Systems and methods for small molecule encapsulation

Abstract

Disclosed herein are systems and methods for encapsulating small molecules (e.g., ketone molecules) with plant-derived proteins, such as for dietary supplements.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 340,820, filed on May 11, 2022, and U.S. Provisional Application No. 63 / 453,958, filed on March 22, 2023, which are incorporated herein by reference in their entirety. Background Art

[0003] The health and environmental benefits of plant-derived protein foods are widely recognized. In order to meet the growing demand for vegetarian and vegan dietary products, scientists have been working to derive plant-derived protein products to replace animal-derived protein products, such as incorporating dietary supplements into foods to enhance bioavailability. There is an unmet need for cost-effective methods for delivering dietary supplements such as ketones, nicotinamide riboside (NR), caffeine or theophylline without using animal-derived protein products. Summary of the invention

[0004] Disclosed herein is a method for encapsulating multiple small molecules, the method comprising: obtaining a plant-derived protein and one or more phytochemicals; mixing the plant-derived protein and the one or more phytochemicals with the multiple small molecules in water to produce a mixture comprising multiple particles, the particles comprising multiple encapsulated small molecules. The multiple small molecules may include multiple ketone molecules. The multiple small molecules may include multiple nicotinamide riboside molecules or nicotinamide riboside analogs. The multiple small molecules may include multiple caffeine molecules. The multiple small molecules may include multiple theophylline molecules. The plant-derived protein may be from a first source, and the one or more phytochemicals may be from a second source different from the first source. The first source may be from a first plant species, and the second source may be from a second plant species. The plant-derived protein may be pea protein. The plant-derived protein may be water-soluble. The plant-derived protein may be banana protein, okra protein, or bean protein. The bean protein may be soy protein. The phytochemical may be derived from mango, tea, okra, berry, or grape. The phytochemical may be derived from mango peel powder, dry tea leaves, okra extract powder, berry extract, or grape extract. The one or more phytochemicals may include mangiferin, catechin or quercetin. The one or more phytochemicals may include quercetin, and the weight ratio of the one or more phytochemicals to the protein of plant origin may be between 1:10000 and 1:40000. The one or more phytochemicals may include quercetin, and the weight ratio of the one or more phytochemicals to the protein of plant origin may be between 1:22000 and 1:23000. The one or more phytochemicals may include mangiferin, and the weight ratio of the one or more phytochemicals to the protein of plant origin may be between 1:20 and 1:60. The one or more phytochemicals may include mangiferin, and the weight ratio of the one or more phytochemicals to the protein of plant origin may be between 1:30 and 1:50. The one or more phytochemicals may include catechin, wherein the catechin is epigallocatechin gallate. The weight ratio of epigallocatechin gallate to the protein of plant origin may be between 1:200 and 1:400. The one or more phytochemicals may include polyphenols. The polyphenols may include one or more of flavonoids and resveratrol. The weight ratio of the one or more phytochemicals to the plant-derived protein may be between 1:300 and 1:500. The mixture may include a dry weight ratio of multiple ketone molecules to the plant-derived protein between 1:1 and 1:5. The mixture may include a weight ratio of the plant-derived protein to water between 1:50 and 1:100 or between 1:10 and 1:100.

[0005] The method may further include mixing the mixture with ethanol. Mixing the mixture with ethanol to produce particles containing ketone molecules or NR molecules can be performed by adding ethanol to the soluble portion at a volume ratio of ethanol to the soluble portion of 1:10 to 1:1. Mixing the mixture with ethanol to produce particles containing ketone molecules or NR molecules can be performed by adding ethanol to the soluble portion at a volume ratio of ethanol to the soluble portion of 1:10 to 1:5. Mixing the mixture with ethanol to produce particles containing ketone or NR molecules can be performed by adding ethanol to the soluble portion at a volume ratio of ethanol to the soluble portion of 1:4 to 1:2. The method may include removing ethanol. Removing ethanol may include evaporating ethanol at 25°C to 40°C.

[0006] The particles may have an average diameter greater than 80 nm. The particles may have an average diameter less than 700 nm. The particles may have an average diameter between 100 nm and 500 nm. The particles may have an average diameter between 300 nm and 500 nm. Removing ethanol may include evaporating ethanol at 25° C. to 40° C.

[0007] The method may further include cross-linking the phytochemical to the plant protein. The phytochemical may be cross-linked to the plant protein via an imine linkage. The cross-linking may be performed without the addition of an aldehyde. The aldehyde may be glutaraldehyde. The cross-linking may occur at one or more functional amino acid groups of the plant protein. The functional amino acid group may comprise a primary amino group. The functional amino acid group may comprise lysine.

[0008] The mixture may comprise a soluble portion and an insoluble portion. The method may comprise separating the soluble portion from the insoluble portion. The soluble portion may be mixed with ethanol. Separating the soluble portion from the insoluble portion may comprise filtering the insoluble portion from the soluble portion. A plurality of small molecules may comprise (R)-1,3 dihydroxypropane-2-yl 3-hydroxybutyrate. At least 50% of the small molecules may be (R)-1,3 dihydroxypropane-2-yl 3-hydroxybutyrate. At least 50% of the small molecules may comprise a glyceryl backbone conjugated to 3-hydroxybutyrate.

[0009] In some embodiments, a plurality of small molecules may comprise nicotinamide riboside. In some embodiments, at least 50% of the small molecules are nicotinamide riboside. In some embodiments, a plurality of small molecules may comprise caffeine. In some embodiments, at least 50% of the small molecules are caffeine. In some embodiments, a plurality of small molecules may comprise theophylline. In some embodiments, at least 50% of the small molecules are theophylline.

