Systems and methods for encapsulating small molecules

Abstract

Systems and methods for encapsulation of small molecules (e.g., ketone molecules) with plant-derived proteins for nutritional supplements and the like are disclosed herein. The method can further include cross-linking a phytochemical to a plant protein. The phytochemical can be cross-linked to the plant protein via an imine bond. The cross-linking can be performed without the addition of an aldehyde. The aldehyde can be glutaraldehyde. The cross-linking can occur at one or more functional amino acid groups of the plant protein. The functional amino acid group can include a primary amino group. The functional amino acid group can include lysine.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 340,820, filed May 11, 2022, and U.S. Provisional Application No. 63 / 453,958, filed Mar. 22, 2023, which are hereby incorporated by reference in their entirety.

Background Art

[0002] Background of the Invention The health and environmental benefits of plant-derived protein foods are widely recognized. To meet the growing demand for food products for vegetarians and vegans, scientists have, for example, made efforts to derive plant-derived protein products to replace animal-derived protein products in the incorporation of dietary supplements into foods to enhance bioavailability. There is an unmet need for a cost-effective way to deliver dietary supplements such as ketones, nicotinamide riboside (NR), caffeine, or theacrine without using animal-derived protein products.

Summary of the Invention

Means for Solving the Problems

[0003] Gist of the Invention A method for encapsulating a plurality of small molecules, comprising obtaining a plant-derived protein and one or more phytochemicals, and mixing the plant-derived protein and the one or more phytochemicals with the plurality of small molecules in water to produce a mixture comprising a plurality of particles containing a plurality of encapsulated small molecules, is disclosed herein. The plurality of small molecules can include a plurality of ketone molecules. The plurality of small molecules can include a plurality of nicotinamide riboside molecules or nicotinamide riboside analogs. The plurality of small molecules can include a plurality of caffeine molecules. The plurality of small molecules can include a plurality of theacrine molecules. The plant-derived protein can be derived from a first source, and the one or more phytochemicals can be derived from a second source different from the first source. The first source can be derived from a first plant species, and the second source can be derived from a second plant species. The plant-derived protein can be pea protein. The plant-derived protein can be water-soluble. The plant-derived protein can be banana protein, okra protein, or legume protein. The legume protein can be soy protein. The phytochemicals can be derived from mango, tea leaves, okra, berries, or grapes. The phytochemicals can be derived from mango peel powder, dried tea leaves, okra extract powder, berry extract, or grape extract. The one or more phytochemicals can include mangiferin, catechin, or quercetin. The one or more phytochemicals can include quercetin, and the weight ratio of the one or more phytochemicals to the plant-derived protein can be between 1:10000 and 1:40000. The one or more phytochemicals can include quercetin, and the weight ratio of the one or more phytochemicals to the plant-derived protein can be between 1:22000 and 1:23000. The one or more phytochemicals can include mangiferin, and the weight ratio of the one or more phytochemicals to the plant-derived protein can be between 1:20 and 1:60. The one or more phytochemicals can include mangiferin, and the weight ratio of the one or more phytochemicals to the plant-derived protein can be between 1:30 and 1:50.One or more phytochemicals can include catechins, and the catechin is epigallocatechin gallate. The weight ratio of epigallocatechin gallate to plant-derived protein can be between 1:200 and 1:400. One or more phytochemicals can include polyphenols. The polyphenols can include one or more of flavonoids and resveratrol. The weight ratio of one or more phytochemicals to plant-derived protein can be between 1:300 and 1:500. The mixture can include a dry weight ratio of a plurality of ketone molecules to plant-derived protein between 1:1 and 1:5. The mixture can include a weight ratio of plant-derived protein to water between 1:50 and 1:100 or between 1:10 and 1:100.

[0004] This method can further include mixing the mixture with ethanol. Mixing the mixture with ethanol to produce particles containing ketone molecules or NR molecules can be carried out by adding ethanol to the soluble fraction at a volume ratio of ethanol to the soluble fraction of 1:10 to 1:1. Mixing the mixture with ethanol to produce particles containing ketone molecules or NR molecules can be carried out by adding ethanol to the soluble fraction at a volume ratio of ethanol to the soluble fraction of 1:10 to 1:5. Mixing the mixture with ethanol to produce particles containing ketones or NR molecules can be carried out by adding ethanol to the soluble fraction at a volume ratio of ethanol to the soluble fraction of 1:4 to 1:2. This method can include removing ethanol. Removing ethanol can include evaporation of ethanol at 25°C to 40°C.

[0005] The particles can have an average diameter greater than 80 nm. The particles can have an average diameter less than 700 nm. The particles can have an average diameter between 100 nm and 500 nm. The particles can have an average diameter between 300 nm and 500 nm. Removing ethanol can include evaporation of ethanol at 25°C to 40°C.

[0006] This method can further include cross-linking a phytochemical to a plant protein. The phytochemical can be cross-linked to the plant protein via an imine bond. The cross-linking can be performed without the addition of an aldehyde. The aldehyde can be glutaraldehyde. The cross-linking can occur at one or more functional amino acid groups of the plant protein. The functional amino acid group can include a primary amino group. The functional amino acid group can include lysine.

[0007] The mixture can include a soluble fraction and an insoluble fraction. This method can include separating the soluble fraction from the insoluble fraction. The soluble fraction can be mixed with ethanol. Separating the soluble fraction from the insoluble fraction can include filtering the insoluble fraction from the soluble fraction. The plurality of small molecules can include 1,3-dihydroxypropan-2-yl (R)-3-hydroxybutanoate. At least 50% of the small molecules can be 1,3-dihydroxypropan-2-yl (R)-3-hydroxybutanoate. At least 50% of the small molecules can include a glycerol backbone conjugated to 3-hydroxybutanoate.

[0008] In some embodiments, the plurality of small molecules can include nicotinamide riboside. In some embodiments, at least 50% of the small molecules are nicotinamide riboside. In some embodiments, the plurality of small molecules can include caffeine. In some embodiments, at least 50% of the small molecules are caffeine. In some embodiments, the plurality of small molecules can include theacrine. In some embodiments, at least 50% of the small molecules are theacrine.

[0009] Mixing can be performed at 20°C to 30°C. Mixing can be performed at a pH of 3.5 to 11.0. Mixing can be performed at a pH of 3.5 to 7.0, 5.0 to 9.0, or 7.0 to 11.0.

[0010] Plant-derived proteins can be water-soluble. Plant-derived proteins can be at least 95% water-soluble. Plant-derived proteins can be at least 98% water-soluble. Plant-derived proteins can be at least 99% water-soluble.

[0011] This specification discloses a nanocapsulated composition formed by the above method. This specification discloses a composition containing small molecules encapsulated within a plant protein. The plant protein can be pea protein. The plant protein can be okra protein, banana protein, or legume protein. The plant protein can be crosslinked with one or more phytochemicals. One or more phytochemicals can be derived from mango, tea leaves, okra, berries, or grapes. One or more phytochemicals can be derived from mango peel powder, dried leaves, okra extract powder, berry extract, or grape extract. One or more phytochemicals can include mangiferin, catechin, or quercetin. Catechin can be epigallocatechin gallate. One or more phytochemicals can include polyphenols. Polyphenols can include one or more of flavonoids and resveratrol. One or more phytochemicals can be crosslinked to the plant protein via an imine bond. The crosslinking can occur at the functional amino acid groups of the plant protein. The functional amino acid groups can include primary amines. The small molecule can include a ketone molecule. The ketone molecule can include 1,3-dihydroxypropan-2-yl (R)-3-hydroxybutanoate. The small molecule can include a nicotinamide riboside molecule or an analog thereof. The small molecule can include a caffeine molecule. The small molecule can include a theacrine molecule. The particles can have an average diameter greater than 80 nm. The particles can have an average diameter less than 700 nm. The particles can have an average diameter between 100 nm and 500 nm. The particles can have a diameter between 300 nm and 500 nm. The composition can have a phenol content greater than 10 mg GAE / g. The composition can have a phenol content less than or equal to 350 mg GAE / g. The composition can have a phenol content between 10 and 500 mg GAE / g. The composition can have a phenol content between 100 and 400 mg GAE / g.The composition can have a phenolic content between 200 and 300 mg GAE / g. In some embodiments, the composition is free of glutaraldehyde.

[0012] Incorporation by reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Brief Description of the Drawings

[0013] The various features of the invention are described in detail in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description, which illustrates exemplary embodiments in which the principles of the invention are utilized, and to the appended drawings.

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[0087] Plant-based proteins are biocompatible, economically feasible, and generally safe for use as nutraceuticals or smart foods / drugs. As used herein, systems and methods are disclosed for encapsulating ketone molecules (KM), nicotinamide riboside (NR) molecules, and analogs thereof to enhance stability, enhance biocompatibility, and convert these types of nutraceuticals / nutraceuticals in a biocompatible, targeted, and cell-specific manner in vivo. As used herein, methods and systems are disclosed for enhancing the ability of KM molecules to penetrate biological domain barriers, including the blood-brain barrier, for rapid and sustained release of KM molecules to achieve immediate and sustainable energy in the human body. Methods and systems for the delivery of NR molecules and analogs thereof are also disclosed herein. Analogue systems suitable for the encapsulation of other small molecules are also disclosed herein.

[0088] As used herein, the term "small molecule" refers to a molecule having a molecular weight of less than or equal to 900 Daltons.

[0089] As used herein, the term "ketone molecule" refers to a molecule having the structure of Compound I below, wherein R 1 , R 2 and R 3 are H or -C4 H 6 O 2 may be, and at least one R 1 , R 2 and R 3 is -C 4 H 6 O 2 .

[0090] [Chemical formula]

[0091] Compound I can include any one of the following Compounds II - V.

[0092] [Chemical formula]

[0093] [Chemical formula]

[0094] [Chemical formula]

[0095] [Chemical formula]

[0096] In some embodiments, -C 4 H 6 O 2 is [Chemical formula] .

[0097] The term "nicotinamide riboside" as described herein refers to a molecule having the structure of the following Compound VI.

[0098] [Chemical formula]

[0099] Analogues of the nicotinamide riboside molecule can include any of the following compounds VII - IX.

[0100]

Chem.

[0101]

Chem.

[0102]

Chem.

[0103] The term "caffeine" as described in this specification refers to a molecule having the structure of the following compound X.

[0104]

Chem.

[0105] The term "theacrine" as described in this specification refers to a molecule having the structure of the following compound XI.

[0106]

Chem.

[0107] The term "plurality of ketone molecules" as described herein can refer to any plurality of ketone molecules. In some examples, the plurality of ketone molecules is at least 70% compound II. The plurality of ketone molecules can be 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% or more of compound II. The plurality of ketone molecules can be 50%, 40%, 30%, 20%, 10%, 5%, or less than 1% or equal to compound III, compound IV, or compound V.

[0108] The molar ratio of the mixture of compounds III-V to compound II in solution can be between 80:20 and 99:1. The molar ratio of the mixture of compounds III-V to compound II in solution can be about 80:20, 85:15, 90:10, 95:5 or 99:1 or more. The molar ratio of the mixture of compounds III-V to compound II in solution can be 99:1, 95:5, 90:10, 85:15 or less than 80:20 or equal.

[0109] The term "plurality of nicotinamide riboside molecules" as described herein can refer to any plurality of nicotinamide riboside molecules. In some cases, the plurality of NR molecules can be nicotinamide riboside hydrogen maleate. In some examples, the plurality of NR molecules can be nicotinamide riboside chloride. In some examples, the plurality of NR molecules is at least 70% compound VI. The plurality of NR molecules can be 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% or more of compound VI. The plurality of NR molecules can be 50%, 40%, 30%, 20%, 10%, 5%, or less than 1% or equal to compound VII, compound VIII, or compound IX.

[0110] As used herein, the term "encapsulating" refers to incorporating small molecules, such as ketones, NR, caffeine, or theacrine molecules, into cross-linked particles, even if the small molecules are not completely surrounded by plant-derived proteins or other non-ketone molecules.

[0111] One embodiment of the invention encapsulates ketone, NR, caffeine, or theacrine molecules within plant-based proteins derived from peas, okra, or bananas. Ketone, NR, caffeine, or theacrine molecules can be encapsulated within plant-based proteins derived from peas, okra, or bananas using plant-based cross-linking agents that include various polyphenols, flavonoids, xanthonoids, and functionalized glucose moieties. The methods and systems disclosed herein can utilize components of plant-based proteins and phytochemicals derived from peas, okra, bananas, mangoes, tea leaves, juicy fruits, and grapes to produce particles that contain plant-based proteins loaded with ketone, NR, caffeine, or theacrine molecules. The methods and systems disclosed herein can be used to produce particles that provide rapid and sustainable energy along with a rich group of important nutrients that include potassium, dietary fiber, polyunsaturated fats, essential amino acids, vitamin B6, vitamin B12, magnesium, potassium, fiber, and protein.

[0112] Fibers derived from peas, bananas, and okra can contribute to gastrointestinal function and health. The amylose content in such plant-based protein extracts can help lower the glycemic index. Additionally, hydrolysis products of pea protein-encapsulated ketone, NR, caffeine, or theacrine molecules can generate bioactive peptides, such as peptides having angiotensin I-converting enzyme inhibitory activity and antioxidant activity. The plant-derived protein particles described herein can provide polyphenols having antioxidant activity, anti-cancer activity, or hypocholesterolemic activity. Plant-derived protein-encapsulated ketone particles can provide a prebiotic effect from galactooligosaccharides and the like in the large intestine.

[0113] Ketone molecule (KM)

[0114] Systems and methods for encapsulating ketone molecules within plant - derived proteins cross - linked with phytochemicals are disclosed herein. In some embodiments, the ketone molecules bind to the plant - derived proteins and phytochemicals via hydrogen bonds, as can be seen in FIG. 39.

[0115] The structure of the ketone molecule (KM) and the respective Fischer esterification schemes involving dehydration of the parent carboxylic acid [(R) - 3 - hydroxybutyric acid] and the corresponding alcohol (glycerol) are shown below.

[0116] Fischer esterification scheme

[0117]

Chemical formula

[0118] The parent alcohol glycerol can contain both primary and secondary alcohol groups. Various primary and secondary monoglycerides, diglycerides, and triglyceride reaction products are possible by the Fischer esterification scheme (below). The ester functional group has the possibility of conformational isomerism (cis / trans). The carboxylic acid (3 - hydroxybutyric acid) is a chiral compound with the possibility of two enantiomers (D and L).

[0119] Fischer esterification product:

[0120]

Chemical formula

[0121] Nicotinamide riboside molecule

[0122] Nicotinamide riboside (NR) is a precursor of nicotinamide adenine dinucleotide (NAD+). NAD+ is an essential coenzyme involved in various metabolic pathways, and an increase in the body's NAD+ content can provide health benefits.

[0123] Systems and methods for encapsulating NR molecules within plant-derived proteins crosslinked with phytochemicals are disclosed herein.

