-2-1) and total antioxidant capacity assay kit (ABTS method) (A015-2-1) are available from Nanjing Jiancheng Biotechnology Institute (Nanjing, Jiangsu, China). Several methods can be used to determine GSH content. The following procedure provides the determination of undigested GSH content.

[0168] 2 g of PWPC-based GSH powder was dissolved in 20 mL of PBS buffer and then sonicated for 20 min for complete extraction. After sonication, 1 mL of the supernatant was collected and diluted 800-fold. Then, 1 mL of the diluted solution was mixed with 1 mL of protein removal agent and centrifuged at 3500 rpm for 10 min. The GSH content of the supernatant was then determined using a GSH assay kit.

[0169] Alternatively, GSH content can be determined after trypsin digestion.

[0170] A release solution with an enzyme activity of 1:250 was prepared by dissolving trypsin (10 g) in 1 L of NaCl solution (0.5%, w / v) and adjusting the pH to 8 using 0.1 M NaOH solution. PWPC-based GSH powder (0.3 g) was added to 30 mL of the release solution and incubated at 37 °C with shaking at 100 rpm for 6 h. The mixture was then centrifuged at 5,000 × g for 20 min, and the supernatant was diluted 100-fold. The diluted suspension was then mixed with a protein removal solution at a 1:1 (v / v) ratio and centrifuged. The GSH content of the supernatant was then determined using a GSH assay kit.

[0171] refer to Figure 11 The standard curves show that, without digestion, the OD values ​​of the two samples were 0.2883 and 0.2862, corresponding to 43.2% and 42.9% (w / w) GSH content in the PWPC-GSH product, respectively. After trypsin digestion, the OD values ​​of the two samples were 0.2654 and 0.2627, corresponding to 49.8% and 49.2% (w / w) GSH content in the PWPC-GSH product, respectively.

[0172] Therefore, a processing technology for manufacturing whey protein-encapsulated GSH (PWP-GSH) has been established, and the recovery rate of glutathione in the matrix is ​​99.2%, indicating that the loss of this heat-sensitive compound is only 0.8% throughout the process.

[0173] Example 4

[0174] Chemical characterization of PWP-GSH samples. It is fully understood that samples prepared according to the principles of this disclosure can be characterized by a variety of methods well known in the art, including but not limited to viscosity measurements and other rheological measurements, FT-IR, TEM / SEM microscopy, microstructure and morphological studies, stability studies (solid phase, solution phase, humidity, heat), particle size, zeta potential, etc. The chemical analyses described herein are expected to further reveal the unique qualities and properties of the compositions described herein. See Khan et al., 2019.

[0175] Example 4A

[0176] 1. Preparation of PWPC-GSH using whey protein concentrate (PWC)

[0177] Compared to other supports based on isobutyl cyanoacrylate, Eudragit RS 100 / cyclodextrin, and montmorillonite, the prepared PWPC-GSH system offers advantages such as simplicity, mildness, and absence of organic solvents. In a characterization test according to Zhang et al. (2021), PWPC exhibited a bimodal pattern with two peaks at 594 nm and 4580 nm, and a broad particle size distribution (span of 9.22), consistent with previous studies. Combining with GSH (287.83 ± 6.18 nm) slightly increased the particle size (D50) from 1085 ± 35.35 nm to 1115 ± 7.07 nm, while decreasing the span from 9.22 ± 0.22 to 6.86 ± 0.19. The zeta potential of PWPC-GSH was found to be 30.37 ± 0.75 mV. The high surface charge imparts high stability to the PWPC-GSH system, as strong electrostatic repulsion between molecules prevents polymerization, precipitation, and flocculation. Furthermore, due to the negative charge carried by the cell membrane, the positive surface charge of PWPC-GSH facilitates in vivo absorption. The PWPC-GSH system exhibits shear-thinning behavior in the range of 1–300 s⁻¹, indicating that the interaction between droplets weakens at higher shear rates.