[0010] The mixing may be performed at 20° C. to 30° C. The mixing may be performed at a pH of 3.5 to 11.0. The mixing may be performed at a pH of 3.5 to 7.0, 5.0 to 9.0, or 7.0 to 11.0.

[0011] The protein of vegetable origin may be water soluble. The protein of vegetable origin may be at least 95% water soluble. The protein of vegetable origin may be at least 98% water soluble. The protein of vegetable origin may be at least 99% water soluble.

[0012] Disclosed herein is a nanoencapsulation composition formed by the above method. Disclosed herein is a composition comprising a small molecule encapsulated in a plant protein. The plant protein may be pea protein. The plant protein may be okra protein, banana protein, or soy protein. The plant protein may be cross-linked with one or more phytochemicals. The one or more phytochemicals may be derived from mango, tea, okra, berries, or grapes. The one or more phytochemicals may be derived from mango peel powder, dry leaves, okra extract powder, berry extract, or grape extract. The one or more phytochemicals may include mangiferin, catechins, or quercetin. The catechins may be epigallocatechin gallate. The one or more phytochemicals may include polyphenols. The polyphenols may include one or more of flavonoids and resveratrol. The one or more phytochemicals may be cross-linked to the plant protein via an imine linkage. The cross-linking may occur at a functional amino acid group of the plant protein. The functional amino acid group may include a primary amine. The small molecule may include a ketone molecule. The ketone molecule may include (R)-3-hydroxybutyric acid 1,3 dihydroxypropane-2-yl ester. The small molecule may include a nicotinamide riboside molecule or an analog thereof. The small molecule may include a caffeine molecule. The small molecule may include a theophylline molecule. The particles may have an average diameter greater than 80 nm. The particles may have an average diameter less than 700 nm. The particles may have an average diameter between 100 nm and 500 nm. The particles may have a diameter between 300 nm and 500 nm. The composition may have a phenol content greater than 10 mg GAE / g. The composition may have a phenol content less than or equal to 350 mg GAE / g. The composition may have a phenol content between 10 and 500 mg GAE / g. The composition may have a phenol content between 100 and 400 mg GAE / g. The composition may have a phenol content between 200 and 300 mg GAE / g. In some embodiments, the composition does not include glutaraldehyde.

[0013] Incorporation by reference

[0014] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference 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

[0015] Various features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description which sets forth exemplary embodiments in which the principles of the present invention are utilized, and the accompanying drawings, in which:

[0016] Figure 1 The UV-Visible spectrum of 2 mg / mL of ketone molecules is shown.

[0017] Figure 2 The UV-visible absorption spectrum of 0.2 mg / mL NR is shown.

[0018] Figure 3 Shows the ketone molecule in D2O 1 H NMR.

[0019] Figure 4 The NR molecules in D2O 1 H NMR.

[0020] Figure 5 Shows the ketone molecule in D2O 13 C NMR.

[0021] Figure 6 Shows the ketone molecule in D2O 13 C NMR.

[0022] Figure 7 Shows the ketone molecule in D2O 13 C NMR.

[0023] Figure 8 The NR molecules in D2O 13 C NMR.

[0024] Fig. 9 An electrospray ionization mass spectrometry (ESI-MS) spectrum of a ketone molecule is shown.

[0025] Fig.10 The electrospray ionization mass spectrometry (ESI-MS) spectrum of the NR molecule is shown.

[0026] Fig.11 A diagram showing GC-MS analysis of ketone molecules.

[0027] Fig.12 A diagram showing LC-MS analysis of ketone molecules.

[0028] Fig.13 The CD spectrum (wavelength versus ellipticity) of a ketone molecule is shown, where the positive peak indicates the R / D configuration of the ketone molecule.

[0029] Fig.14A The NR molecule CD spectrum (wavelength versus ellipticity) is shown, where the negative peak indicates the S / L configuration of the NR molecule.

[0030] Fig. 14B A graph showing LC-MS analysis of the caffeine (CA) molecule is shown.

[0031] Fig. 14C A graph showing LC-MS analysis of theophylline (TH) molecule.

[0032] Fig.15 A schematic diagram of the preparation of KM-protein nanoparticles by the desolvation method is shown.

[0033] Fig.16 A schematic diagram of the preparation of NR-protein nanoparticles by the desolvation method is shown.

[0034] Fig.17A The UV-visible absorption spectrum of 0.2 mg / mL water-soluble pea protein is shown.

[0035] Fig. 17B The fluorescence emission spectrum of 0.2 mg / mL water-soluble pea protein under 285 nm excitation is shown.

[0036] Fig.18 MALDI-MS analysis of water-soluble pea proteins is shown.

[0037] Fig.19 Shown are TEM images of ketone molecules encapsulated by pea protein nanoparticles produced by ethanol desolvation, cross-linked with mango peel-derived phytochemicals.

[0038] Fig. 20A Shown are TEM images of NR molecules encapsulated by pea protein nanoparticles produced by ethanol desolvation, cross-linked with mango peel-derived phytochemicals.

[0039] Fig. 20B Shown are TEM images of CA molecules encapsulated by pea protein nanoparticles cross-linked with mango peel-derived phytochemicals.

[0040] Fig. 20C Shown are TEM images of TH molecules encapsulated by pea protein nanoparticles cross-linked with mango peel-derived phytochemicals.

[0041] Fig.21 The fluorescence emission spectra of KM-PP-MP-NPs and PP-MP-NPs under 285 nm excitation are shown.

[0042] Fig.22A Cell viability of human aortic endothelial cells (HAEC) after 24, 48 and 72 hours of incubation with KM is shown. Controls were untreated cells.