[0124] The structures of NR and NR analogs are shown below:

Chem.

[0125] Caffeine

[0126] Caffeine (CA) is a central nervous system (CNS) stimulant. It belongs to the methylxanthine class of purines. Performance of wakefulness and attention is improved. Coffee beans are the main source of CA and have a bitter taste. CA is classified as GRAS by the US FDA. A maximum of 400 mg of caffeine per day is safe. The toxic dose is over 10 grams per day for adults.

[0127] Systems and methods for encapsulating CA molecules within plant-derived proteins crosslinked with phytochemicals are disclosed herein.

[0128] The structure of CA is shown below.

Chem.

[0129] Theacrine

[0130] Theacrine (TH) is a purine alkaloid that is structurally similar to caffeine. It improves mood, energy, and concentration. Cupuas (trees of the tropical rainforest) and Chinese tea (kuchas) are the main sources of TH. It affects adenosine signaling, similar to caffeine. It is clinically safe and has non-habit-forming effects in humans. The daily use of TH up to 300 mg / day is safe.

[0131] Disclosed herein are systems and methods for encapsulating TH molecules within plant-derived proteins crosslinked with phytochemicals.

[0132] The structure of TH is shown below.

Chemical formula

[0133] Phytochemicals for crosslinking plant-derived proteins:

[0134] Crosslinking agents can be beneficial for the synthesis of ketone molecules (KM), NR, caffeine, or theacrine-encapsulated protein nanoparticles. The crosslinking agent crosslinks protein amino groups to form denser particles.

[0135] A common chemical crosslinking agent used in protein nanoparticle synthesis is glutaraldehyde. This is an effective crosslinking agent that imparts nanoparticle stability and sustained drug release. Although effective, it can cause measurable systemic toxicity to humans, especially when used for long periods. Due to the lack of alternative non-toxic crosslinking agents, the food, beverage, and pharmaceutical industries continue to use glutaraldehyde at lower concentrations, despite the continuing risk of long-term systemic toxicity with irreversible harmful toxic effects on human health.

[0136] As an effective and natural cross-linking agent for protein nanoparticle formulations, biocompatible and non-toxic plant-based substances are required (Figure 38). Under oxidative conditions, polyphenols including mangiferin, epigallocatechin gallate (EGCG), quercetin, and resveratrol interact with the amino groups of pea proteins to produce covalently bonded cross-links, thereby resulting in the bioconjugation of various polyphenols with the protein network. In the present invention, as outlined in Figure 36, the hydroxyl and oxo groups on the polyphenols can cross-link proteins. Plant-based substances including mangiferin (MGF), catechin, epigallocatechin gallate (EGCG), quercetin (QUE), and resveratrol (RESV) can be utilized for the effective cross-linking and encapsulation of ketone molecules (KM) (Figure 23), NR molecules (Figure 24A), CA molecules (Figure 20B and Figure 24B), and TH molecules (Figure 20C and Figure 24C).

[0137] Disclosed herein are plant-based substances for effective cross-linking and encapsulation. Naturally available plant-based substances including mangiferin, EGCG, quercetin, and resveratrol were utilized for the effective protein cross-linking and encapsulation of KM (Figure 37), NR, CA, and TH. The cross-linking agent can bind to the protein amino groups to form particles. A common chemical cross-linking agent used in protein nanoparticle synthesis is glutaraldehyde. Methods and systems for cross-linking plant-derived proteins without using glutaraldehyde are disclosed herein. Methods and products free of glutaraldehyde are disclosed herein.

[0138] Disclosed herein are biocompatible plant-based cross-linking agents including naturally available cross-linking agents derived from tea (epigallocatechin gallate; EGCG), mango (mangiferin; MGF and various families of polyphenols and xanthonoids), berry (quercetin; QUE), and grape (resveratrol; RESV).

[0139] As can be seen in Figure 58, the main phytochemical of mango peel extract is mangiferin. Mangiferin, a xanthonoid, is a glucoside of nor-ethyliorinol and a natural phenolic compound. Mangiferin can be isolated from the leaves and bark of Mangifera indica (mango). It can be extracted from mango peel (MP) and kernels.

[0140] Catechin, a natural phenol, belongs to the flavonoid family. It is found in tea and fruits and is an antioxidant.

[0141] As can be seen in Figure 59, the main phytochemical of tea leaf extract is epigallocatechin gallate (EGCG). EGCG is a catechin formed by the esterification of gallic acid and epigallocatechin. Polyphenols such as EGCG and catechin are abundantly found in tea leaves.

[0142] As can be seen in Figure 60, the main phytochemical of blackberry extract is quercetin. Quercetin, a polyphenol belonging to the flavonoid group, is widely found in fruits and vegetables. Quercetin is the most abundant polyphenol in blackberry extract.

[0143] As can be seen in Figure 61, the main phytochemical of grape extract is trans-resveratrol. Resveratrol, a stilbenoid plant phenol, is found in grape extract. Resveratrol (RESV) is a stilbenoid, a form of natural phenol, and is a phytoalexin mainly found in the skin of grapes.

[0144] The highly acidic phenolic functional groups found in these plant-derived phytochemicals are effective in cross-linking with amino groups in proteins. Figure 38 shows the construction of a plant protein-polyphenol platform through the interaction between polyphenol compounds and protein amino side chains.

[0145]

Chemical Structure

[0146] The interaction between phytochemicals and water-soluble pea protein was studied using nuclear magnetic resonance (NMR) spectroscopy. The reactions of mangiferin, epigallocatechin gallate (EGCG), quercetin, and resveratrol were carried out individually with pea protein, and the changes in phytochemicals and pea protein were monitored. NMR analysis showed distinct changes in phytochemicals indicating covalent binding interactions with the protein (Figs. 41 - 43, Figs. 45 - 46, Figs. 48 - 49, and Figs. 51 - 52). Then, KM was added to the reaction mixture and further NMR analysis was performed. In the presence of KM, there were no changes in the phytochemicals or the protein, indicating the stability of KM in their presence as well (Figs. 41 - 53). Plant extracts (mango peel, tea, blackberry, or grape extract) are used for the synthesis of KM nanoparticles, and it should be noted that each plant extract is a phytochemical cocktail containing various other active ingredients that can also play a role in the cross-linking and encapsulation processes while adding nutraceutical value to the formulation.

[0147] Under oxidative conditions, polyphenols react with protein amino groups (forming cross-links and a network). The reaction of ortho-quinones with proteins to form C-N or C-S bonds is possible in the cross-linking process (Fig. 38). Plant proteins such as pea and soybean proteins have many lysine amino acid residues. These contain primary amino groups for cross-linking with phytochemicals. Phytochemicals conjugate to the primary (ε-) amino groups of lysine residues to generate a protein-polyphenol cross-linking platform. The sulfur-containing amino acid cysteine is present in small amounts in pea and soybean proteins. Phytochemicals also cross-link proteins by binding to thiol groups. Pea protein-mangiferin and pea protein-EGCG cross-link products are shown below.

[0148] The structure of mangiferin crosslinked with pea protein can be seen on the left and bottom. The structure of EGCG crosslinked with pea protein can be seen in the lower right.

Chem.

[0149] Plant-derived protein: The proteins described in this specification can be derived from plants such as beans, okra, and peas. The legume protein can be soy protein. The plant-derived protein can be water-soluble. The water-soluble protein can be obtained by hydrolyzing plant protein. Figure 57 shows a comparison of the amino acid compositions of pea protein, okra extract, and soy protein isolate.

Table 1-1

Table 1-2

[0150] Water-soluble pea protein can be derived from pea protein. Pea protein can be purified using enzymatic digestion, filtration, or spray drying. The protein content of pea protein can be over 85%. Pea protein can exist as globulin (65 - 80%) and can be composed of a small contribution from legumin, vicilin, and lectin proteins. Pea protein can contain less than 7% moisture, less than 7% ash, and less than 7% crude fiber. Pea protein can have a pH between 5.0 and 6.0 when dissolved in water. The amino acid composition of pea protein can be found in Figure 54.

[0151] Okra extract (Abelmoschus esculentus L.) can contain water (90%), protein (2%), carbohydrates (7%) and trace amounts of fat. Okra extract is a source of dietary fiber, vitamin C and vitamin K. Okra mucilage or gel from pods is a natural polysaccharide and consists of D-galactose, L-rhamnose and L-galacturonic acid. Okra extract can be extracted in water and can form a gelatinous viscous solution. The amino acid composition of okra extract can be found in Figure 55.

[0152] Soy protein isolate (SPI) is derived from soybeans. Soy protein isolate can be composed of β-conglycinin, glycinin, and lipophilic proteins. Soy protein can have a protein content of 92% (dry basis). Furthermore, it can contain 6% moisture, 4.1% ash, 0.8% fat (PE extract), 0.25% crude fiber (coarse), 0.15% calcium, 0.8% phosphorus, 1.3% sodium, 0.05% potassium, and can exhibit a pH (water slurry) of 7.1. The amino acid composition of soy protein isolate can be found in Figure 56.

[0153] Kit

[0154] A kit can include one or more containers that house one or more components provided in this disclosure and instructions for use. Specifically, such a kit can include one or more compositions described herein, along with instructions that describe the intended use and the proper use and / or placement of these compositions. The kit can contain appropriate concentrations or amounts of components for performing various experiments.

[0155] Method for manufacturing nanoparticles

[0156] Disclosed herein are systems and methods for producing nanoparticles comprising plant-derived proteins crosslinked with one or more phytochemicals described herein. The nanoparticles can encapsulate one or more small molecules such as ketone molecules, nicotinamide riboside molecules, caffeine molecules, and / or theacrine molecules. In some embodiments, the plant-derived protein, the phytochemical-containing powder, and the ketone molecule are combined in water. In some embodiments, the plant-derived protein, the phytochemical-containing powder, and the NR molecule are combined in water. In some embodiments, the plant-derived protein, the phytochemical-containing powder, and the caffeine molecule are combined in water. In some embodiments, the plant-derived protein, the phytochemical-containing powder, and the theacrine molecule are combined in water.

[0157] In some embodiments, ethanol is added to a mixture of plant-derived protein, phytochemical-containing powder, and ketone molecules. In some embodiments, ethanol is added to a mixture of plant-derived protein, phytochemical-containing powder, and NR molecules. In some embodiments, ethanol is added to the mixture at a volume ratio of the mixture to ethanol of 1:10 to 1:1. In some embodiments, the volume ratio of the mixture to ethanol is greater than or equal to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the volume ratio of the mixture to ethanol is less than or equal to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the volume ratio of the mixture to ethanol is between 1:4 and 1:7, 1:5 and 1:9, or 1:6 and 1:10. In some embodiments, the volume ratio of the mixture to ethanol is less than or equal to 1:7. In some embodiments, ethanol is removed from a mixture of plant-derived protein, phytochemical-containing powder, and ketone molecules. In some embodiments, ethanol is removed from a mixture of plant-derived protein, phytochemical-containing powder, and NR molecules. In some embodiments, ethanol is removed from the mixture by incubating the mixture at a temperature greater than or equal to 37°C. In some embodiments, ethanol is removed by rotary vapor evaporation.

[0158] In some embodiments, the phytochemical-containing powder includes tea leaves, blackberries, grapes, mangoes, or okra. In some embodiments, the phytochemical-containing powder includes rooibos tea, blackberry powder, grapes, mango peel powder, or okra extract powder. In some embodiments, the concentration of phytochemicals in the phytochemical-containing powder is greater than or equal to 90 μg / g dry matter, 1 mg / g dry matter, 170 mg / g dry matter, or 950 mg / g dry matter.

[0159] In some embodiments, the phytochemical-containing powder is mango peel powder. In some embodiments, the phytochemical of the mango peel powder is mangiferin. In some embodiments, the concentration of the phytochemical in the mango peel powder is less than or equal to 200 mg / g dry matter, 190 mg / g dry matter, 180 mg / g dry matter, 170 mg / g dry matter, 160 mg / g dry matter, 150 mg / g dry matter, 140 mg / g dry matter, 130 mg / g dry matter, 120 mg / g dry matter, 110 mg / g dry matter, or 100 mg / g dry matter. In some embodiments, the concentration of the phytochemical in the mango peel powder is greater than or equal to 100 mg / g dry matter, 110 mg / g dry matter, 120 mg / g dry matter, 130 mg / g dry matter, 140 mg / g dry matter, 150 mg / g dry matter, 160 mg / g dry matter, 170 mg / g dry matter, 180 mg / g dry matter, 190 mg / g dry matter, or 200 mg / g dry matter. In some embodiments, the concentration of the phytochemical in the mango peel powder is between 100 mg / g and 120 mg / g dry matter, between 110 mg / g and 130 mg / g dry matter, between 120 mg / g and 140 mg / g dry matter, between 150 mg / g and 170 mg / g dry matter, between 160 mg / g and 180 mg / g dry matter, between 170 mg / g and 190 mg / g dry matter, or between 180 mg / g and 200 mg / g dry matter.

[0160] In some embodiments, the weight ratio of mangiferin to plant-derived protein is greater than or equal to 1:60, 1:55, 1:50, 1:45, 1:40, 1:35, or 1:30. In some embodiments, the weight ratio of mangiferin to plant-derived protein is less than or equal to 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, or 1:60. In some embodiments, the weight ratio of mangiferin to plant-derived protein is between 1:30 and 1:40, between 1:35 and 1:45, between 1:40 and 1:50, between 1:45 and 1:55, or between 1:50 and 1:60. In some embodiments, the weight ratio of mangiferin to plant-derived protein is 1:40.

[0161] In some embodiments, the phytochemical-containing powder includes tea leaves. In some embodiments, the phytochemical of the tea leaves is epigallocatechin 3-O-gallate (EGCG). In some embodiments, the concentration of the phytochemical in the tea leaves is less than or equal to 1050 mg / g dry matter, 1000 mg / g dry matter, 950 mg / g dry matter, 900 mg / g dry matter, 850 mg / g dry matter, 800 mg / g dry matter, 750 mg / g dry matter, 700 mg / g dry matter, 650 mg / g dry matter, 600 mg / g dry matter, 550 mg / g dry matter, or 500 mg / g dry matter. In some embodiments, the concentration of the phytochemical in the tea leaves is greater than or equal to 500 mg / g dry matter, 550 mg / g dry matter, 600 mg / g dry matter, 650 mg / g dry matter, 700 mg / g dry matter, 750 mg / g dry matter, 800 mg / g dry matter, 850 mg / g dry matter, 900 mg / g dry matter, 950 mg / g dry matter, 1000 mg / g dry matter, or 1050 mg / g dry matter. In some embodiments, the concentration of the phytochemical in the tea leaves is between 500 mg / g and 600 mg / g dry matter, between 550 mg / g and 650 mg / g dry matter, between 600 mg / g and 700 mg / g dry matter, between 650 mg / g and 750 mg / g dry matter, between 700 mg / g and 800 mg / g dry matter, between 750 mg / g and 850 mg / g dry matter, between 800 mg / g and 900 mg / g dry matter, between 850 mg / g and 950 mg / g dry matter, between 900 mg / g and 1000 mg / g dry matter, or between 950 mg / g and 1050 mg / g dry matter.