[0178] The DSC thermogram of GSH showed an exothermic peak at 198 °C, and this melting peak disappeared in the PWPC-GSH system, indicating that GSH was molecularly dispersed in PWPC particles. FTIR spectroscopy analysis showed that PWPC exhibited an amide I (C=O vibration) spectral peak at 1654.39 cm⁻¹, and a redshift occurred after binding with GSH, indicating a structural change in PWPC and the formation of intermolecular hydrogen bonds. The PWPC-GSH system exhibited a worm-like aggregate morphology, with most aggregates approximately 200 nm in size, and some larger aggregates measuring approximately 400 nm, as determined by standard TEM imaging techniques. Figure 12 ).

[0179] 2. In vivo pharmacokinetics and antioxidant activity of PWPC-GSH

[0180] Whey protein has been widely studied as an effective means of nutrient delivery due to its resistance to pepsin digestion, non-toxicity, wide availability, and broad biocompatibility. Pharmacokinetic studies were conducted on the PWPC-GSH delivery system and free GSH, and plasma GSH concentration-time curves were determined for all groups. The highest plasma GSH concentration was observed in the PWPC-GSH group, followed by free GSH, PWPC, and the control group. The plasma GSH concentration in mice administered PWPC-GSH by gavage was higher than that in the free GSH group, possibly due to the protective effect of the highly viscous PWPC by embedding GSH internally and preventing damage to gastrointestinal enzymes and the acidic environment. These results are consistent with previous studies that whey protein encapsulation improves the bioavailability of quercetin and vitamin D.

[0181] Pharmacokinetic parameters were calculated using a mouse model. (The text then abruptly shifts to a seemingly unrelated topic: "and free GSH (maximum concentration (C...)...") 最大 Compared to (7.37 mg / L) and (AUC) of 19.23 hx mg / L), a higher C was observed. 最大 The values ​​of (19.41 mg / L) and AUC (48.63 hx mg / L) indicate that the rate and extent of GSH absorption in the bloodstream were greater in mice after administration of PWP-GSH. The C values ​​in the PWP-GSH group... 最大 The AUC was 2.5-fold and 2.6-fold higher, indicating that the PWPC-GSH delivery system effectively improves the in vivo bioavailability of GSH compared to its pure form alone. During absorption into the intestine, whey protein, acting as a carrier, also appears to protect GSH, likely due to resistance to pepsin digestion. In addition to the delivery of GSH itself, whey protein supplementation contributes to increased in vivo GSH levels through the abundance of cysteine ​​residues inherent in whey protein, which, as rate-limiting amino acids, have the ability to promote GSH biosynthesis. The time to reach maximum concentration (Tmax) in the PWPC-GSH group was lower (1 h) compared to the time to reach maximum concentration in free GSH (2 h), indicating a shorter time required to reach maximum concentration after administration. Plasma GSH concentrations in the GSH group reached peak levels after 1.5 to 2 h, consistent with data reported in earlier literature relative to oral administration of free GSH.

[0182] The total antioxidant capacity of samples was measured at different time points using an assay kit. Throughout the entire time period, the plasma antioxidant capacity of mice administered PWPC-GSH via gavage was significantly higher than that of mice administered free GSH via gavage (p<0.05). The first reason for the increased plasma antioxidant capacity after PWPC-GSH administration in mice is that the use of PWPC as a delivery carrier improved the plasma GSH concentration. The second reason may be due to the antioxidant properties of whey protein. As measured by T-AOC (mM), the plasma antioxidant capacity of mice administered PWPC via gavage also showed a slight improvement, the extent of which may be consistent with or inconsistent with the additive effect.

[0183] Example 4B

[0184] Based on Examples 3, 4, and 4A, various parameters of PWPC-GSH and PWPI-GSC were measured compared to whey protein concentrate (WPC) and whey protein isolate (WPI) standards.

[0185] Figure 13A and 13B The particle size distributions of glutathione nanoparticles encapsulated with whey protein from WPC and WPI starting materials are shown, respectively.

[0186] Figure 14 The polydispersity index (PDI) of whey protein-encapsulated glutathione nanoparticles for both WPC and WPI starting materials is shown.

[0187] Figure 15 The measured zeta potential (mV) of whey protein-encapsulated glutathione nanoparticles for both WPC and WPI starting materials is shown.