[0043] Fig. 22B Cell viability of human aortic endothelial cells (HAEC) after incubation with KM-encapsulated pea protein nanoparticles (KM-PP-MP-NPs) with mango peel crosslinking for 24, 48, and 72 hours is shown. The KM concentration in the KM-PP-MP-NPs was used for plotting. The control was untreated cells.

[0044] Fig.23 TEM images of KM-encapsulated pea protein nanoparticles (KM-PP-TE-NPs) obtained by ethanol desolvation method and tea extract cross-linking are shown.

[0045] Fig.24A TEM images of NR encapsulated pea protein nanoparticles (NR-PP-TE-NPs) obtained by ethanol desolvation method and tea extract cross-linking are shown.

[0046] Fig. 24B TEM images of CA encapsulated pea protein nanoparticles (CA-PP-TE-NPs) obtained by cross-linking with tea extract are shown.

[0047] Fig.24C TEM images of TH-encapsulated pea protein nanoparticles (TH-PP-TE-NPs) obtained by cross-linking of tea extract are shown.

[0048] Fig.25 The fluorescence emission spectra of KM-PP-TE-NPs and PP-TE-NPs under 285 nm excitation are shown.

[0049] Fig.26A Cell viability of human aortic endothelial cells (HAEC) after 24, 48 and 72 hours of incubation with KM is shown. Controls were untreated cells.

[0050] Fig.26B Cell viability of human aortic endothelial cells (HAEC) after incubation with KM-encapsulated pea protein nanoparticles (KM-PP-TE-NPs) cross-linked with tea extract for 24, 48 and 72 hours is shown. The KM concentration in KM-PP-MP-NPs was used for plotting. The control was untreated cells.

[0051] Fig.27A TEM images of KM-encapsulated pea protein nanoparticles (KM-PP-BE-NPs) obtained by cross-linking with berry extracts are shown.

[0052] Fig.27B TEM images of KM-encapsulated pea protein nanoparticles (KM-PP-GE-NPs) obtained by grape extract cross-linking are shown.

[0053] Fig.27C TEM images of CA encapsulated pea protein nanoparticles (CA-PP-BE-NPs) obtained by cross-linking with berry extracts are shown.

[0054] Fig.27D TEM images of TH-encapsulated pea protein nanoparticles (TH-PP-BE-NPs) obtained by cross-linking with berry extracts are shown.

[0055] Fig.28 The total polyphenol content of plant-based cross-linked pea protein nanoparticles encapsulating KM is shown. Gallic acid standard curve (a) and total polyphenol content determined by the Folin-Ciocalteu method (b) Bars represent the mean ± SEM of three independent experiments performed in duplicate. GAE = Gallic acid equivalent

[0056] Fig.29 Shown is the MALDI-MS analysis of okra mucus.

[0057] Fig.30 The UV-visible absorption spectrum of okra mucilage is shown.

[0058] Fig.31 TEM images of okra extract nanoparticles encapsulating KM (KM-OE-NPs) are shown.

[0059] Fig.32 TEM images of okra extract nanoparticles encapsulating NR (NR-OE-NPs) are shown.

[0060] Fig.33 The UV-visible absorption spectrum of water-soluble soy protein isolate (SPI) (0.2 mg / mL) is shown.

[0061] Fig.34 Soy protein isolate (SPI) MALDI-MS analysis is shown.

[0062] Fig.35 Shown are TEM images of KM-encapsulated soy protein isolate nanoparticles (KM-SPI-NPs) obtained by ethanol desolvation method.

[0063] Fig.36 The synthesis of pea protein KM nanoparticles using a biocompatible plant-based cross-linker is shown.

[0064] Fig.37TEM images of pea protein nanoparticles encapsulating KM obtained by ethanol desolvation method and plant-based cross-linking (α-mangiferin from mango peel extract, b-EGCG from tea extract, c-quercetin from blackberry extract, and d-resveratrol from grape extract) are shown.

[0065] Fig.38 The generation of a plant protein-phytochemical platform through the interaction of phytochemicals with protein amino side chains is shown.

[0066] Fig.39 Ketone molecules bound to plant-derived proteins and phytochemicals are shown.

[0067] Fig.40 Shows the KM in D2O 1 H NMR.

[0068] Fig.41 The results show that water-soluble pea protein (PP) in D2O 1 H NMR.

[0069] Fig.42 It shows that mangiferin in CD3OD 1 H NMR.

[0070] Fig.43 The results show that the reaction mixture of mangiferin and water-soluble pea protein (PP) in CD3OD / D2O 1 H NMR (expanded) (arrows indicate proton changes).

[0071] Fig.44 The results show that mangiferin, water-soluble pea protein (PP) and KM reaction mixture in CD3OD / D2O 1 H NMR (extended).

[0072] Fig.45 The results show that epigallocatechin gallate (EGCG) in D2O 1 HNMR.

[0073] Fig.46 The results show that the reaction mixture of epigallocatechin gallate (EGCG) and water-soluble pea protein (PP) in D2O 1 H NMR (expanded) (arrows indicate proton changes).

[0074] Fig.47 1H NMR (expanded) of the reaction mixture of epigallocatechin gallate (EGCG), water-soluble pea protein (PP) and KM in D2O is shown.

[0075] Fig.48 The results showed that quercetin in CD3OD 1 H NMR.

[0076] Fig.49 H NMR (expanded) of a reaction mixture of quercetin and water-soluble pea protein (PP) in CD3OD / D2O is shown (arrows indicate changes in protons).

[0077] Fig.50 The results show the reaction mixture of quercetin, water-soluble pea protein (PP) and KM in CD3OD / D2O. 1 H NMR (extended).