[0162] In some embodiments, the weight ratio of EGCG to plant-derived protein exceeds or is equal to 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:150, or 1:100. In some embodiments, the weight ratio of EGCG to plant-derived protein is less than or equal to 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, or 1:500. In some embodiments, the weight ratio of EGCG to plant-derived protein is between 1:100 and 1:200, between 1:150 and 1:250, between 1:200 and 1:300, between 1:250 and 1:350, between 1:300 and 1:400, between 1:350 and 1:450, or between 1:400 and 1:500. In some embodiments, the weight ratio of EGCG to plant-derived protein is 1:265.

[0163] In some embodiments, the phytochemical-containing powder comprises a grape extract. In some embodiments, the phytochemical of the grape extract is trans-resveratrol. In some embodiments, the concentration of the phytochemical in the grape extract is less than or equal to 40 mg / g dry matter, 35 mg / g dry matter, 30 mg / g dry matter, 25 mg / g dry matter, 20 mg / g dry matter, 19 mg / g dry matter, 18 mg / g dry matter, 17 mg / g dry matter, 16 mg / g dry matter, 15 mg / g dry matter, 14 mg / g dry matter, 13 mg / g dry matter, 12 mg / g dry matter, 11 mg / g dry matter, 10 mg / g dry matter, 9 mg / g dry matter, 8 mg / g dry matter, 7 mg / g dry matter, 6 mg / g dry matter, 5 mg / g dry matter, 4 mg / g dry matter, 3 mg / g dry matter, 2 mg / g dry matter, or less than 1 mg / g dry matter. In some embodiments, the concentration of the phytochemical in the grape extract is greater than or equal to 1 mg / g dry matter, 2 mg / g dry matter, 3 mg / g dry matter, 4 mg / g dry matter, 5 mg / g dry matter, 6 mg / g dry matter, 7 mg / g dry matter, 8 mg / g dry matter, 9 mg / g dry matter, 10 mg / g dry matter, 11 mg / g dry matter, 12 mg / g dry matter, 13 mg / g dry matter, 14 mg / g dry matter, 15 mg / g dry matter, 16 mg / g dry matter, 17 mg / g dry matter, 18 mg / g dry matter, 19 mg / g dry matter, 20 mg / g dry matter, 25 mg / g dry matter, 30 mg / g dry matter, 35 mg / g dry matter, or 40 mg / g dry matter.In some embodiments, the concentration of the phytochemicals in the grape extract is between 1 mg / g and 3 mg / g of dry matter, between 2 mg / g and 4 mg / g of dry matter, between 5 mg / g and 7 mg / g of dry matter, between 6 mg / g and 8 mg / g of dry matter, between 7 mg / g and 9 mg / g of dry matter, between 8 mg / g and 10 mg / g of dry matter, between 9 mg / g and 11 mg / g of dry matter, between 10 mg / g and 12 mg / g of dry matter, between 11 mg / g and 13 mg / g of dry matter, between 12 mg / g and 14 mg / g of dry matter, between 13 mg / g and 15 mg / g of dry matter, between 14 mg / g and 16 mg / g of dry matter, between 15 mg / g and 17 mg / g of dry matter, between 16 mg / g and 18 mg / g of dry matter, between 17 mg / g and 19 mg / g of dry matter, between 18 mg / g and 20 mg / g of dry matter, between 19 mg / g and 25 mg / g of dry matter, between 20 mg / g and 30 mg / g of dry matter, between 25 mg / g and 35 mg / g of dry matter, or between 30 mg / g and 40 mg / g of dry matter.

[0164] In some embodiments, the weight ratio of resveratrol to the plant-derived protein is greater than or equal to 1:500, 1:450, 1:400, 1:350, or 1:300. In some embodiments, the weight ratio of resveratrol to the plant-derived protein is less than or equal to 1:300, 1:350, 1:400, 1:450, or 1:500. In some embodiments, the weight ratio of resveratrol to the plant-derived protein is between 1:300 and 1:400, between 1:350 and 1:450, or between 1:400 and 1:500. In some embodiments, the weight ratio of resveratrol to the plant-derived protein is 1:400.

[0165] In some embodiments, the phytochemical-containing powder comprises blackberries. In some embodiments, the phytochemical of the blackberries is quercetin. In some embodiments, the concentration of the phytochemical in the blackberries is less than or equal to 10000 μg / 100 g dry matter, 95000 μg / 100 g dry matter, 90000 μg / 100 g dry matter, 85000 μg / 100 g dry matter, 80000 μg / 100 g dry matter, 75000 μg / 100 g dry matter, 70000 μg / 100 g dry matter, 65000 μg / 100 g dry matter, 60000 μg / 100 g dry matter, 55000 μg / 100 g dry matter, or 50000 μg / 100 g dry matter. In some embodiments, the concentration of the phytochemical in the blackberries is greater than or equal to 50000 μg / 100 g dry matter, 55000 μg / 100 g dry matter, 60000 μg / 100 g dry matter, 65000 μg / 100 g dry matter, 70000 μg / 100 g dry matter, 75000 μg / 100 g dry matter, 80000 μg / 100 g dry matter, 85000 μg / 100 g dry matter, 90000 μg / 100 g dry matter, 95000 μg / 100 g dry matter, or 100000 μg / 100 g dry matter. In some embodiments, the concentration of the phytochemical in the blackberries is between 50000 μg / 100 g and 60000 μg / 100 g dry matter, between 55000 μg / 100 g and 65000 μg / 100 g dry matter, between 60000 μg / 100 g and 70000 μg / 100 g dry matter, between 65000 μg / 100 g and 75000 μg / 100 g dry matter, between 70000 μg / 100 g and 80000 μg / 100 g dry matter, between 75000 μg / 100 g and 85000 μg / 100 g dry matter, between 80000 μg / 100 g and 90000 μg / 100 g dry matter, between 85000 μg / 100 g and 95000 μg / 100 g dry matter, or between 90000 μg / 100 g and 100000 μg / 100 g dry matter.

[0166] In some embodiments, the weight ratio of quercetin to plant-derived protein is greater than or equal to 1:40000, 1:35000, 1:30000, 1:25000, 1:24000, 1:23500, 1:23,000, 1:22,750, 1:22,500, 1:22,250, or 1:22,000. In some embodiments, the weight ratio of quercetin to plant-derived protein is less than or equal to 1:22,000, 1:22,250, 1:22,500, 1:22,750, 1:23,000, 1:23500, 1:24000, 1:25000, 1:30000, or 1:40000. In some embodiments, the weight ratio of quercetin to plant-derived protein is between 1:22,000 and 1:22,500, between 1:22,250 and 1:22,750, between 1:22,500 and 1:23,000, between 1:22,750 and 1:23,500, between 1:23,000 and 1:24,000, between 1:23,500 and 1:25,000, between 1:24,000 and 1:30,000, or between 1:25,000 and 1:40,000. In some embodiments, the weight ratio of quercetin to plant-derived protein is 1:22,222.

[0167] In some embodiments, the water has undergone one or more processes such as distillation or filtration. In some embodiments, the water is distilled water.

[0168] In some embodiments, the dry weight ratio of the ketone molecule to the plant-derived protein is less than or equal to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the dry weight ratio of the ketone molecule to the plant-derived protein is greater than or equal to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the dry weight ratio of the ketone molecule to the plant-derived protein is between 1:10 and 1:1, between 1:5 and 1:2, or between 1:3 and 1:2. In some embodiments, the dry weight ratio of the ketone molecule to the plant-derived protein is less than or equal to 1:2. In some embodiments, the dry weight ratio of the ketone molecule to the plant-derived protein is 1:2.5.

[0169] In some embodiments, the dry weight ratio of the NR molecule to the plant-derived protein is less than or equal to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the dry weight ratio of the NR molecule to the plant-derived protein is greater than or equal to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the dry weight ratio of the NR molecule to the plant-derived protein is between 1:10 and 1:1, between 1:5 and 1:2, or between 1:3 and 1:2. In some embodiments, the dry weight ratio of the NR molecule to the plant-derived protein is less than or equal to 1:2. In some embodiments, the dry weight ratio of the NR molecule to the plant-derived protein is 1:3.2.

[0170] In some embodiments, the dry weight ratio of caffeine molecules to plant-derived proteins is less than or equal to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the dry weight ratio of caffeine molecules to plant-derived proteins is greater than or equal to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the dry weight ratio of caffeine molecules to plant-derived proteins is between 1:10 and 1:1, between 1:5 and 1:2, or between 1:3 and 1:2. In some embodiments, the dry weight ratio of caffeine molecules to plant-derived proteins is less than or equal to 1:2. In some embodiments, the dry weight ratio of caffeine molecules to plant-derived proteins is 1:2.

[0171] In some embodiments, the dry weight ratio of TH molecules to plant-derived proteins is less than or equal to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the dry weight ratio of TH molecules to plant-derived proteins is greater than or equal to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the dry weight ratio of TH molecules to plant-derived proteins is between 1:10 and 1:1, between 1:5 and 1:2, or between 1:3 and 1:2. In some embodiments, the dry weight ratio of TH molecules to plant-derived proteins is less than or equal to 1:2. In some embodiments, the dry weight ratio of TH molecules to plant-derived proteins is 1:2.

[0172] In some embodiments, the weight ratio of the plant-derived protein to water exceeds or is equal to 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, or 1:10. In some embodiments, the weight ratio of the plant-derived protein to water is less than or equal to 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100. In some embodiments, the weight ratio of the plant-derived protein to water is between 1:40 and 1:70, between 1:50 and 1:90, or between 1:60 and 1:100. In some embodiments, the weight ratio of the plant-derived protein to water is less than or equal to 1:70. In some embodiments, the weight ratio of the plant-derived protein to water is 1:66.7. In some embodiments, the weight ratio of the plant-derived protein to water is 1:20.

[0173] Nanoparticles

[0174] Disclosed herein are nanoparticles comprising plant-derived proteins crosslinked with one or more phytochemicals. In some embodiments, the nanoparticles can comprise one or more ketone molecules.

[0175] In some embodiments, the average diameter of the nanoparticles is less than or equal to 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, or 50 nm. In some embodiments, the average diameter of the nanoparticles is greater than or equal to 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, or 700 nm. In some embodiments, the average diameter between the nanoparticles is between 50 nm and 70 nm, between 60 nm and 80 nm, between 70 nm and 90 nm, between 80 nm and 100 nm, between 90 nm and 200 nm, between 100 nm and 300 nm, between 100 nm and 400 nm, between 100 nm and 500 nm, between 200 nm and 400 nm, between 200 nm and 50 nm, between 300 nm and 500 nm, between 400 nm and 600 nm, or between 500 nm and 700 nm.

[0176] In some embodiments, the nanoparticles contain phenol. In some embodiments, the phenol content of the nanoparticles is quantified using total gallic acid equivalents (GAE). In some embodiments, the phenol content of the nanoparticles described herein is greater than 5 mg GAE / g, 10 mg GAE / g, 20 mg GAE / g, 30 mg GAE / g, 40 mg GAE / g, 50 mg GAE / g, 60 mg GAE / g, 70 mg GAE / g, 80 mg GAE / g, 90 mg GAE / g, 100 mg GAE / g, 150 mg GAE / g, 200 mg GAE / g, 250 mg GAE / g, 300 mg GAE / g, or 350 mg GAE / g. In some embodiments, the phenol content of the nanoparticles described herein is less than 400 mg GAE / g, 350 mg GAE / g, 300 mg GAE / g, 250 mg GAE / g, 200 mg GAE / g, 150 mg GAE / g, 100 mg GAE / g, 90 mg GAE / g, 80 mg GAE / g, 70 mg GAE / g, 60 mg GAE / g, 50 mg GAE / g, 40 mg GAE / g, 30 mg GAE / g, 20 mg GAE / g, 10 mg GAE / g, or 5 mg GAE / g. In some embodiments, the phenol content of the nanoparticles described herein is between 5 mg GAE / g and 20 mg GAE / g, between 10 mg GAE / g and 30 mg GAE / g, between 20 mg GAE / g and 40 mg GAE / g, between 30 mg GAE / g and 50 mg GAE / g, between 40 mg GAE / g and 60 mg GAE / g, between 50 mg GAE / g and 70 mg GAE / g, between 60 mg GAE / g and 80 mg GAE / g, between 70 mg GAE / g and 90 mg GAE / g, between 80 mg GAE / g and 100 mg GAE / g, between 90 mg GAE / g and 150 mg GAE / g, between 100 mg GAE / g and 200 mg GAE / g, between 150 mg GAE / g and 250 mg GAE / g, between 200 mg GAE / g and 300 mg GAE / g, between 250 mg GAE / g and 350 mg GAE / g, or between 300 mg GAE / g and 400 mg GAE / g.

Example

[0177] Example

[0178] Example 1 - Synthesis of Ketone Molecule

[0179] The ketone molecule described in this specification was prepared by the Fischer esterification reaction of (R)-3-hydroxybutyric acid and glycerol described in this specification to obtain a mixture of Compounds II-V described in this specification. Compound II was purified by fractional distillation according to methods known in the art. (See, for example, www.sciencedirect.com / topics / chemistry / fractional-distillation.) The resulting ketone molecule contained more than an 80:20 molar ratio of Compound II to Compounds II-V.

[0180] Example 2a - Synthesis of Nicotinamide Riboside Molecule

[0181] The nicotinamide riboside molecule and Compound IV were obtained from Thorne Healthcare.

[0182] Example 2b - Synthesis of Caffeine Molecule

[0183] The caffeine molecule was obtained from Sigma-Aldrich.

[0184] Example 2c - Synthesis of Theacrine Molecule

[0185] The theacrine molecule was obtained from BulkStimulants.com.

[0186] Example 3 - Characterization of Ketone Molecule (KM):

[0187] Appearance: KM was a slightly turbid, oily, highly viscous liquid.

[0188] Odor: The odor of KM was neutral.

[0189] Solubility: KM was water-soluble and miscible with ethanol. KM did not dissolve in methylene chloride.

[0190] pH: KM showed a pH of 4.5 in water, indicating an acidic aqueous solution.

[0191] UV-Visible Absorption Spectroscopy: The electronic transitions of KM were related to the ultraviolet (UV) region based on UV-visible absorption measurements. KM showed broad absorption up to approximately 245 nm in the UV region (Figure 1). There was no absorption peak characteristic of a chromophore in the visible region. Therefore, UV-visible spectroscopy was not utilized for the tracking of KM or KM-encapsulated protein nanoparticles.