[0188] Figure 16A and 16B The apparent viscosity versus shear rate of whey protein-encapsulated glutathione nanoparticles based on WPC and WPI starting materials are shown, respectively.

[0189] Figure 17A and 17B The circular dichroism of whey protein-encapsulated glutathione nanoparticles based on WPC and WPI starting materials was shown.

[0190] Figure 18A and 18B The FT-IR spectra of glutathione nanoparticles encapsulated with whey protein from WPC and WPI starting materials are shown, respectively.

[0191] Example 5

[0192] Preparation of PWP-DIM. The procedure of Khan et al. (2019) was modified as follows.

[0193] The components are added to a continuously scraped, temperature- and pH-controlled tank or equivalent continuously stirred reactor (CSTR) system. Specifically, the tank is jacketed and connected to a steam source. The temperature is controlled by a thermocouple. Other components include a side pH probe, a bottom thermometer, and a mechanical stirring system.

[0194] Before adding DIM, disperse and polymerize the whey protein in the tank.

[0195] The temperature range is 70°C to 95°C, and the pH range is 6.5 to 9.0.

[0196] The viscosity measurements of the mixture of materials before and after polymerization ranged from 100-300 mPas to 3000 mPas.

[0197] Results and discussion.

[0198] DIM and whey protein concentrate have opposite hydrophobic / hydration properties. This method is designed to encapsulate a dispersed phase (DIM) in a continuous phase with a polymerized whey protein polymer. During stirring and heating, when the system viscosity reaches the desired range, the dispersed phase is suspended and encapsulated by the polymer. The two phases of the material form continuous and consistent microgels or aggregates.

[0199] Finally, the reaction product was spray-dried using standard methods and collected as an encapsulated powder.

[0200] Example 6

[0201] Chemical characterization of PWP-DIM samples. It is fully understood that samples prepared according to the principles of this disclosure can be characterized by a variety of methods well known in the art, including but not limited to viscosity measurements and other rheological measurements, FT-IR, TEM / SEM microscopy, microstructure and morphological studies, stability studies (solid phase, solution phase, humidity, heat), particle size, zeta potential, etc. The chemical analyses described herein are expected to further reveal the unique qualities and properties of the compositions described herein. See Khan et al., 2019.

[0202] Example 7

[0203] Similarly, coenzyme Q10 encapsulated with polymerized whey protein (PWP-CoQ10) was prepared using the method described above with whey protein isolate (WPI) and characterized as an orange flake powder. Determination (HPLC): 20.69% by weight.

[0204] like Figure 19 , 20As shown in Figures 21 and 22, PWP-CoQ10 with various ratios ranging from 20:1 (PWPI:CoQ10) to 100:1 (PWPI:CoQ10) was characterized by particle size (nm), polydispersity index (PDI), and zeta potential (mV).

[0205] In the context of describing the invention currently claimed (especially in the context of the claims), the use of the terms “a,” “the,” and similar pronouns should be interpreted as encompassing both the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise stated herein, the description of ranges of values ​​is intended only as a shorthand method of individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were stated separately herein. The use of the term “about” is intended to describe values ​​that are higher or lower than the stated value within a range of about ±10%; in other embodiments, these values ​​may be higher or lower than the stated value within a range of about ±5%; in other embodiments, these values ​​may be higher or lower than the stated value within a range of about ±2%; in other embodiments, these values ​​may be higher or lower than the stated value within a range of about ±1%. The foregoing ranges are intended to be determined according to the context and do not imply further limitations. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better describe the invention and does not constitute a limitation on the scope of the invention, unless otherwise stated. No language in the specification should be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0206] Although certain embodiments of the invention have been described in the foregoing specification, and many details have been set forth for illustrative purposes, it will be apparent to those skilled in the art that the invention is susceptible to other embodiments and that some details described herein may be modified considerably without departing from the basic principles of the invention.

[0207] All references cited herein are incorporated herein by reference in their entirety. The invention may be presented in other specific forms without departing from the spirit or essential attributes of the invention, and therefore reference should be made to the appended claims that define the scope of the invention rather than to the foregoing description.