[0078] Fig.51 The results showed that resveratrol in CD3OD 1 H NMR.

[0079] Fig.52 The results show that the reaction mixture of resveratrol and water-soluble pea protein (PP) in CD3OD / D2O 1 H NMR (expanded) (arrows indicate proton changes).

[0080] Fig.53 The results show the reaction mixture of resveratrol, water-soluble pea protein (PP) and KM in CD3OD / D2O. 1 HNMR (extended).

[0081] Fig.54 The amino acid composition of pea protein is shown (grams / 100 g protein).

[0082] Fig.55 The amino acid composition of okra extract is shown (grams / 100 g protein).

[0083] Fig.56 The amino acid composition of soy protein isolate is shown (grams / 100 g protein).

[0084] Fig.57 A comparison of the amino acid composition (grams / 100 g protein) of pea protein, okra extract, and soy protein isolate is shown.

[0085] Fig.58 Shown is the polyphenol composition profile (mg / g dry matter) of mango peel powder obtained from mango fruits.

[0086] Fig.59 The polyphenol composition distribution of black tea leaves is shown (mg / g dry matter).

[0087] Fig.60The anthocyanin and non-anthocyanin phenolic compound composition (μg / 100 g dry matter) of blackberry extract (BE) is shown.

[0088] Fig.61 The polyphenol composition distribution of grape extracts is shown (mg / g dry matter). DETAILED DESCRIPTION

[0089] Plant-based proteins are biocompatible, economically viable, and generally safe to use as nutraceuticals or smart foods / drugs. Disclosed herein are systems and methods for encapsulating ketone molecules (KM), nicotinamide riboside (NR) molecules, and their analogs to enhance stability, enhance biocompatibility, and convert these types of food supplements / nutraceuticals into biocompatible, targeted, and cell-specific in vivo. Disclosed herein are methods and systems 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 to the human body. Also disclosed herein are methods and systems for delivering NR molecules and their analogs. Also disclosed herein are similar systems suitable for encapsulating other small molecules.

[0090] As used herein, the term "small molecule" refers to a molecule less than or equal to 900 Daltons.

[0091] As used herein, the term "ketone molecule" refers to a molecule having the structure of the following compound I, wherein R 1 , R 2 and R 3 can be H or -C4H6O2, and R 1 , R 2 and R 3 At least one of them is -C4H6O2

[0092]

[0093] Compound I may comprise any one of the following compounds II-V.

[0094]

[0095]

[0096] In some embodiments, -C4H6O2 is

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

[0098]

[0099] The analog of the nicotinamide riboside molecule may include any one of the following compounds VII-IX.

[0100]

[0101]

[0102] The term "caffeine" as used herein refers to a molecule having the structure of Compound X below.

[0103]

[0104] The term "theophylline" as used herein refers to a molecule having the structure of the following compound XI.

[0105]

[0106] The term "multiple ketone molecules" as described herein can refer to any multiple ketone molecules. In some cases, the multiple ketone molecules are at least 70% of compound II. The multiple ketone molecules can be greater than or equal to 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% of compound II. The multiple ketone molecules can be less than or equal to 50%, 40%, 30%, 20%, 10%, 5% or 1% of compound III, compound IV, or compound V.

[0107] The molar ratio of the mixture of Compound II and Compound III-V in the solution may be between 80:20 and 99:1. The molar ratio of the mixture of Compound II and Compound III-V in the solution may be greater than or equal to about 80:20, 85:15, 90:10, 95:5, or 99:1. The molar ratio of the mixture of Compound II and Compound III-V in the solution may be less than or equal to 99:1, 95:5, 90:10, 85:15, or 80:20.

[0108] As used herein, the term "plurality of nicotinamide riboside molecules" may refer to any plurality of nicotinamide riboside molecules. In some cases, the plurality of NR molecules may be nicotinamide riboside hydrogen maleate. In some cases, the plurality of NR molecules may be nicotinamide riboside chloride. In some cases, the plurality of NR molecules are at least 70% of compound VI. The plurality of NR molecules may be greater than or equal to 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% of compound VI. The plurality of NR molecules may be less than or equal to 50%, 40%, 30%, 20%, 10%, 5% or 1% of compound VII, compound VIII, or compound IX.

[0109] As used herein, the term "encapsulation" refers to the incorporation of small molecules (such as ketone, NR, caffeine or theophylline molecules) into cross-linked particles, even if the small molecules are not completely surrounded by plant-derived proteins or other non-ketone molecules.

[0110] One embodiment of the present invention encapsulates ketone, NR, caffeine or theophylline molecules in plant-based proteins from peas, okra or bananas. Plant-based crosslinkers can be used to encapsulate ketone, NR, caffeine or theophylline molecules in plant-based proteins from peas, okra or bananas, and the plant-based crosslinkers include various polyphenols, flavonoids, xanthonoids and functionalized glucose moieties. The methods and systems disclosed herein can utilize plant-based proteins and phytochemical ingredients from peas, okra, bananas, mangoes, tea leaves, berries and grapes to produce particles, and the particles include plant-based proteins loaded with ketone, NR, caffeine or theophylline molecules. The methods and systems disclosed herein can be used to produce particles, and the particles provide fast and sustainable energy and a rich set of key nutrients, including potassium, dietary fiber, polyunsaturated fats, essential amino acids, vitamin B6, vitamin B12, magnesium, potassium, fiber and protein.