[0192] Nuclear Magnetic Resonance Spectroscopy: 1H nuclear magnetic resonance (NMR) spectra were recorded at 300 MHz on a Bruker 300 MHz spectrometer. 13C NMR spectra were recorded at 151 MHz on a Bruker 600 MHz spectrometer. NMR spectra were recorded in deuterated water (D2O). 1H chemical shifts were reported relative to internal D2O (Figure 3). 13C NMR chemical shifts were reported relative to an external tetramethylsilane (TMS) standard (Figures 5 - 7). KM is a carboxylate ester containing the parent carboxylic acid (3-hydroxybutyric acid) and the corresponding alcohol (glycerol). Various glycerides are possible products during the synthesis of KM. NMR showed approximately 77% of KM, i.e., monoglyceride. Glycerol (11%) and 3-hydroxybutyric acid (12%) of the starting materials were also seen in the NMR. Figure 42 shows the 1H NMR of KM in D2O. 2 O of KM 1 1H NMR is shown.

[0193] Mass spectrometry: A series of advanced mass spectrometry techniques have been utilized to develop highly reliable quality control parameters for ketone molecules (KM) and their protein-encapsulated products. Analysis of KM was performed using electrospray ionization mass spectrometry (ESI-MS) technology. Apart from obtaining the elemental / isotope signature of KM using MS, the [M+H]+ peak was used for molecular weight estimation. The calculated molecular weight of KM based on its chemical structure was 178.18 g / mol. The [M+Na]+ peak at 201.07 was detected as the main peak indicating the presence of monoglyceride (KM) (Figure 9).

[0194] Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS): In addition to mass spectrometry, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were utilized for quality control confirmation of KM. GC-MS and LC-MS techniques are excellent analytical and quality control tools for detecting KM purity and the presence of starting materials. KM is a carboxylate ester containing the parent carboxylic acid (3-hydroxybutyric acid) and the corresponding alcohol (glycerol). In the GC-MS analysis (Figure 11), 3-hydroxybutyric acid (about 10%) and glycerol (about 23%) were seen at retention RTs of 17.1 minutes and 20.2 minutes, respectively. Two monoglyceride peaks were seen at retention times (RT) of 30.4 minutes and 30.9 minutes. The peak at 30.4 minutes (about 16%) and the peak at 30.9 minutes (about 50%) gave a combined KM monoglyceride rate of about 66%. Diglyceride and triglyceride peaks were not present (which should have been seen after about 30 minutes RT). In the LC-MS analysis (Figure 12), the main peak corresponded to the loss of water from the monoglyceride. The main peak at 1.4 minutes RT corresponded to the [M-H2O]+ peak of 161, which corroborated the results from NMR as well as the results from GC-MS.

[0195] Circular Dichroism (CD): Chiral molecules are mirror images or enantiomers. The physical properties of enantiomers are identical in all respects except for how the molecules interact with polarized light and how they interact with other chiral molecules. Enantiomers are a special form of isomers. Circular Dichroism (CD) is measured as a function of wavelength and is the difference in absorbance between left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL). When a chiral chromophore is present, one state of circular polarization is absorbed more or less than the other. Thus, over the corresponding wavelengths, the circular dichroism signal can be positive or negative depending on whether L-CPL is absorbed to a greater extent than R-CPL (CD signal positive for the R / D configuration) or to a lesser extent (CD signal negative for the S / L configuration). KM was used for CD measurements. The concentration of KM was 7 mg / ml in water. The starting material (R)-3-hydroxybutyric acid, also known as D-β-hydroxybutyric acid, is chiral and introduced chirality into KM. The circular dichroism (CD) spectrum of KM was positive, indicating the R / D configuration in KM (Figure 13).

[0196] KM Evaluation: The analysis of KM showed 80 - 70% monoglycerides based on NMR and GC-MS, respectively. Diglycerides and triglycerides based on GC-MS analysis were not present. 10 - 20% glycerol and 10% 3-hydroxybutyric acid starting material were also seen by NMR and GC-MS, respectively. No other impurities were seen.

[0197] Quality Control Parameters: Based on the study of the quality control parameters of the ketone molecule (KM), GC-MS and LC-MS analyses were important quality control and characterization tools in the complete characterization of green nanotechnology-based KM encapsulation within plant proteins.

[0198] Example 4a - NR Property Evaluation:

[0199] Appearance: NR was a fine white powder.

[0200] Odor: The odor of NR was neutral.

[0201] Solubility: NR was water-soluble and miscible with ethanol. The chloride salt of nicotinamide riboside had good solubility in water.

[0202] pH: NR showed a pH of 4.0 in water, indicating an acidic aqueous solution.

[0203] UV-Visible Absorption Spectroscopy: The electronic transitions of NR were related to the ultraviolet region (UV) based on UV-visible absorption measurements. NR showed broad absorption up to about 300 nm in the UV region with peaks at 220 nm, 230 nm, and 265 nm (Figure 2). There were no absorption peaks characteristic of chromophores in the visible region. Therefore, UV-visible spectroscopy was not utilized for the tracking of NR-encapsulated protein nanoparticles.

[0204] Nuclear Magnetic Resonance Spectroscopy: 1 1H Nuclear Magnetic Resonance (NMR) spectra were recorded at 500 MHz on a Bruker 500 MHz spectrometer. 13 13C NMR spectra were recorded at 126 MHz on a Bruker 500 MHz spectrometer. The NMR spectra were recorded in deuterated water (D 2 2O). 1 1H chemical shifts were reported relative to internal D 2 2O (Figure 4). 13 13C NMR chemical shifts were reported relative to an external tetramethylsilane (TMS) standard (Figure 8). The proton and carbon NMR of NR showed clean spectra without impurities.

[0205] Mass Spectrometry: A series of advanced mass spectrometry techniques have been utilized to develop reliable quality control parameters for NR and its protein-encapsulated products. NR was analyzed using electrospray ionization mass spectrometry (ESI-MS) technology. Apart from obtaining the elemental / isotope signature of NR using MS, the [M+H]+ peak was used for molecular weight estimation. The nicotinamide riboside anion of 255.09 [M]+ The peak was detected as the main peak (Figure 10). The peak at 123.05 correlated with the nicotinamide [M+H] fragment peak of NR. + It was correlated with the fragment peak.

[0206] Circular dichroism (CD): NR was evaluated by circular dichroism. The concentration of NR was 0.2 mg / ml in water. The circular dichroism spectrum of NR was negative, indicating an S / L configuration (Figure 14). The anionic part of NR can introduce chirality.

[0207] NR evaluation: The analysis of NR showed a nicotinamide riboside-containing compound. NMR showed a clean spectrum without starting materials or solvent impurities. Mass spectrometry showed peaks correlating with nicotinamide riboside and nicotinamide units. The circular dichroism (CD) spectrum of NR was negative, indicating an S / L configuration.

[0208] Quality control parameters: Based on the study of the quality control parameters of nicotinamide riboside (NR), the above analytical tools were important for the development of various NR-related products by NR encapsulation based on green nanotechnology.

[0209] Characterization of Example 4b - CA

[0210] Appearance: CA was a fine white powder.

[0211] Odor: The odor of CA was neutral and the taste was bitter.

[0212] Solubility: CA was water-soluble (25 mg / ml).

[0213] pH: CA showed a pH of 7.0 in water indicating a neutral aqueous solution.

[0214] Liquid Chromatography-Mass Spectrometry (LC-MS): For the quality control confirmation of CA, Liquid Chromatography-Mass Spectrometry (LC-MS) was utilized. LC-MS technology is an excellent analytical and quality control tool for detecting CA purity, quantity, and the presence of other substances. LC-MS analysis (Figure 14B). The dominant peak at 3.0 minutes RT corresponded to the peak of [M+H] of 195, which verified the results from the literature on caffeine. + The peak corresponded to the results from the literature on caffeine.

[0215] CA Evaluation: The LC-MS analysis of CA showed a pure substance. CA is a well-established compound, and other sophisticated characterization data can be obtained from the websites of the National Institute of Standards and Technology (NIST) and the Spectral Database for Organic Compounds (SDBS).

[0216] Quality Control Parameters: Based on the research on the quality control parameters of caffeine (CA), the LC-MS analysis tool was important for the development of various CA-related products by CA encapsulation based on green nanotechnology.

[0217] Example 4b - TH Characterization

[0218] Appearance: TH was a fine white powder.

[0219] Odor: The odor of TH was neutral, and the taste was very bitter.

[0220] Solubility: CA was water-soluble (25 mg / ml).

[0221] pH: CA showed a pH of 7.0 in water, indicating a neutral aqueous solution.

[0222] Liquid Chromatography-Mass Spectrometry (LC-MS): Liquid Chromatography-Mass Spectrometry (LC-MS) was used for the quality control verification of TH. LC-MS technology is an excellent analytical and quality control tool for detecting TH purity, quantity, and the presence of other substances. LC-MS analysis (Figure 14C). The dominant peak at 3.0 min RT corresponded to the [M+H] peak of 225, which supported the results from the literature on teacrine. + The peak corresponded to the results from the literature on teacrine.

[0223] TH Evaluation: The LC-MS analysis of TH showed a pure substance. TH is a well-established compound, and other sophisticated characterization data can be obtained from the website of the National Institute of Standards and Technology (NIST).

[0224] Quality Control Parameters: Based on the study of the quality control parameters of teacrine (TH), the LC-MS analysis tool was important for the development of various TH-related products by green nanotechnology-based TH encapsulation.

[0225] Example 5 - Characterization of Plant-Derived Proteins

[0226]

Table 2

[0227] Pea Protein

[0228] Water-soluble pea protein (PP) was purchased from the website of WaterSolubleProtein.com. The plant source was peas, natural and non-GMO yellow peas, which were hydrolyzed to be highly water-soluble (100 mg / ml). The protein solution was a clear yellow liquid without precipitate or residue. The protein content was 80%. This is edible, immediately water-soluble, and forms a stable solution.

[0229] UV-Visible Absorption Spectroscopy of Pea Protein: The electronic transitions of water-soluble pea protein (PP) are related to the ultraviolet (UV) region based on UV-visible absorption measurements. PP showed a weak absorption peak at 265 nm and broad absorption in the UV region of 250 nm - 200 nm (Figure 17A). There was no characteristic absorption peak in the visible region.

[0230] Fluorescence Spectroscopy of Pea Protein: Phenylalanine, tyrosine, and tryptophan amino acids in the protein gave rise to intrinsic protein fluorescence. The fluorescence emission spectrum of water-soluble pea protein (PP) was recorded by excitation at 285 nm corresponding to tryptophan amino acid. A broad emission peak was seen at 352 nm in the emission spectrum of PP (Figure 17B). Changes in the tryptophan microenvironment could be monitored by fluorescence spectroscopy. Shifts in the fluorescence maximum peak and variations in fluorescence intensity may provide insights into the pea protein interaction with ligands (References: Akbar, S.M. et al, Journal of Bioenergetics and Biomembranes, 2016, 48, 241 - 247 and Vivian, J.T. et al, Biophysical Journal 2001, 80, 2093 - 2109).

[0231] Matrix-Assisted Laser Desorption / Ionization Mass Spectrometry (MALDI-MS) of Pea Protein: Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) was used to analyze water-soluble pea protein (PP) to confirm the molecular weights of the protein components. Sinapinic acid matrix was used. A mass range of 2 kDa - 400 kDa was studied with Bruker AutoFlex Speed MALDI-MS, but no significant peaks were seen. Then the mass range was decreased to 5 kDa, and peaks around 877 and 993 m / z were seen, indicating a protein molecular weight of less than about 1 kDa (Figure 18).

[0232] NMR of Pea Protein. Figure 41 shows the 1H NMR of water-soluble pea protein (PP) in D2O.

[0233] Soy protein isolate

[0234] The soy protein isolate was obtained from MP Biomedicals (catalog number ICN90545605). Soy protein is an abundant and inexpensive plant protein derived from soybeans. Consumption of soybeans is beneficial for heart health and reduces inflammation. Therefore, soybeans have important uses for the treatment of diabetes, atherosclerosis, and cancer. Soy protein isolate (SPI) is a soy food with a maximum soy protein content of 92%. SPI is water-soluble and a useful drug delivery vehicle for water-soluble pharmaceuticals. SPI is generally regarded as a safe pharmaceutical and is considered to be biodegradable, inexpensive, and abundantly available. The anti-inflammatory properties of soy protein can also be utilized in nanotherapeutic formulations.

[0235] Soy protein isolate UV-visible absorption spectroscopy: Electronic transitions SPI is related to the ultraviolet region (UV) based on UV-visible absorption measurements. SPI showed broad absorption in the UV region from 240 nm to 200 nm (Figure 42). There was no absorption peak characteristic of the visible region.

[0236] Soy protein isolate Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS): Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) was used to analyze water-soluble pea protein (PP) to confirm the molecular weights of the protein components. A sinapic acid matrix was used. The mass range from 2 kDa to 400 kDa was studied with a Bruker AutoFlex Speed MALDI-MS. A significant peak was observed at 8960 m / z, indicating a protein molecular weight of approximately 9 kDa (Figure 34).

[0237] Okra extract

[0238] The okra extract was obtained from Bulk Supplements (SKU OKR25kg). Okra is a flowering plant belonging to the mallow family. Its green seed pods are edible and consist of water (90%), protein (2%), carbohydrates (7%) and trace amounts of fat. It is a rich source of dietary fiber, vitamin C and vitamin K. Okra mucilage or gel from the pods is a natural polysaccharide and contains D-galactose, L-rhamnose and L-galacturonic acid. Okra mucilage is extracted in water and forms a gelatinous viscous solution. Natural polysaccharides are biocompatible, non-toxic and have applications in biopolymer forms such as microspheres, nanomatrices and nanoparticles. Okra extract (OE), a finely ground dry powder, was purchased from the website of BulkSupplements.com. This is edible, water-soluble and can be stored at room temperature. OE produced a slightly turbid brown solution of okra mucilage or gel in water. Natural polysaccharides such as okra gel can be used for encapsulation, stabilization and drug delivery applications.

[0239] Okra mucilage matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS): Okra mucilage or gel from the green seed pods was extracted in water. This was a gelatinous viscous solution. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) was used to analyze okra mucilage to confirm the molecular weights of the protein components. A sinapic acid matrix was used. The mass range of 2 kDa to 400 kDa was studied with a Bruker AutoFlex Speed MALDI-MS. A peak was seen at around 8 kDa for okra polysaccharide mucilage (Figure 29).

[0240] Okra mucilage UV-visible absorption spectroscopy: The electronic transitions in okra mucilage are mainly related to the ultraviolet region (UV) based on UV-visible absorption measurements. Okra mucilage showed a weak broad absorption between 400 nm and 300 nm. Furthermore, in the UV region, a stronger broad absorption was shown in the region of 300 nm to 200 (Figure 30). There was no characteristic absorption peak in the visible region.