[0208] This invention also provides the following items:

[0209] 1. A composition comprising polymeric whey protein, said polymeric whey protein encapsulating an antioxidant compound selected from the group consisting of 3,3'-diindolemethane (DIM), glutathione (GSH) and coenzyme Q10 (CoQ10).

[0210] 2. The composition as described in Project 1, wherein the polymeric whey protein is a polymeric whey protein concentrate.

[0211] 3. The composition as described in Project 2, wherein the ratio of the polymerized whey concentrate to the antioxidant compound is in the range of about 2:1 (wt / wt) to about 10:1 (wt / wt).

[0212] 4. A method for preparing polymeric whey protein microencapsulated 3,3'-diindolemethane (DIM), the method comprising the following steps:

[0213] (a) Dissolve whey protein concentrate powder in water at 10% w / v to provide an aqueous solution;

[0214] (b) Heating the whey protein concentrate solution to at least about 70°C-80°C to provide a polymerized whey protein solution;

[0215] (c) Add DIM to the polymerized whey protein solution;

[0216] (d) Adjust the pH to a range of approximately 6.5 to approximately 9.0;

[0217] (e) Cool the polymerized whey protein solution;

[0218] (f) Stirring during cooling from about 80°C to about 45°C to provide a clear, homogeneous solution; and

[0219] (g) Separation of polymerized whey protein microencapsulated DIM.

[0220] 5. The method as described in Project 4, wherein whey protein concentrate and DIM are used in equivalent amounts by weight.

[0221] 6. The method as described in Project 4, wherein the weight ratio of whey protein concentrate to DIM is approximately 1:1 to 20:1.

[0222] 7. The method as described in Project 4, wherein the separation step is spray drying.

[0223] 8. The method as described in Project 4, wherein the separation step is freeze drying.

[0224] 9. A method for preparing polymerized whey protein microencapsulated glutathione, the method comprising the following steps:

[0225] (a) Dissolve whey protein concentrate powder in water at 10% w / v to provide an aqueous solution;

[0226] (b) Heating the whey protein concentrate solution to approximately 70°C-80°C for approximately 15 minutes to provide a polymerized whey protein solution;

[0227] (c) Add glutathione to the polymerized whey protein solution;

[0228] (d) Stirring to provide a clear, homogeneous solution; and

[0229] (e) Isolation of polymerized whey protein microencapsulated glutathione.

[0230] 10. The method as described in Project 9, wherein whey protein concentrate and glutathione are used in equivalent amounts by weight.

[0231] 11. The method as described in Project 9, wherein the weight ratio of whey protein concentrate to glutathione is about 1:1 to 1:2.

[0232] 12. The method as described in Project 9, wherein the separation step is spray drying.

[0233] 13. The method as described in Project 9, wherein the separation step is freeze drying.

Claims

1. A method for preparing microencapsulated 3,3'-diindolemethane (DIM) powder of polymerized whey protein concentrate, the method comprising the following steps: (a) Dissolve whey protein concentrate (WPC) powder in water at 10% w / v to provide an aqueous WPC solution; (b) Heating the WPC aqueous solution to 70°C-80°C to provide a WPC polymerization solution; (c) Add DIM powder to the polymerized WPC solution to provide a mixture; (d) Adjust the pH of the mixture to a range of 6.5 to 9.0; (e) The mixture is stirred during cooling from 80°C to 45°C to provide a clear, homogeneous solution for the microencapsulation of DIM via polymerized whey protein; and (f) Microencapsulated DIM by WPC polymerization is separated by drying and treated as a solid powder; The weight ratio of WPC powder to DIM powder is 1:

1.

2. The method of claim 1, wherein the viscosity of the WPC aqueous solution after step (a) is 100 mPas to 300 mPas, and wherein the viscosity of the mixture after step (e) is 3000 mPas.

3. The method of claim 1, wherein the drying is spray drying.

4. The method of claim 1, wherein the drying is freeze drying.

5. A composition comprising 3,3'-diindolemethane microencapsulated from a polymeric whey protein concentrate prepared by any one of claims 1-4.

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