[0111] Fiber from peas, bananas, and okra can contribute to gastrointestinal function and health. The amylose content in such plant-based protein extracts can contribute to lowering the glycemic index. In addition, the hydrolyzates of pea protein-encapsulated ketones, NR, caffeine, or theophylline molecules can produce biologically active peptides, such as peptides that can have angiotensin I converting enzyme inhibitor activity and antioxidant activity. The plant-derived protein particles described herein can provide polyphenols with antioxidant, anticancer, or hypocholesterolemia activity. Plant-derived protein-encapsulated ketone particles can provide prebiotic effects in the large intestine, such as prebiotic effects from galacto-oligosaccharides.

[0112] Ketone molecule (KM)

[0113] Disclosed herein are systems and methods for encapsulating ketone molecules within plant-derived proteins cross-linked with phytochemicals. In some embodiments, the ketone molecules are bound to the plant-derived proteins and phytochemicals via hydrogen bonds, such as those found in Fig.39 It can be seen in.

[0114] Shown below is the structure of the ketone molecule (KM) and the corresponding Fischer esterification scheme involving dehydration of the parent carboxylic acid [(R)-3-hydroxybutyric acid] and the corresponding alcohol (glycerol):

[0115] Fischer esterification scheme

[0116]

[0117] The parent alcohol glycerol can contain primary and secondary alcohol groups. Various primary and secondary glycerol mono- and di- and triglyceride reaction products are possible via the Fischer esterification protocol (below). The ester functional group has the possibility of conformational isomerism (cis / trans). Carboxylic acid (3-hydroxybutyric acid) is a chiral compound with the possibility of two enantiomers (D and L).

[0118] Fischer esterification products:

[0119]

[0120] Nicotinamide riboside molecule

[0121] Nicotinamide riboside (NR) is a precursor to nicotinamide adenine dinucleotide (NAD+). NAD+ is an essential coenzyme involved in various metabolic pathways, and elevated NAD+ levels in the body can provide health benefits.

[0122] Disclosed herein are systems and methods for encapsulating NR molecules within plant-derived proteins cross-linked with phytochemicals.

[0123] The structures of NR and NR analogs are shown below:

[0124]

[0125] caffeine

[0126] Caffeine (CA) is a central nervous system (CNS) stimulant. It belongs to the methylxanthine class of purines. It increases alertness and attention performance. Coffee beans are the main source of CA, and it tastes bitter. CA is classified as GRAS by the US FDA. Up to 400 mg of caffeine per day is safe. The toxic dose for adults is more than 10 grams per day.

[0127] Disclosed herein are systems and methods for encapsulating CA molecules within plant-derived proteins cross-linked with phytochemicals.

[0128] The structure of a CA is as follows:

[0129]

[0130] Theophylline

[0131] Theophylline (TH) is a purine alkaloid similar in structure to caffeine. It improves mood, energy, and concentration. Theobroma cacao (tropical rainforest tree) and Chinese tea (kuchas) are the main sources of TH. It affects adenosine signaling similar to caffeine. It is clinically safe and has a non-habituating effect in humans. Daily use of up to 300 mg / day of TH is safe.

[0132] Disclosed herein are systems and methods for encapsulating TH molecules within plant-derived proteins cross-linked with phytochemicals.

[0133] The structure of TH is as follows:

[0134]

[0135] Phytochemicals used to cross-link plant-derived proteins:

[0136] Cross-linking agents can be beneficial for the synthesis of protein nanoparticles encapsulating ketone molecules (KM), NR, caffeine or theophylline. Cross-linking agents cross-link protein amino groups to form denser particles.

[0137] A common chemical cross-linker used in the synthesis of protein nanoparticles is glutaraldehyde. It is an effective cross-linker that imparts stability and sustained drug release to nanoparticles. Although effective, it can produce measurable systemic toxicity in humans, especially when used for extended periods of time. In the absence of alternative non-toxic cross-linkers, the food, beverage, and pharmaceutical industries have continued to use lower concentrations of glutaraldehyde, despite persistent concerns about long-term systemic toxicity with irreversible adverse toxic effects on human health.

[0138] Biocompatible and nontoxic plant-based materials are needed as effective and natural cross-linkers for protein nanoparticle formulations ( Fig.38 ). Under oxidative conditions, polyphenols (including mangiferin, epigallocatechin gallate (EGCG), quercetin, and resveratrol) interact with amino groups of pea proteins to produce covalently bound crosslinks, resulting in bioconjugation of various polyphenols to the protein network. In the present invention, Fig.36 As shown, the hydroxyl and oxo groups on polyphenols can cross-link proteins. Plant-based materials including mangiferin (MGF), catechins, epigallocatechin gallate (EGCG), quercetin (QUE), and resveratrol (RESV) can be used to synthesize ketone molecules (KM) ( Fig.23 ), NR molecules ( Fig.24A )、CA molecule( Fig. 20B and Fig. 24B ) and TH molecules ( Fig. 20C and Fig.24C ) for effective cross-linking and encapsulation.

[0139] Disclosed herein are plant-based materials for effective crosslinking and encapsulation. Naturally available plant-based materials (including mangiferin, EGCG, quercetin and resveratrol) are used for KM ( Fig.37), NR, CA and TH. The cross-linking agent can be bound to the protein amino groups to form particles. The common chemical cross-linking agent used for protein nanoparticle synthesis is glutaraldehyde. Disclosed herein are methods and systems for cross-linking plant-derived proteins in the absence of glutaraldehyde. Disclosed herein are methods and products that do not include glutaraldehyde.

[0140] Disclosed herein are biocompatible, plant-based cross-linking agents, including naturally available cross-linking agents from tea (epigallocatechin gallate; EGCG), mango (mangiferin; MGF and various families of polyphenols and xanthones), berries (quercetin; Que), and grapes (resveratrol; RESV).