[0241] Example 6 - Evaluation of Phytochemical Properties

[0242] The proton (1H) NMR spectrum was recorded at 600 MHz on a Bruker 600 MHz spectrometer. The carbon (13C) NMR spectrum was recorded at 151 MHz on a Bruker 600 MHz spectrometer. The NMR spectra were recorded in deuterated water (D2O) or deuterated methanol (CD3OD) or a combination of both. The 1H chemical shifts were reported relative to internal D2O or CD3OD. The 13C NMR chemical shifts were reported relative to an external tetramethylsilane (TMS) standard. The carbon NMR showed corresponding changes.

[0243] Hydrogen bonding is a characteristic of KM, protein, and phytochemical structures. The opportunities for both intermolecular and intramolecular hydrogen bonding are extensive. Hydrogen bonding can facilitate KM interactions with proteins and phytochemicals for the purposes of nanoparticle synthesis and encapsulation. The extensive hydrogen bonding characteristics promoted KM nanocapsulation, as well as the interaction of KM with proteins and phytochemicals (Figure 30). It was also beneficial for the interaction between proteins and phytochemicals.

[0244] Figure 42 is CD 3 of mangiferin in 1 H NMR is shown.

[0245] Figure 45 is D 2 of epigallocatechin gallate (EGCG) in 1 H NMR is shown.

[0246] Figure 48 is CD 3 of quercetin in 1 H NMR is shown.

[0247] Figure 51 is CD 3 of resveratrol in 1 H NMR is shown.

[0248] The phytochemicals were supplied from the following.

[0249]

Table 3

[0250] Example 7 - Crosslinking of Pea Protein and Mangiferin and Encapsulation of Ketone Molecules into Mangiferin - Crosslinked Pea Protein

[0251] Synthesis of KM - Encapsulated Pea Protein Nanoparticles (KM - PP - MP - NP) by Mango Peel Crosslinking: 15 g of water - soluble pea protein (PP), 15 g of mango peel (MP) powder, and 6 g of ketone molecule (KM) were weighed and transferred to a 2 L conical flask equipped with a stir bar. 1 L of distilled water was added to the flask, and the contents of the reaction mixture were stirred overnight. Stirring was stopped, and then the insoluble residue was allowed to settle. The insoluble residue was separated from the solution by first decantation and then filtration (75 - micron size). 625 mL of the filtered solution was transferred to a 2 L round - bottom flask equipped with a stir bar. 210 mL of anhydrous ethanol (1 / 3 volume of the filtered solution) was added to the contents of the flask and stirred vigorously. Then, the reaction mixture was stirred overnight. Then, the KM - encapsulated pea protein nanoparticles (KM - PP - MP - NP) obtained by mango peel crosslinking were characterized by TEM, DLS size, and ZP measurements. The KM content was estimated by GC - MS analysis and neat KMst. Gas Chromatography-Mass Spectrometry (GC-MS) Procedure: 1 μL of the KM sample to be analyzed was diluted 1000-fold by adding 1000 μL of pyridine. 25 μL of this solution was trimethylsilylated by incubating with 25 μL of MSTFA [N-methyl-N-(trimethyl-silyl)trifluoroacetamide] + 1% TMCS (chlorotrimethylsilane) reagent at 50 °C for 1 hour. The derivatized sample was then analyzed using an Agilent 6890 GC coupled to a 5973N MSD mass spectrometer in a scan range of 50 - 650 m / z (Agilent Technologies, Inc., Santa Clara, CA). 1 μl of the sample was injected into the GC column at a split ratio of 1:1 with a run time of 60 minutes. Separation was achieved with a temperature program of 80 °C for 2 minutes at a constant flow rate of 1.0 ml / min of helium gas, then rising to 315 °C at 5 °C / min and holding at 315 °C for 12 minutes using a 60 m DB-5MS column (J&W Scientific, inner diameter 0.25 mm, film thickness 0.25 μm). A standard alkane mixture was used for GC-MS quality control and calculation of retention indices. Chromatographic peaks in the sample were deconvoluted using AMDIS and annotated by mass spectrum and retention index matching with an in-house constructed spectral library and the commercially available NIST17 mass spectral library. Five different concentrations of neat KM standards prepared in pyridine were analyzed in the same way. Peak areas were calculated using Agilent's Enhanced Data Analysis (EDA) software to create a calibration curve. The concentration of monoglycerides in the KM solution was accurately calculated using the calibration curve and the peak areas of the GC peaks of the sample solution at RT~30 minutes.

[0252] Standard Calibration. To extend the shelf life, the KM-PP-MP-NP solution was refrigerated (5 °C ± 3 °C). Blank pea protein nanoparticles (PP-MP-NP) crosslinked with mango peel were synthesized following the same procedure without the addition of KM.

[0253] Figure 43 shows the 1H NMR (expanded) of mangiferin and water-soluble pea protein (PP) reaction mixture in CD3OD / D2O (arrows indicate the changes in protons).

[0254] Figure 44 shows the 3 OD / D 2 O of mangiferin, water-soluble pea protein (PP) and KM reaction mixture 1 H NMR (expanded).

[0255] The aromatic ring protons of phytochemicals showed distinct changes indicating covalent binding interactions with the protein. These changes led to crosslinking with the protein amino functional groups. The mangiferin proton NMR showed peaks equivalent at 7.5, 6.8 and 6.4 ppm. However, after reaction with pea protein, there were significant changes in the 6.8 and 6.4 ppm peaks.

[0256] KM encapsulated pea protein nanoparticles (KM-PP-MP-NP) by mango peel crosslinking: Naturally available plant-based substances were investigated for crosslinking. Mangiferin (MGF), a phytochemical found in mango, was extracted from mango peel. Multiple hydroxyl groups in MGF were effective for protein crosslinking. KM encapsulated water-soluble pea protein nanoparticles (KM-PP-MP-NP) using mango peel crosslinking were attempted by ethanol desolvation method (Figure 15). After nanoparticle synthesis, the insoluble residue was removed by centrifugation / filtration. The KM-PP-MP-NP size was between 336 - 422 nm, with an average of 367 nm (using DLS) and showed an average zeta potential of -15 mV (Table 4), indicating reasonable stability. A polydispersity index (PDI) of 0.2 indicates nanoparticles of uniform size (monomodal distribution). The core size of the nanoparticles was investigated by TEM (Figure 23) using negative staining technique. This showed good particles with a size in the range of about 400 nm. KM-PP-MP-NP was formulated and fully characterized to define quality control parameters for scale-up and commercial level production.

[0257] The particles showed an average size of 428 nm (using DLS) and an average zeta potential of -16 mV (Table 4), indicating reasonable stability. A PDI of 0.2 indicated nanoparticles with a uniform size (monomodal distribution). The nanoparticle core size was investigated by TEM (Figure 19) using negative staining techniques. This showed good particles with sizes in the range of approximately 400 nm. The blank nanoparticles were equivalent to the KM-encapsulated nanoparticles, with a slight increase in size. Blank pea protein nanoparticles cross-linked with mango peel were synthesized without KM for comparison (Accession No. 5).

[0258]

Table 4

[0259] Fluorescence spectroscopy of KM-PP-MP-NP and PP-MP-NP: The fluorescence emission spectra of KM-PP-MP-NP and PP-MP-NP were recorded by excitation at 285 nm corresponding to the tryptophan amino acid. A broad emission peak at 351 nm with a fluorescence intensity of 40 was seen in the emission spectrum of (Figure 21). Changes in the tryptophan microenvironment could be monitored by fluorescence spectroscopy. The fluorescence maximum peak and intensity of KM-PP-MP-NP and PP-MP-NP were equivalent, indicating no significant change in the tryptophan region of the pea protein nanoparticles. Since KM is water-soluble and hydrophilic, it did not interact with the hydrophobic tryptophan microenvironment of the pea protein. Fluorescence measurements provided insights into the interaction between pea protein and KM.

[0260] GC-MS analysis of KM-encapsulated pea protein nanoparticles (KM-PP-MP-NP) cross-linked with mango peel: GC-MS analysis was used to establish the amount of KM in the nanoparticle formulation. This was found to be quantitative, indicating 100% KM encapsulation. The high KM encapsulation efficiency may be important for the biological delivery of KM.

[0261] Extensive hydrogen bonding is a characteristic of KM, protein, and phytochemical structures. This is not surprising considering the ester, hydroxyl, amine, and oxo functional groups in their structures. The opportunities for both intermolecular and intramolecular hydrogen bonding are extensive. Hydrogen bonding is very useful for the interaction of KM with proteins and phytochemicals for the purposes of nanoparticle synthesis and encapsulation. The extensive hydrogen bonding characteristics promoted KM nanocapsulation, as well as the interaction of KM with proteins and phytochemicals (Figure 39). It was also advantageous for the interaction between proteins and phytochemicals.

[0262] Example 8a - Crosslinking of pea protein with mangiferin and encapsulation of nicotinamide riboside molecules into pea protein crosslinked with mangiferin

[0263] Synthesis of NR-encapsulated pea protein nanoparticles (NR-PP-MP-NP) by mango peel crosslinking: 15 g of water-soluble pea protein (PP), 16 g of mango peel (MP) powder, and 5 g of nicotinamide riboside (NR) were weighed and transferred to a 2 L Erlenmeyer flask equipped with a stir bar. 1 L of distilled water was added to the flask, and the contents of the reaction mixture were stirred overnight. Stirring was stopped, and then the insoluble residue was allowed to precipitate. The insoluble residue was separated from the solution by decantation and then filtration (75 micron size). 700 mL of the filtered solution was transferred to a 2 L round-bottom flask equipped with a stir bar. 234 mL of absolute ethanol (1 / 3 volume of the filtered solution) was added to this flask and stirred vigorously. The reaction mixture was then stirred overnight, and the insoluble residue was removed by centrifugation / filtration (75 micron size). The NR-encapsulated pea protein nanoparticles (NR-PP-MP-NP) obtained by mango peel crosslinking were characterized by TEM, DLS size, and ZP measurements (Table 5). To extend the shelf life, the NR-PP-MP-NP solution was refrigerated (5°C ± 3°C).

[0264] Standard calibration. Blank pea protein nanoparticles (PP-MP-NP) crosslinked with mango peel were synthesized following the same procedure without the addition of NR.

[0265] NR-encapsulated pea protein nanoparticles crosslinked with mango peel (NR-PP-MP-NP): Natural and available plant-based substances were investigated for crosslinking. Mangiferin (MGF), a phytochemical found in mango, was extracted from mango peel. Multiple hydroxyl groups in MGF were effective for protein crosslinking. As described above, NR-encapsulated water-soluble pea protein nanoparticles crosslinked with mango peel (NR-PP-MP-NP) were attempted by the ethanol desolvation method (Figure 16). After nanoparticle synthesis, insoluble residues larger than 75 microns in size were removed by centrifugation / filtration. The NR-PP-MP-NP size was between 286 and 369 nm, with an average of 297 nm (using DLS), showing an average zeta potential of -18 mV (Table 5a), indicating reasonable stability. A polydispersity index (PDI) of 0.2 indicated nanoparticles with a uniform size (monomodal distribution). The core size of the nanoparticles was investigated by TEM (Figure 20) using negative staining techniques. This showed particles with a size in the range of approximately 300 nm. NP-PP-MP-NP was formulated and fully characterized to define quality control parameters for use in scale-up and commercial-level production. The blank nanoparticles were comparable to the NR-encapsulated nanoparticles with a slightly increased size.

[0266]

Table 5-1

[0267] Example 8b - Crosslinking of pea protein and mangiferin and encapsulation of caffeine molecules into mangiferin-crosslinked pea protein

[0268] Synthesis of CA-encapsulated pea protein nanoparticles (CA-PP-MP-NP) by mango peel cross-linking: 25 g of water-soluble pea protein (PP), 8 g of mango peel (MP) powder, and 13 g of caffeine (CA) were weighed and transferred to a 1 L Erlenmeyer flask equipped with a stir bar. 500 mL of distilled water was added to the flask, and the contents of the reaction mixture were stirred overnight. The reaction mixture was then stirred overnight, and the insoluble residue was removed by centrifugation / filtration (75 micron size). The CA-encapsulated pea protein nanoparticles (CA-PP-MP-NP) obtained by mango peel cross-linking were characterized by TEM, DLS size, and ZP measurements (Table 5b). To extend the shelf life, the CA-PP-MP-NP solution was refrigerated (5 °C ± 3 °C).

[0269] CA-encapsulated pea protein nanoparticles (CA-PP-MP-NP) by mango peel cross-linking: Natural and available plant-based substances were investigated for cross-linking. Mangiferin (MGF), a phytochemical found in mangoes, could be extracted from mango peels. Multiple hydroxyl groups in MGF were effective for protein cross-linking. CA-PP-MP-NP had an average size of 652 nm (using DLS) and showed an average zeta potential of -16 mV (Table 5b), indicating reasonable stability. A polydispersity index (PDI) of approximately 0.3 indicated nanoparticles with a nearly uniform size (monomodal distribution). The nanoparticle core size was investigated by TEM (Figure 20B) using negative staining techniques. This showed longitudinally oriented particles with sizes in the range of approximately 200 nm. CA-PP-MP-NP was formulated, fully characterized, and quality control parameters were defined for scale-up and commercial-level production.

[0270]

Table 5-2

[0271] Example 8c - Cross-linking of pea protein by mangiferin and encapsulation of theacrine molecules into theacrine-cross-linked pea protein

[0272] Synthesis of TH-encapsulated pea protein nanoparticles (TH-PP-MP-NP) by mango peel crosslinking: 25 g of water-soluble pea protein (PP), 8 g of mango peel (MP) powder, and 13 g of theacrine (TH) were weighed and transferred to a 1 L Erlenmeyer flask equipped with a stir bar. 500 mL of distilled water was added to the flask, and the contents of the reaction mixture were stirred overnight. The reaction mixture was then stirred overnight, and the insoluble residue was removed by centrifugation / filtration (75 micron size). The TH-encapsulated pea protein nanoparticles (TH-PP-MP-NP) obtained by mango peel crosslinking were characterized by TEM, DLS size, and ZP measurements (Table 5c). To extend the shelf life, the TH-PP-MP-NP solution was refrigerated (5 °C ± 3 °C).

[0273] TH-encapsulated pea protein nanoparticles (TH-PP-MP-NP) by mango peel crosslinking: Naturally available plant-based substances were investigated for crosslinking. Mangiferin (MGF), a phytochemical found in mango, could be extracted from mango peel. Multiple hydroxyl groups in MGF were effective for protein crosslinking. TH-PP-MP-NP had an average size of 563 nm (using DLS) and showed an average zeta potential of -17 mV (Table 5c), indicating reasonable stability. A polydispersity index (PDI) of approximately 0.5 indicated nanoparticles with a slightly broad size distribution. The nanoparticle core size was investigated by TEM (Figure 20C) using negative staining technique. This showed particles with sizes in the range of approximately 100 nm. TH-PP-MP-NP was formulated, fully characterized, and quality control parameters were defined for use in scale-up and commercial-level production.

Table 5-3

[0274] Example 9 - Crosslinking of pea protein by EGCG and encapsulation of ketone molecules in pea protein crosslinked with EGCG.

[0275] KM-encapsulated pea protein nanoparticles synthesized by tea extract cross-linking (KM-PP-TE-NP): For nanoparticle synthesis, a tea extract (TE) solution was prepared. 6 g of Darjeeling loose leaf tea (Table 3) was weighed and transferred to a 1 L Erlenmeyer flask. 300 mL of distilled water was added to the flask and boiled at 100 °C for 10 minutes. Then, the heating switch was turned off and the tea extract solution was cooled to room temperature. The insoluble tea leaf residue from the solution was separated using Whatman paper filtration. The filtered clear dark brown TE solution was used for nanoparticle synthesis. 8 g of water-soluble pea protein (PP) was weighed and transferred to a 2 L Erlenmeyer flask equipped with a stir bar. 500 mL of distilled water was added to the flask and the pea protein solution was stirred for 30 minutes. Then, 3 g of ketone molecule (KM) was weighed and transferred to the flask. The solution was stirred for an additional 30 minutes. 100 mL of the TE solution was added and the reaction mixture was stirred for an additional 2 hours. Then, while stirring vigorously, 170 mL of absolute ethanol (1 / 3 volume of 500 ml of water) was added to the contents of the flask. The reaction mixture was stirred overnight. Then, the KM-encapsulated pea protein nanoparticles (KM-PP-TE-NP) obtained by tea extract cross-linking were characterized by TEM, DLS size, and ZP measurements. The KM content was estimated by GC-MS analysis and the neat KM standard calibration described above. To extend the shelf life, the KM-PP-TE-NP solution was refrigerated (5 °C ± 3 °C). Blank pea protein nanoparticles (PP-TE-NP) by tea extract cross-linking were synthesized following the same procedure without the addition of KM.

[0276] Figure 46 shows the 1H NMR (expanded) of the reaction mixture of epigallocatechin gallate (EGCG) and water-soluble pea protein (PP) in D 2 2O (the arrow indicates the change in protons). 1

[0277] Figure 47 shows the 1H NMR (expanded) of the reaction mixture of epigallocatechin gallate (EGCG), water-soluble pea protein (PP), and KM in D 2 2O. 1 ​​

[0278] The aromatic ring protons of phytochemicals showed distinct changes indicating covalent interaction with proteins. These changes led to cross-linking with protein amino functional groups. In the EGCG proton NMR, after reacting with pea protein, the doublet peak at 6.1 ppm disappeared.

[0279] Tea extracts contain various polyphenols including flavonoids, epigallocatechin gallate (EGCG), and other catechins. Therefore, tea extracts may be effective in cross-linking protein nanoparticles. Using the ethanol desolvation method, KM encapsulated water-soluble pea protein nanoparticles cross-linked with tea extracts (KM-PP-TE-NP) were prepared. After nanoparticle synthesis, insoluble residues were removed by centrifugation / filtration. Ethanol was removed from the formulation by rotary evaporation at 37 °C. The KM-PP-TE-NP size was between 316 - 373 nm, with an average size of 338 nm (using DLS) and showed an average zeta potential of -13 mV (Table 6), indicating stability. A polydispersity index (PDI) of approximately 0.2 indicated nanoparticles with a uniform size (monomodal distribution). Using negative staining techniques, the nanoparticle core size was estimated by TEM (Figure 23). This showed stable particles with sizes in the range of approximately 300 nm. KM-PP-TE-NP was formulated and characterized to define quality control parameters for use in scale-up and commercial-level production. First, small-scale (about 10 mL and 50 mL) nanoparticles were formulated. After establishing characterization and quality control parameters, it was scaled up to about 1 L. KM from iterative batches was utilized for large-scale synthesis. The large-scale batches showed reliable scale-up and characterization parameters.

[0280] GC-MS Analysis of KM-Encapsulated Pea Protein Nanoparticles (KM-PP-TE-NP) by Tea Extract Crosslinking: The amount of KM in the nanoparticle formulation was established using GC-MS analysis. This was found to be quantitative, indicating 100% KM encapsulation. The high KM encapsulation efficiency can be important for the biological delivery of KM. The GC-MS column provided good resolution of the starting material and various glycerides. Quantification of KM could be done by obtaining a calibration curve using pure and neat KM. The curve had good linearity and the analytical concentrations were within the calibration range. GC-MS could be used for quality control of the KM encapsulation process.

[0281] Blank Protein Nanoparticles (PP-TE-NP) by Tea Extract Crosslinking: For comparison, blank pea protein nanoparticles by tea extract crosslinking were synthesized without KM. This showed a size of 362 nm (using DLS) and a zeta potential of -14 mV (Table 6), indicating reasonable stability. A PDI of 0.1 indicates nanoparticles of uniform size (monomodal distribution). The nanoparticle core size was investigated by TEM (Figure 23) using negative staining techniques. This showed good particles with a size in the range of approximately 400 nm. The blank nanoparticles were comparable to the KM-encapsulated nanoparticles with a slightly increased size and potential.

[0282] [Table 6]

[0283] KM-PP-TE-NP and PP-TE-NP Fluorescence Spectroscopy: The fluorescence emission spectra of KM-PP-TE-NP and PP-TE-NP were recorded by excitation at 285 nm corresponding to the tryptophan amino acid. A broad emission peak at 353 nm with a fluorescence intensity of 30 was seen in the emission spectrum of (Figure 25). Changes in the tryptophan microenvironment could be monitored by fluorescence spectroscopy. The fluorescence maximum peaks and intensities of KM-PP-TE-NP and PP-TE-NP were equivalent, indicating that there were no significant changes in the tryptophan region of the pea protein nanoparticles. Since KM is water-soluble and hydrophilic, it did not interact with the hydrophobic tryptophan microenvironment of the pea protein. Fluorescence measurements provided insights into the interaction between the pea protein and KM.

[0284] Example 10 - Crosslinking of Pea Protein by EGCG and Encapsulation of NR Molecules in Pea Protein Crosslinked with EGCG.

[0285] NR-encapsulated pea protein nanoparticles synthesized by tea extract cross-linking (NR-PP-TE-NP): 5 g of Darjeeling loose leaf tea was weighed and transferred to a 1 L conical flask to prepare a tea extract (TE) solution for nanoparticle synthesis. 250 mL of distilled water was added to the flask and boiled at 100 °C for 10 minutes. The heating switch was turned off and the tea extract solution was cooled to room temperature. Insoluble tea leaf residues from the solution were separated using Whatman paper filtration. The filtered clear dark brown TE solution was used for nanoparticle synthesis. 8 g of water-soluble pea protein (PP) was weighed and transferred to a 2 L Erlenmeyer flask equipped with a stir bar. 500 mL of distilled water was added to the flask and the pea protein solution was stirred for 30 minutes. 3 g of NR was weighed and transferred to the flask. The solution was stirred for an additional 30 minutes. An additional 100 mL of TE solution was added and the mixture was stirred for an additional 2 hours. Then, while stirring vigorously, 170 mL of absolute ethanol (1 / 3 volume of 500 ml of water) was added to the contents of the flask. The reaction mixture was then stirred overnight. The NR-encapsulated pea protein nanoparticles (NR-PP-TE-NP) obtained by tea extract cross-linking were characterized by TEM, DLS size, and ZP measurements. The NR content was estimated by GC-MS analysis and the above neat KM standard calibration. To extend the shelf life, the NR-PP-TE-NP solution was refrigerated (5 °C ± 3 °C). Blank pea protein nanoparticles (PP-TE-NP) by tea extract cross-linking were synthesized following the same procedure without the addition of NR.

[0286] Tea extracts contain various polyphenols such as flavonoids, epigallocatechin gallate (EGCG), and other catechins. Therefore, tea extracts may be effective in cross-linking protein nanoparticles. NR-encapsulated water-soluble faba bean protein nanoparticles (NR-PP-TE-NP) using tea extract cross-linking were prepared by the ethanol desolvation method. After nanoparticle synthesis, insoluble residues larger than 75 microns in size were removed by centrifugation / filtration. The size of NR-PP-TE-NP was between 370 and 429 nm, with an average size of 415 nm (using DLS), showing an average zeta potential of -14 mV (Table 7), indicating stability. A polydispersity index (PDI) of approximately 0.2 indicated nanoparticles with a uniform size (monomodal distribution). The negative staining technique was used to estimate the nanoparticle core size by TEM (Figure 24). This showed stable particles with sizes in the range of approximately 400 nm. NR-PP-TE-NP was formulated and fully characterized to define quality control parameters for use in scale-up and commercial-level production. First, small-scale (approximately 10 mL) nanoparticles were formulated. After establishing the characterization and quality control parameters, it was scaled up to approximately 1 L. NR from iterative batches was utilized for large-scale synthesis. The large-scale batches showed reliable scale-up and characterization parameters.

[0287] Blank protein nanoparticles (PP-TE-NP) by tea extract cross-linking: For comparison, blank faba bean protein nanoparticles by tea extract cross-linking were synthesized without NR. This showed a size of 370 nm (using DLS) and a zeta potential of -19 mV (Table 7a), indicating reasonable stability. A PDI of 0.1 indicated nanoparticles with a uniform size (monomodal distribution). The negative staining technique was used to investigate the nanoparticle core size by TEM (Figure 16). This showed good particles with sizes in the range of approximately 300 nm. The blank nanoparticles were comparable to the NR-encapsulated nanoparticles with slightly increased size and potential.

[0288]

Table 7-1

[0289] Example 11 - Crosslinking of Pea Protein by EGCG and Encapsulation of Caffeine Molecules into EGCG - Crosslinked Pea Protein

[0290] CA - encapsulated pea protein nanoparticles were synthesized by tea extract crosslinking (CA - PP - TE - NP): To prepare a tea extract (TE) solution for nanoparticle synthesis, 5 g of Darjeeling loose leaf tea was weighed and transferred to a 1 L conical flask. 250 mL of distilled water was added to the flask and boiled at 100 °C for 10 minutes. The heating switch was turned off and the tea extract solution was cooled to room temperature. Insoluble tea leaf residues from the solution were separated using Whatman paper filtration. The filtered clear dark - brown TE solution was used for nanoparticle synthesis. 20 g of water - soluble pea protein (PP) was weighed and transferred to a 1 L Erlenmeyer flask equipped with a stir bar. 400 mL of distilled water was added to the flask and the pea protein solution was stirred for 30 minutes. 10 g of CA was weighed and transferred to the flask. The solution was stirred for an additional 30 minutes. Further, 100 mL of the TE solution was added and the mixture was stirred overnight. Next, the CA - encapsulated pea protein nanoparticles (CA - PP - TE - NP) obtained by tea extract crosslinking were characterized by TEM, DLS size, and ZP measurements. To extend the shelf life, the CA - PP - TE - NP solution was refrigerated (5 °C ± 3 °C).

[0291] The tea extract contains various polyphenols such as flavonoids, epigallocatechin gallate (EGCG), and other catechins. Therefore, the tea extract may be effective in cross-linking protein nanoparticles. CA-capsulated water-soluble pea protein nanoparticles (CA-PP-TE-NP) using tea extract cross-linking were carried out. After nanoparticle synthesis, insoluble residues larger than 75 microns in size were removed by centrifugation / filtration. CA-PP-TE-NP had an average size of 330 nm and a zeta potential of -13 mV (Table 7b) as measured by (using DLS). A polydispersity index (PDI) of approximately 0.4 indicated nanoparticles with a fairly uniform size (monomodal distribution). The nanoparticle core size was estimated by TEM (Figure 24B) using negative staining techniques. This showed longitudinal particles with sizes in the range of approximately 500 nm. CA-PP-TE-NP was formulated and characterized to define quality control parameters for use in scale-up and commercial-level production. First, small-scale (approximately 10 mL) nanoparticles were formulated. After establishing the property evaluation and quality control parameters, it was scaled up to approximately 500 mL. The large-scale batch showed reliable scale-up and property evaluation parameters.

[0292]

Table 7-2

[0293] Example 12 - Cross-linking of pea protein with EGCG and encapsulation of theacrine molecules into EGCG-cross-linked pea protein

[0294] TH-encapsulated pea protein nanoparticles synthesized by tea extract cross-linking (TH-PP-TE-NP): 5 g of Darjeeling loose leaf tea was weighed and transferred to a 1 L conical flask to prepare a tea extract (TE) solution for nanoparticle synthesis. 250 mL of distilled water was added to the flask and boiled at 100 °C for 10 min. The heating switch was turned off and the tea extract solution was cooled to room temperature. Insoluble tea leaf residues from the solution were separated using Whatman paper filtration. The filtered clear dark brown TE solution was used for nanoparticle synthesis. 20 g of water-soluble pea protein (PP) was weighed and transferred to a 1 L Erlenmeyer flask equipped with a stir bar. 400 mL of distilled water was added to the flask and the pea protein solution was stirred for 30 min. 10 g of TH was weighed and transferred to the flask. The solution was stirred for an additional 30 min. Further, 100 mL of the TE solution was added and the mixture was stirred overnight. Next, the TH-encapsulated pea protein nanoparticles (TH-PP-TE-NP) obtained by tea extract cross-linking were characterized by TEM, DLS size, and ZP measurements. To extend the shelf life, the TH-PP-TE-NP solution was refrigerated (5 °C ± 3 °C).

[0295] Tea extracts contain various polyphenols such as flavonoids, epigallocatechin gallate (EGCG), and other catechins. Therefore, tea extracts may be effective in crosslinking protein nanoparticles. TH-capsulated water-soluble pea protein nanoparticles (TH-PP-TE-NP) using tea extract crosslinking were carried out. After nanoparticle synthesis, insoluble residues larger than 75 microns in size were removed by centrifugation / filtration. TH-PP-TE-NP had an average size of 322 nm (using DLS) and a zeta potential of -12 mV (Table 7c). A polydispersity index (PDI) of approximately 0.4 indicated nanoparticles of fairly uniform size (monomodal distribution). The nanoparticle core size was estimated by TEM (Figure 24C) using negative staining techniques. This showed particles with sizes in the range of approximately 100 nm. TH-PP-TE-NP was formulated and characterized to define quality control parameters for use in scale-up and commercial-level production. First, small-scale (approximately 50 mL) nanoparticles were formulated. After establishing property evaluation and quality control parameters, it was scaled up to approximately 500 mL. The large-scale batch showed reliable scale-up and property evaluation parameters.