[0141] The main phytochemical in mango peel extract is mangiferin, which can be found in Fig.58 Mangiferin, a xanthonoid, is a glucoside of mangiferin and a natural phenolic compound. Mangiferin can be isolated from the leaves and bark of Mangifera indica (Mango). It can also be extracted from mango peel (MP) and kernel.

[0142] Catechins, a natural phenol, belong to the flavonoid family. It is found in tea and fruits and is an antioxidant.

[0143] The main phytochemical in tea extract is epigallocatechin gallate (EGCG), which can be found in Fig.59 EGCG is a catechin formed by the esterification of gallic acid and epigallocatechin. Polyphenols such as EGCG and catechin are found in large quantities in tea leaves.

[0144] The main phytochemical in blackberry extract is quercetin, which can be found in Fig.60 Quercetin, a polyphenol belonging to the flavonoid group, is widely found in fruits and vegetables. Quercetin is the most abundant polyphenol in blackberry extract.

[0145] The main phytochemical in grape extract is trans-resveratrol, which can be found in Fig.61 Resveratrol, a stilbene-based plant phenol, is found in grape extracts. Resveratrol (RESV) is a stilbene, a form of natural phenol and a phytoalexin found primarily in grape skins.

[0146] The strongly acidic phenolic functional groups found in these plant-derived phytochemicals effectively cross-link with amino groups in proteins. Fig.38 The generation of a plant protein-polyphenol platform through the interaction of polyphenolic compounds with protein amino side chains is shown in .

[0147]

[0148] The interaction of phytochemicals with water-soluble pea protein was investigated using nuclear magnetic resonance (NMR) spectroscopy. Mangiferin, epigallocatechin gallate (EGCG), quercetin, and resveratrol were reacted with pea protein separately to monitor the changes in phytochemicals and pea protein. NMR analysis showed that different changes in phytochemicals indicated covalent interactions with protein ( Figures 41-43 , 45-46, 48-49 and 51-52). KM was then added to the reaction mixture and further NMR analysis was performed. In the presence of KM, there was no change in phytochemicals or proteins, also indicating the stability of KM in their presence ( Figure 41-53 ). When plant extracts (mango peel, tea, blackberry or grape extracts) are used for KM nanoparticle synthesis, it should be noted that the corresponding plant extracts are mixtures of phytochemicals containing various other active ingredients that can also play a role in the cross-linking and encapsulation process while adding nutritional value to the formulation.

[0149] Under oxidative conditions, polyphenols react with protein amino groups (forming crosslinks and creating networks). The crosslinking process is made possible by the reaction of o-quinones with proteins to form CN or CS bonds ( Fig.38 ). Plant proteins such as pea and soy have a large number of lysine amino acid residues. These contain primary amino groups for cross-linking with phytochemicals. Plant-based materials are conjugated to the primary (ε-) amino groups of lysine residues to produce a protein-polyphenol cross-linking platform. The sulfur-containing amino acid cysteine ​​is present in small amounts in pea and soy proteins. Phytochemicals also cross-link proteins by conjugating to thiol groups. Pea protein-mangiferin and pea protein-EGCG cross-linking products are shown below.

[0150] The structure of mangiferin cross-linked to pea protein can be seen below on the left. The structure of EGCG cross-linked to pea protein can be seen below on the right.

[0151]

[0152] Protein from plant sources:

[0153] The protein described herein may be derived from plants, such as beans, okra and peas. The bean protein may be soy protein. The plant-derived protein may be water-soluble. The water-soluble protein may be a hydrolyzed plant protein. Fig.57 A comparison of the amino acid composition of pea protein, okra extract, and soy protein isolate is shown.

[0154] Table 1. Comparison of amino acid composition of water-soluble pea protein (PP), soy protein isolate (SPI) and okra extract (OE) proteins

[0155]

[0156] Methionine 0.9 1.2 2.2 Phenylalanine 4.6 4.9 4.9 Proline 4.0 6.4 5.0 Serine 5.7 5.2 5.o Threonine 4.0 3.9 2.9 Tryptophan 0.4 1.3 2.0 Tyrosine 3.7 3.1 4.0 Valine 3.9 3.9 4.3

[0157] Water-soluble pea protein can be derived from pea protein. Pea protein can be refined using enzymatic digestion, filtration or spray drying. The protein content of pea protein can be >85%. Pea protein can exist as globulin (65-80%) and can be composed of legumin, pea globulin and a small contribution from convicilin. Pea protein can contain less than 7% moisture, less than 7% ash and less than 7% crude fiber. Pea protein can have a pH of 5.0 to 6.0 when dissolved in water. It can be Fig.54 Find the amino acid composition of pea protein in.

[0158] Okra extract (Abelmoschus esculentus L.) may 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 mucus or gel from the pod is a natural polysaccharide and is composed of D-galactose, L-rhamnose, and L-galacturonic acid. Okra extract can be extracted in water and forms a gelatinous viscous solution. It can be Fig.55 Find the amino acid composition of okra in.

[0159] Soy protein isolate (SPI) is derived from soybeans. Soy protein isolate may consist of β-conglycinin, glycinin, and lipophilic proteins. Soy protein may have a protein content of 92% (on a dry basis). In addition, it may contain 6% moisture, 4.1% ash, 0.8% fat (PE extract), 0.25% crude fiber (crude), 0.15% calcium, 0.8% phosphorus, 1.3% sodium, 0.05% potassium, and may show a pH of 7.1 (water slurry). It may be Fig.56 Find the amino acid profile of soy protein isolate in.

[0160] Reagent test kit

[0161] The kit may include one or more containers and instructions for use containing one or more components provided in the present disclosure. Specifically, such kits may include one or more compositions described herein, and instructions describing the intended application and appropriate use and / or disposal of these compositions. The kit may contain components in appropriate concentrations or amounts for running various experiments.

[0162] Methods of making nanoparticles

[0163] Disclosed herein are systems and methods for producing nanoparticles comprising plant-derived proteins cross-linked with one or more phytochemicals as described herein. The nanoparticles can encapsulate one or more small molecules, such as ketone molecules, nicotinamide riboside molecules, caffeine molecules, and / or theophylline molecules. In some embodiments, plant-derived proteins, powders containing phytochemicals, and ketone molecules are combined in water. In some embodiments, plant-derived proteins, powders containing phytochemicals, and NR molecules are combined in water. In some embodiments, plant-derived proteins, powders containing phytochemicals, and caffeine molecules are combined in water. In some embodiments, plant-derived proteins, powders containing phytochemicals, and theophylline molecules are combined in water. In some embodiments, plant-derived proteins, powders containing phytochemicals, and theophylline molecules are combined in water.

[0164] In some embodiments, ethanol is added to a mixture of plant-derived protein, powder containing phytochemicals, and ketone molecules. In some embodiments, ethanol is added to a mixture of plant-derived protein, powder containing phytochemicals, and NR molecules. In some embodiments, ethanol is added to the mixture at a volume ratio of ethanol to the mixture of 1:10 to 1:1. In some embodiments, the volume ratio of ethanol to the mixture 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 ethanol to the mixture 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 ethanol to the mixture 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 ethanol to the mixture is less than or equal to 1:7. In some embodiments, ethanol is removed from the mixture of plant-derived protein, phytochemical-containing powder, and ketone molecules. In some embodiments, ethanol is removed from the 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 evaporation.

[0165] In some embodiments, the powder containing phytochemicals comprises tea leaves, blackberries, grapes, mangoes, or okra. In some embodiments, the powder containing phytochemicals comprises loose leaf tea, blackberry powder, grapes, mango peel powder, or okra extract powder. In some embodiments, the concentration of phytochemicals in the powder containing phytochemicals 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.

[0166] In some embodiments, the powder containing phytochemicals is mango peel powder. In some embodiments, the phytochemical of mango peel powder is mangiferin. In some embodiments, the concentration of phytochemicals in 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 phytochemicals 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 phytochemicals in 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.

[0167] 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.

[0168] In some embodiments, the powder containing phytochemicals comprises tea leaves. In some embodiments, the phytochemical of tea leaves is epigallocatechin 3-O-gallate (EGCG). In some embodiments, the concentration of phytochemicals in 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 phytochemicals 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 phytochemicals 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.

[0169] In some embodiments, the weight ratio of EGCG to plant-derived protein is greater than or 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.

[0170] In some embodiments, the powder containing phytochemicals comprises grape extract. In some embodiments, the phytochemical of grape extract is trans-resveratrol. In some embodiments, the concentration of phytochemicals in 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 1 mg / g dry matter. In some embodiments, the concentration of phytochemicals 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 phytochemicals in the grape extract is between 1 mg / g and 3 mg / g dry matter, between 2 mg / g and 4 mg / g dry matter, between 5 mg / g and 7 mg / g dry matter, between 6 mg / g and 8 mg / g dry matter, between 7 mg / g and 9 mg / g dry matter, between 8 mg / g and 10 mg / g dry matter, between 9 mg / g and 11 mg / g dry matter, between 10 mg / g and 12 mg / g dry matter, between 11 mg / g and 13 mg / g dry matter, between 12 mg / g and 14 mg / g dry matter. dry matter, between 13 mg / g and 15 mg / g dry matter, between 14 mg / g and 16 mg / g dry matter, between 15 mg / g and 17 mg / g dry matter, between 16 mg / g and 18 mg / g dry matter, between 17 mg / g and 19 mg / g dry matter, between 18 mg / g and 20 mg / g dry matter, between 19 mg / g and 25 mg / g dry matter, between 20 mg / g and 30 mg / g dry matter, between 25 mg / g and 35 mg / g dry matter, or between 30 mg / g and 40 mg / g dry matter.

[0171] In some embodiments, the weight ratio of resveratrol to 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 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 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 plant-derived protein is 1:400.

[0172] In some embodiments, the powder containing phytochemicals comprises blackberry. In some embodiments, the phytochemical of blackberry is quercetin. In some embodiments, the concentration of phytochemicals in blackberry 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 phytochemicals in blackberries is greater than or equal to 50,000 μg / 100 g dry matter, 55,000 μg / 100 g dry matter, 60,000 μg / 100 g dry matter, 65,000 μg / 100 g dry matter, 70,000 μg / 100 g dry matter, 75,000 μg / 100 g dry matter, 80,000 μg / 100 g dry matter, 85,000 μg / 100 g dry matter, 90,000 μg / 100 g dry matter, 95,000 μg / 100 g dry matter, or 100,000 μg / 100 g dry matter. In some embodiments, the concentration of phytochemicals in blackberries is between 50,000 μg / 100 g and 60,000 μg / 100 g dry matter, between 55,000 μg / 100 g and 65,000 μg / 100 g dry matter, between 60,000 μg / 100 g and 70,000 μg / 100 g dry matter, between 65,000 μg / 100 g and 75,000 μg / 100 g dry matter, between 70,000 μg / 100 g and 80,000 μg / 100 g dry matter, between 80,000 μg / 100 g and 90,000 μg / 100 g dry matter, between 90,000 μg / 100 g and 100,000 μg / 100 g dry matter, between 100,000 μg / 100 g and 110,000 μg / 100 g dry matter, between 120,000 μg / 100 g and 130,000 μg / 100 g dry matter, between 140,000 μg / 100 g and 150,000 μg / 100 g dry matter, between 160,000 μg / 100 g and 170,000 μg / 100 g dry matter, between 180,000 μg / 100 g and 190,000 μg / 100 g dry matter, between 190,000 μg / 100 g and 200,000 μg / 100 g

[0026] In some embodiments, the present invention relates to an aqueous phase having an amount of at least 200 μg / 100 g of dry matter and a concentration of at least 200 μg / 100 g of dry matter. In some embodiments, the aqueous phase having an amount of at least 200 μg / 100 g of dry matter and a concentration of at least 200 μg / 100 g of dry matter is at least 300 μg / 100 g of dry matter.