[0296]

Table 7-3

[0297] Example 13 - Crosslinking of Pea Protein with Quercetin and Encapsulation of Ketone Molecules into Quercetin-Crosslinked Pea Protein

[0298] Synthesis of Kidney Bean Protein Nanoparticles Encapsulated with KM by Berry Extract Crosslinking (KM-PP-BE-NP): An aqueous solution of blackberry extract (BE) for nanoparticle synthesis was prepared. 100 g of dried purple berry powder was weighed and transferred to a 1 L Erlenmeyer flask. 1000 ml of distilled water was added to the flask, and the contents were boiled at 100 °C for 15 minutes. Then, the heating was stopped, the solution was cooled to room temperature, and the remaining insoluble berry fruit powder residue was allowed to settle. The insoluble residue from the purple solution was separated by decantation and then filtration (using a 75 micron mesh strainer or by centrifugation). The filtered clear purple BE aqueous solution was used for nanoparticle synthesis. 50.0 g of water-soluble kidney bean protein (PP) and 300.0 g of ketone molecule (KM) were weighed and transferred to a 2 L Erlenmeyer flask equipped with a stir bar. 1000 mL of the BE extract aqueous solution was added to the flask, and the contents were boiled at 100 °C for 15 minutes. Then, 333 mL of absolute ethanol was added to the flask with stirring, and the contents of the reaction mixture were stirred overnight. The KM-encapsulated kidney bean protein nanoparticles (KM-PP-BE-NP) obtained by berry extract crosslinking were characterized by TEM, DLS size, and ZP measurements. To extend the shelf life, the KM-PP-BE-NP solution was refrigerated (5 ± 3 °C). Blank kidney bean protein nanoparticles (PP-BE-NP) by berry extract crosslinking were synthesized following the same procedure without the addition of KM.

[0299] Quercetin proton NMR showed peaks equivalent to 7.7, 7.6, 6.9, 6.4, and 6.2 ppm. After reaction with pea protein, the doublet peak at 6.4 ppm decreased significantly (Figures 48 and 49). NMR analysis clearly showed a change in quercetin, indicating a covalent interaction with the protein cross-linking pathway. The aromatic ring protons of the phytochemical showed distinct changes indicating covalent interaction with the protein. These changes led to cross-linking with the protein amino functional groups. Quercetin proton NMR showed peaks equivalent to 7.7, 7.6, 6.9, 6.4, and 6.2 ppm. However, after reaction with pea protein, the doublet peak at 6.4 ppm decreased significantly.

[0300] The blackberry extract containing quercetin was a phytochemical cocktail containing various other active ingredients that could also play a role in the cross-linking and encapsulation processes while adding nutritional supplement value to the formulation.

[0301] Figure 49 shows the 1 1H NMR (expanded) of the quercetin and water-soluble pea protein (PP) reaction mixture in CD3OD / D2O (arrows indicate changes in protons).

[0302] Figure 50 shows 3 the 2 1H NMR (expanded) of the quercetin, water-soluble pea protein (PP), and KM reaction mixture in CD 1 OD / D

[0303] Crosslinking was studied using naturally occurring plant-derived substances from blackberries. Quercetin (QUE), a phytochemical present in the juice fruits, could be extracted from the berry extract. The hydroxyl groups in QUE are effective in crosslinking proteins. Using the ethanol desolvation method, KM-capsulated water-soluble pea protein nanoparticles (KM-PP-BE-NP) with berry extract crosslinking were prepared. Centrifugation / filtration was used to remove the insoluble residue after nanoparticle formation. The KM-PP-BE-NP size between an average of 392 - 474 nm was 433 nm (determined by DLS), and the average zeta potential was -9 mV (Table 8a), showing reasonable stability. A polydispersity index (PDI) of 0.3 suggested nanoparticles of nearly uniform size (monomodal distribution). Transmission electron microscopy (TEM) was used to study the nanoparticle size using the negative staining technique for nanoparticles (Figure 27A). This showed a particle size in the range of approximately 100 - 500 nm. KM-PP-BE-NP was formulated and fully characterized to define quality control parameters for scale-up and commercial-level production. First, the nanoparticles were formulated on a small scale (about 10 mL). After establishing the characterization and quality control parameters, it was successfully scaled up to about 1 L to test large-scale production. The large-scale batch showed reproducible scale-up and characterization parameters. These experiments helped define the quality control parameters for use in scale-up and commercial-level production.

[0304]

Table 8-1

[0305] Example 14 - Crosslinking of Pea Protein with Quercetin and Encapsulation of Caffeine Molecules into Pea Protein Crosslinked with Quercetin

[0306] Synthesis of CA-encapsulated pea protein nanoparticles (CA-PP-BE-NP) by berry extract crosslinking: An aqueous solution of blackberry extract (BE) for nanoparticle synthesis was prepared. 25 g of dried purple berry powder was weighed and transferred to a 1 L Erlenmeyer flask. 500 ml of distilled water was added to the flask, and the contents were boiled at 100 °C for 15 minutes. Then, the heating was stopped, the solution was cooled to room temperature, and the remaining insoluble berry fruit powder residue was allowed to settle. The insoluble residue from the purple solution was separated by decantation and then filtration (using a 75 micron mesh strainer or by centrifugation). The filtered clear purple BE aqueous solution was used for nanoparticle synthesis. 25 g of water-soluble pea protein (PP) and 13 g of caffeine (CA) were weighed and transferred to a 1 L Erlenmeyer flask equipped with a stir bar. 250 mL of the BE extract aqueous solution and 250 mL of distilled water were added to the flask, and the mixture was stirred overnight. The CA-encapsulated pea protein nanoparticles (CA-PP-BE-NP) obtained by berry extract crosslinking were characterized by TEM, DLS size, and ZP measurements. To extend the shelf life, the CA-PP-BE-NP solution was refrigerated (5 ± 3 °C).

[0307] Crosslinking was studied using naturally occurring phytochemicals derived from blackberries. Quercetin (QUE), a phytochemical present in the juice fruits, was extracted from the berry extract. The hydroxyl groups in QUE are effective in crosslinking proteins. Calcium alginate encapsulated water-soluble pea protein nanoparticles (CA-PP-BE-NP) with berry extract crosslinking were prepared. Centrifugation / filtration was used to remove the insoluble residue after nanoparticle formation. The average size of CA-PP-BE-NP was 386 nm (determined by DLS), and the average zeta potential was -14 mV (Table 8b), indicating reasonable stability. A polydispersity index (PDI) of 0.4 suggested nanoparticles of fairly uniform size (monomodal distribution). Transmission electron microscopy (TEM) was used to study the nanoparticle size using the negative staining technique for nanoparticles (Figure 27C). This showed a particle size in the range of approximately 200 nm. CA-PP-BE-NP was formulated and fully characterized to define quality control parameters for scale-up and commercial-level production. First, the nanoparticles were formulated on a small scale (about 10 mL). After establishing the characterization and quality control parameters, it was successfully scaled up to about 500 mL to test large-scale production. The large-scale batch showed reproducible scale-up and characterization parameters. These experiments helped to define the quality control parameters for use in scale-up and commercial-level production.

[0308]

Table 8-2

[0309] Example 15 - Crosslinking of Pea Protein with Quercetin and Encapsulation of Theacrine Molecules into Quercetin-Crosslinked Pea Protein

[0310] Synthesis of TH-encapsulated pea protein nanoparticles (TH-PP-BE-NP) by blackberry extract crosslinking: An aqueous solution of blackberry extract (BE) for nanoparticle synthesis was prepared. 25 g of dried purple berry powder was weighed and transferred to a 1 L Erlenmeyer flask. 500 ml of distilled water was added to the flask, and the contents were boiled at 100 °C for 15 minutes. Then, the heating was stopped, the solution was cooled to room temperature, and the remaining insoluble berry fruit powder residue was allowed to settle. The insoluble residue from the purple solution was separated by decantation and then filtration (using a 75 micron mesh strainer or by centrifugation). The filtered clear purple BE aqueous solution was used for nanoparticle synthesis. 25 g of water-soluble pea protein (PP) and 13 g of theacrine (TH) were weighed and transferred to a 1 L Erlenmeyer flask equipped with a stir bar. 230 mL of the BE extract aqueous solution and 230 mL of distilled water were added to the flask, and the mixture was stirred overnight. The TH-encapsulated pea protein nanoparticles (TH-PP-BE-NP) obtained by blackberry extract crosslinking were characterized by TEM, DLS size, and ZP measurements. To extend the shelf life, the TH-PP-BE-NP solution was refrigerated (5 ± 3 °C).

[0311] Crosslinking was studied using naturally occurring plant-derived substances from blackberries. Quercetin (QUE), a phytochemical present in the juice fruits, could be extracted from the berry extract. The hydroxyl groups in QUE are effective in crosslinking proteins. TH-capsulated water-soluble pea protein nanoparticles (TH-PP-BE-NP) with berry extract crosslinking were prepared. Centrifugation / filtration was used to remove the insoluble residue after nanoparticle generation. The average size of TH-PP-BE-NP was 360 nm (determined by DLS), the average zeta potential was -15 mV (Table 8c), showing reasonable stability. A polydispersity index (PDI) of 0.3 suggested nanoparticles with a uniform size (monomodal distribution). TEM was used to study the nanoparticle size using the negative staining technique for nanoparticles (Figure 27D). This showed a particle size in the range of approximately 100 nm. TH-PP-BE-NP was formulated and fully characterized to define quality control parameters for scale-up and commercial production. First, the nanoparticles were formulated on a small scale (about 10 mL). After establishing the characterization and quality control parameters, it was successfully scaled up to about 500 mL to test large-scale production. The large-scale batch showed reproducible scale-up and characterization parameters. These experiments helped to define quality control parameters for use in scale-up and commercial-level production.

[0312]

Table 8-3

[0313] Example 16 - Crosslinking of Pea Protein and Resveratrol and Encapsulation of Ketone Molecules in Pea Protein Crosslinked with Resveratrol

[0314] Synthesis of KM-encapsulated pea protein nanoparticles (KM-PP-GE-NP) by grape extract cross-linking: 50 g of whole grapes and 1000 mL of distilled water were blended and boiled at 100 °C for 15 minutes. Then, the heating was stopped, the solution was cooled to room temperature, and the remaining insoluble grape residue was allowed to settle. The insoluble residue from the clear solution was separated by decantation followed by filtration (using a 75-micron mesh strainer or by centrifugation). The filtered clear aqueous grape extract solution was used for nanoparticle synthesis. 50.0 g of water-soluble pea protein (PP) and 300.0 g of ketone molecule (KM) were weighed and transferred to a 2 L Erlenmeyer flask equipped with a stir bar. 1000 mL of the aqueous GE extract solution was added to this flask and boiled at 100 °C for 15 minutes. Then, while stirring, 333 mL of absolute ethanol was added to the flask. The contents of the reaction mixture were stirred overnight. The KM-encapsulated pea protein nanoparticles (KM-PP-GE-NP) obtained by grape extract cross-linking were characterized by TEM, DLS size, and ZP measurements. To extend the shelf life, the KM-PP-GE-NP solution was refrigerated (5 °C ± 3 °C). Blank pea protein nanoparticles (PP-BE-NP) by grape extract cross-linking were synthesized following the same procedure without the addition of KM.

[0315] Figure 52 shows CD 3 OD / D 2 of resveratrol and water-soluble pea protein (PP) in the reaction mixture of 1 H NMR (expanded) (arrows indicate proton changes).

[0316] Figure 53 shows resveratrol, water-soluble pea protein (PP), and CD 3 OD / D 2 of the KM reaction mixture in 1 H NMR (expanded).

[0317] In resveratrol proton NMR, the peaks in the 6.8 - 6.7 ppm region showed a significant change in the peak splitting pattern and a slight decrease in the peak at 6.2 ppm after reacting with pea protein (Figs. 51 and 52). NMR analysis clearly showed a distinct change in resveratrol, indicating a covalent interaction with the protein cross - linking pathway. The aromatic ring protons of the phytochemical showed distinct changes indicating covalent interaction with the protein. These changes led to cross - linking with the protein amino functional groups. In resveratrol proton NMR, the peaks in the 6.8 - 6.7 ppm region showed a significant change in the peak splitting pattern and a slight decrease in the peak at 6.2 ppm after reacting with pea protein, indicating a covalent interaction with the protein. Carbon NMR showed corresponding changes.

[0318] Resveratrol is a well - known antioxidant with electroactive hydroxy groups promoted by its planar structure due to double bonds. Also, grape extract containing resveratrol is a phytochemical cocktail containing various other active ingredients that can play a role in the cross - linking and encapsulation processes while adding nutritional supplement value to the formulation.

[0319] The framework of grape extract (GE) was investigated for the nanocapsulation of KM. Grape extract contains flavonoids and resveratrol among other polyphenols. KM-encapsulated pea protein grape extract cross-linked nanoparticles (KM-PP-GE-NP) were prepared by stirring an aqueous solution of GE, pea protein, and KM overnight. KM was absorbed or incorporated into the grape skeleton that forms the nanomatrix. The size of KM-PP-GE-NP was between 499 and 607 nm, the average size was 553 nm (using DLS), and the average zeta potential was determined to be -10 mV (Table 9), showing reasonable stability. A polydispersity index (PDI) of 0.2 indicated that the nanoparticles had a uniform size and showed a unimodal distribution. The size and morphology of the nanoparticles were studied using TEM and the negative staining technique (Figure 27B). A good particle size was shown in the range of about 200 - 600 nm. KM-PP-GE-NP was formulated and fully characterized to establish quality control criteria for scale-up and commercial production. First, the nanoparticles were formulated on a small scale (about 10 mL), and then, after establishing the characterization and quality control parameters, the production was scaled up to about 1 L. For large-scale synthesis, KM from several batches was utilized for large-scale synthesis. The large-scale batches showed reliable scale-up and characterization parameters. These studies helped to define the quality control parameters for use in scale-up and commercial-level production.

[0320]

Table 9

[0321] Example 17 - Encapsulation of Ketone Molecules into Soy Protein Isolate

[0322] Synthesis and Characterization of KM-Encapsulated Soy Protein Isolate Nanoparticles: For the nanocapsulation of KM by the desolvation method, the soy protein isolate (SPI) framework was investigated. Since soy protein was investigated first, glutaraldehyde was used for crosslinking to check nanoparticle formation before using plant-based phytochemicals. However, mango peel, tea, berry, and grape extracts could also be used to crosslink the nanoparticles. KM-loaded soy protein isolate nanoparticles (KM-SPI-NP) were prepared by the ethanol desolvation method. KM-SPI-NP was formulated and fully characterized to define quality control parameters for use in scale-up and commercial-level production. Various KM concentrations were investigated for nanoparticle synthesis. The KM-SPI-NP size was between 151 and 230 nm (using DLS). A polydispersity index (PDI) of approximately 0.2 indicated nanoparticles with a uniform size (monomodal distribution). They showed a zeta potential between 35 and 53 mV and very good stability (Table 10). The core size of the nanoparticles was investigated by TEM (Figure 35) using negative staining techniques.

[0323]

Table 10

[0324] Example 18 - Encapsulation of ketone molecules in okra extract crosslinked with okra extract.

[0325] Synthesis of KM-encapsulated okra extract nanoparticles (KM-OE-NP): 10 g of okra extract (OE) powder and 25 g of ketone molecule (KM) were weighed and transferred to a 1 L Erlenmeyer flask equipped with a stir bar. 500 mL of distilled water was added to the flask, and the contents were stirred for 1 hour. 2 g of ascorbic acid, a preservative, was weighed and transferred to the flask and stirred overnight. The resulting KM-encapsulated okra extract (KM-OE-NP) was characterized by TEM, DLS size, and ZP measurements. The KM content was estimated by GC-MS analysis and neat KM standard calibration. To extend the shelf life, the KM-OE-NP solution was refrigerated (5°C ± 3°C). A blank okra extract (OE) without the addition of KM was utilized for comparison with KM-OE-NP.

[0326] Example 19 - Encapsulation of Ketone Molecule in Okra Extract

[0327] The okra extract (OE) framework was investigated for the nanocapsulation of KM. KM okra extract nanoparticles were prepared by mixing aqueous solutions of OE and KM and stirring overnight. KM was absorbed or embedded in okra mucilage or gel, which is in the form of a nanomatrix. The KM-OE-NP size was 158 nm (using DLS) and showed a zeta potential of -22 mV (Table 11), indicating reasonable stability. A polydispersity index (PDI) of 0.6 indicates nanoparticles of a not very uniform size (departing from a unimodal distribution). The core size of the nanoparticles was investigated by TEM (Figure 31) using negative staining techniques. This showed good particles with sizes in the range of about 100 - 500 nm. KM-OE-NP was formulated and fully characterized to define quality control parameters for use in scale-up and commercial-level production. First, the nanoparticles were formulated on a small scale (about 10 mL). After establishing characterization and quality control parameters, it was scaled up to about 1 L. KM from various batches was utilized for large-scale synthesis. Ascorbic acid was also added as a preservative. Consequently, there were changes in size and zeta potential. The size was 280 nm (using DLS) and showed a zeta potential of -9 mV (Table 11). The large-scale batch showed reliable scale-up and characterization parameters. These experiments helped to define quality control parameters for use in scale-up and commercial-level production.

[0328] Blank okra extract: A blank aqueous okra extract without KM was also characterized for comparison. This showed a size of 82 nm (using DLS) and a zeta potential of -27 mV (Table 11), indicating good stability. A PDI of 1.0 indicates an okra gel nanomatrix of non-uniform size (multimodal distribution). The core size of the okra extract was investigated by TEM (Figure 31) using negative staining techniques. This showed sparse particles with sizes in the range of about 100 nm. The KM-encapsulated okra extract nanoparticles (KM-OE-NP) showed more particles with better polydispersity values compared to the blank okra extract.

[0329]

Table 11

[0330] GC-MS analysis of KM-encapsulated okra extract nanoparticles (KM-OE-NP): Using GC-MS analysis, the amount of KM in the nanoparticle formulation was established. This was found to be quantitative, indicating 100% KM encapsulation. High KM encapsulation efficiency may be important for the biological delivery of KM. The GC-MS column provided good resolution of the starting material and various glycerides. Quantification of KM can be performed by obtaining a calibration curve using glyceryl tributyrate (GTB) standard. The curve should have good linearity and the analytical concentration should be within the calibration range. GC-MS could be used for quality control of the KM encapsulation process.

[0331] Example 20 - Encapsulation of NR molecules in okra extract cross-linked with okra extract.

[0332] Synthesis of NR-encapsulated okra extract nanoparticles (NR-OE-NP): 10 g of okra extract (OE) powder (Table 3) and 25 g of NR were weighed and transferred to a 1 L conical flask equipped with a stir bar. 500 mL of distilled water was added to the flask and the contents were stirred for 1 hour. 2 g of ascorbic acid, a preservative, was weighed and transferred to the flask and stirred overnight. The resulting NR-encapsulated okra extract (NR-OE-NP) was characterized by TEM, DLS size, and ZP measurements. To extend the shelf life, the NR-OE-NP solution was refrigerated (5°C ± 3°C). A blank okra extract (OE) without the addition of NR was utilized for comparison with NR-OE-NP.

[0333] The okra extract (OE) framework was investigated for the nanocapsulation of NR. NR okra extract nanoparticles were prepared by mixing an aqueous solution of OE and NR and stirring overnight. NR was absorbed or embedded in okra mucilage or gel, which is in the form of a nanomatrix. The NR-OE-NP size was 149 nm (using DLS) and showed a zeta potential of -17 mV (Table 12), indicating reasonable stability. A polydispersity index (PDI) of 0.5 indicated nanoparticles of a size that was not very uniform (departing from a unimodal distribution). The core size of the nanoparticles was investigated by TEM (Figure 32) using negative staining techniques. This showed good particles with sizes in the range of about 200 nm. NR-OE-NPs were formulated, fully characterized, and quality control parameters were defined for use in scale-up and commercial-level production. First, the nanoparticles were formulated on a small scale (about 10 mL). After establishing the characterization and quality control parameters, it was scaled up to about 1 L. NR from various batches was utilized for large-scale synthesis. Ascorbic acid was also added as a preservative. However, there were no significant changes in size and zeta potential. The size was 162 nm (using DLS) and showed a zeta potential of -9 mV (Table 8). The large-scale batches showed reliable scale-up and characterization parameters. These experiments helped to define the quality control parameters for use in scale-up and commercial-level production.

[0334] Blank okra extract: A blank aqueous okra extract without NR was also characterized for comparison. This showed a size of 82 nm (using DLS) and a zeta potential of -27 mV (Table 12), indicating good stability. A PDI of 1.0 indicated an okra gel nanomatrix of non-uniform size (multimodal distribution). The core size of the okra extract was investigated by TEM (Figure 32) using negative staining techniques. This showed sparse particles with sizes in the range of about 100 nm. The NR-capsulated okra extract nanoparticles (NR-OE-NP) showed more particles with better polydispersity values compared to the blank okra extract.

[0335]

Table 12

[0336] Example 21 - Folin-Ciocalteu Analysis of Total Phenols in KM-Encapsulated Pea Protein Nanoparticles by Crosslinking with Plant-Based (Mango Peel, Tea, Berry, and Grape Extracts)

[0337] Total phenolic content can be an important component in the beverage, supplement, functional food, food additive, and nutraceutical industries, providing flavor, color, and sensory qualities such as bitterness and astringency during storage, aroma, and the development of turbidity. Phenols can exhibit antioxidant activity due to their extensive conjugated π - electron systems that facilitate the donation of an electron or hydrogen atom from the hydroxyl moiety to free radicals. The oxygen radical absorbance capacity (ORAC) of whole foods, juices, and food additives has been previously utilized as an industry standard for determining the antioxidant strength of these foods. The ORAC method utilizes peroxyl radicals, the most abundant free radicals found in the human body. Long - term consumption of plant - based foods rich in phytochemicals / polyphenolic substances has shown physiological functionality in the human diet and is also associated with a reduced risk of developing chronic diseases induced by oxidative stress. As a result, they have the ability to limit the growth of carcinogenic tumors, reduce the risk of cardiovascular diseases, and possess antibacterial, anti - inflammatory, anti - obesity, anti - diabetic, anti - aging, antispasmodic, and antidiarrheal properties. Furthermore, the energy - enhanced KM within the nanocapsulation platform contains many health - promoting components, including antioxidant polyphenols from mango peel, tea, berries, and grape extracts. Products rich in antioxidants are important as they protect the body's cells from the destructive effects of free radicals that can damage proteins, lipids, and DNA, thereby reducing and preventing the onset of various degenerative diseases such as cancer and coronary heart disease. Phenols provide health benefits through several mechanisms, including the removal of free radicals and the protection and regeneration of other dietary antioxidants (e.g., vitamin E) acting as protectants, as well as their high antioxidant activity and healing properties, including a valuable group of plant components. The total phenolic content was determined using the Folin - Ciocalteu method, which involves reducing the Folin - Ciocalteu reagent with polyphenols present in the nanopharmaceuticals. The total phenolic content was quantified using total gallic acid equivalent (GAE), the most frequently used reference molecule.As shown in Fig. 28, the total phenolic content was 236 mg GAE / g for KM-PP-MP-NP, 127 mg GAE / g for KM-PP-TE-NP, 205 mg GAE / g for KM-OE-NP, 324 mg GAE / g for KM-PP-BE-NP, 275 mg GAE / g for KM-PP-GE-NP, and 12 mg GAE / g for KM-PP-NP.

[0338] Example 22: Cytotoxicity investigation of KM-encapsulated pea protein nanoparticles (KM-PP-TE-NP) by KM and tea extract cross-linking

[0339] Using human aortic endothelial cells (HAECs), the cytotoxicity of KM-encapsulated pea protein nanoparticles (KM-PP-TE-NP) by KM and tea extract cross-linking was investigated. The cell proliferation assay kit used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a tetrazolium dye for measuring cell viability. The MTT assay was used to measure the cell viability for both KM and KM-encapsulated nanoparticles. HAECs after 24, 48, and 72 hours (h) of incubation with KM showed no toxicity over a concentration range of 81 - 1300 μg / mL. This is shown in Fig. 26A. KM-encapsulated pea protein isolate nanoparticles (KM-PP-TE-NP) cross-linked with tea extract were also synthesized. HAECs after 24, 48, and 72 hours of incubation with KM-PPI-NP showed no toxicity over a concentration range of 81 - 650 μg / mL. This is shown in Fig. 26B. The toxicity concentration of KM-encapsulated nanoparticles was slightly lower compared to free KM. From this observation, an improvement in the bioavailability of KM in KM-PP-TE-NP was inferred. The KM concentration in KM-PP-MP-NP was used to plot the concentration values.

[0340] Example 23 - Cytotoxicity investigation of KM-encapsulated pea protein nanoparticles (KM-PP-MP-NP) by KM and mango peel cross-linking

[0341] Cytotoxicity investigation of KM-encapsulated pea protein nanoparticles (KM-PP-MP-NP) by KM and mango peel crosslinking was performed using human aortic endothelial cells (HAEC). The cell proliferation assay kit used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a tetrazolium dye for measuring cell viability. The MTT assay was used to measure cell viability for KM and KM-encapsulated nanoparticles. HAEC after 24, 48, and 72 hours (h) of incubation with KM showed no toxicity over a concentration range of 9 - 142 μg / mL. This is shown in Figure 22A. KM-encapsulated pea protein isolate nanoparticles crosslinked with mango peel (KM-PP-MP-NP) were also synthesized. HAEC after 24, 48, and 72 h of incubation with KM-PPI-NP showed no toxicity over a concentration range of 9 - 71 μg / mL. This is shown in Figure 22B. The toxicity concentration of KM-encapsulated nanoparticles was slightly lower compared to free KM. From this observation, an improvement in the bioavailability of KM in KM-PP-MP-NP was inferred. The KM concentration in KM-PP-MP-NP was used to plot the concentration values.

[0342] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous variations, modifications, and substitutions will occur to those skilled in the art. It should be understood that various alternative forms to the embodiments of the present invention described herein may be used in practicing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be encompassed thereby.

Claims

1. A method for encapsulating multiple small molecules, (a) To obtain plant-derived proteins and one or more phytochemicals, (b) Mixing the plant-derived protein and the one or more phytochemicals with the plurality of small molecules in water to produce a mixture containing a plurality of particles including encapsulated small molecules, The plurality of small molecules include a plurality of ketone molecules, If necessary, the plurality of small molecules (i) Multiple nicotinamide riboside molecules or nicotinamide riboside analogs; (ii) Multiple caffeine molecules; or (iii) Multiple theacrin molecules Methods that further include the above.

2. The method according to claim 1, wherein the plant-derived protein is pea protein, banana protein, okra protein, or legume protein.

3. The method according to claim 1 or 2, wherein the 1 or more phytochemicals include mangiferin, catechin, or quercetin.

4. The aforementioned 1 or more phytochemicals include quercetin, and the weight ratio of the aforementioned 1 or more phytochemicals to the plant-derived protein is between 1:10,000 and 1:40,000, or The aforementioned 1 or more phytochemicals include mangiferin, and the weight ratio of the aforementioned 1 or more phytochemicals to the plant-derived protein is between 1:30 and 1:50, or The aforementioned 1 or more phytochemicals include epigallocatechin gallate, and the weight ratio of the aforementioned 1 or more phytochemicals to the plant-derived protein is between 1:200 and 1:

400. The method according to claim 1 or 2.

5. The method according to claim 1 or 2, wherein the 1 or more phytochemicals include polyphenols, and the polyphenols include one or more of flavonoids and resveratrols.

6. The method according to claim 1 or 2, wherein the weight ratio of the 1 or more phytochemicals to the plant-derived protein is between 1:300 and 1:

500.

7. (c) further comprising mixing the mixture with ethanol, where (c) (i) The volume ratio of the ethanol to the mixture is 1:10 to 1:1; (ii) in a volume ratio of ethanol to the mixture of 1:10 to 1:5; or (iii) The volume ratio of ethanol to the mixture is 1:4 to 1:2 This is carried out by adding the ethanol to the mixture. The method according to claim 1 or 2.

8. The method further includes crosslinking the aforementioned plant chemical substance to the aforementioned plant protein, (i) The plant chemical is crosslinked to a water-soluble plant protein via an imine bond; (ii) The crosslinking is carried out without the addition of aldehydes; or (iii) The crosslinking occurs at one or more functional amino acid groups of the plant protein, The method according to claim 1 or 2.

9. The method according to claim 1, wherein the plurality of small molecules include 1,3-dihydroxypropan-2-yl(R)-3-hydroxybutanoate.

10. The method according to claim 1, wherein at least 50% of the small molecule is 1,3-dihydroxypropan-2-yl(R)-3-hydroxybutanoate.

11. The method according to claim 1, wherein at least 50% of the small molecule comprises a glyceryl skeleton conjugated to 3-hydroxybutanoate.

12. A composition comprising small molecules encapsulated within a plant protein crosslinked with one or more phytochemicals, wherein the plurality of small molecules comprise a plurality of ketone molecules.

13. The composition according to claim 12, wherein the plant protein is pea protein, banana protein, okra protein, or legume protein.

14. The composition according to claim 12 or 13, wherein the 1 or more phytochemicals include mangiferin, catechin, or quercetin.

15. The composition according to claim 12 or 13, wherein the 1 or more phytochemicals include polyphenols, and the polyphenols include one or more of flavonoids and resveratrols.

16. The composition according to claim 12 or 13, wherein the 1 or more phytochemicals are crosslinked to the plant protein via imine bonds.

17. The composition according to claim 12 or 13, wherein the ketone molecule comprises 1,3-dihydroxypropan-2-yl(R)-3-hydroxybutanoate.

18. The composition according to claim 12, wherein the small molecule further comprises (i) a nicotinamide riboside molecule or an analog thereof, (ii) a caffeine molecule, or (iii) a theacrin molecule.

19. The composition according to claim 12, wherein the composition has a phenol content of more than 10 mg GAE / g.

20. The composition according to claim 12, wherein the composition has a phenol content of less than or equal to 350 mg GAE / g.

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