[0173] 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.

[0174] In some embodiments, the water has undergone one or more processes, such as distillation or filtration. In some embodiments, the water is distilled water.

[0175] In some embodiments, the dry weight ratio of ketone 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 ketone 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 ketone 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 ketone molecules to plant-derived proteins is less than or equal to 1:2. In some embodiments, the dry weight ratio of ketone molecules to plant-derived proteins is 1:2.5.

[0176] In some embodiments, the dry weight ratio of NR 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 NR 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 NR 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 NR molecules to plant-derived proteins is less than or equal to 1:2. In some embodiments, the dry weight ratio of NR molecules to plant-derived proteins is 1:3.2.

[0177] 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.

[0178] 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.

[0179] In some embodiments, the weight ratio of the plant-derived protein to water is greater than or 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, 1:50 and 1:90, or 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.

[0180] Nanoparticles

[0181] Disclosed herein are nanoparticles comprising plant-derived proteins cross-linked with one or more phytochemicals. In some embodiments, the nanoparticles may comprise one or more ketone molecules.

[0182] In some embodiments, the average diameter of the nanoparticles is less than or equal to 700nm, 600nm, 500nm, 400nm, 300nm, 200nm, 100nm, 90nm, 80nm, 70nm, 60nm, or 50nm. In some embodiments, the average diameter of the nanoparticles is greater than or equal to 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, or 700nm. In some embodiments, the nanoparticles have an average diameter 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.

[0183] In some embodiments, the nanoparticles include phenols. In some embodiments, the phenol content of the nanoparticles is quantified using total gallic acid equivalent (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 phenolic 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 nanoparticles described herein have a phenolic content of 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.

[0184] Example

[0185] Example 1 - Ketone Molecule Synthesis

[0186] As described herein, the ketone molecules as described herein are produced by the Fisher esterification reaction of (R)-3-hydroxybutyric acid and glycerol to obtain a mixture of compounds II-V as described herein. Compound II is purified by fractional distillation according to methods known in the art. (See, for example, www.sciencedirect.com / topics / chemistry / fractional-distillation) The resulting ketone molecules contain a molar ratio of Compound II to Compound II-V greater than 80:20.

[0187] Example 2a-Nicotinamide riboside molecule synthesis

[0188] Nicotinamide riboside molecules and compound IV were obtained from Thorne Healthcare.

[0189] Example 2b - Caffeine Molecular Synthesis

[0190] Caffeine molecule was obtained from Sigma-Aldrich.

[0191] Example 2c-Theophylline Molecular Synthesis

[0192] Theophylline molecule was obtained from BulkStimulants.com.

[0193] Example 3 - Ketone molecule (KM) characterization:

[0194] Appearance: KM is a slightly turbid, oily, highly viscous liquid.

[0195] Smell: The smell of KM is neutral.

[0196] Solubility: KM is water soluble and miscible with ethanol. KM is insoluble in dichloromethane.

[0197] pH: KM shows a pH of 4.5 in water, indicating an acidic aqueous solution.

[0198] UV-visible absorption spectrum: Based on UV-visible absorption measurements, the electronic transitions in KM belong to the ultraviolet region (UV). KM shows a broad absorption up to ~245 nm in the UV region ( Figure 1 ). There is no chromogenic characteristic absorption peak in the visible region. Therefore, UV-visible spectroscopy is not used for tracking KM or protein nanoparticles encapsulating KM.

[0199] Nuclear Magnetic Resonance Spectra: 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 are reported relative to internal D2O ( Figure 3 ). 13C NMR chemical shifts are reported relative to an external tetramethylsilane (TMS) standard ( Figure 5-7 ). KM is a carboxylic acid ester composed of the parent carboxylic acid (3-hydroxybutyric acid) and the corresponding alcohol (glycerol). During the synthesis of KM, various glycerides are possible products. NMR shows ~77% KM, i.e. monoglyceride. In the NMR, the starting materials glycerol (11%) and 3-hydroxybutyric acid (12%) are also seen. Fig.42 Shows the KM in D2O 1 H NMR.

[0200] Mass Spectrometry: A complex array of mass spectrometry techniques have been utilized to develop 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) technique. In addition to obtaining the elemental / isotopic signature of KM using MS, the [M+H]+ peak was also used for molecular weight estimation. Based on the chemical structure, the calculated molecular weight of KM was 178.18 g / mol. The [M+Na]+ peak at 201.07 was detected as the major peak, indicating the presence of monoglyceride (KM) ( Fig. 9 ).

[0201] Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS): In addition to mass spectrometry analysis, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are used for quality control confirmation of KM. GC-MS and LC-MS techniques are excellent analytical and quality control tools for detecting the purity of KM and the presence of starting materials. KM is a carboxylic acid ester composed of a parent carboxylic acid (3-hydroxybutyric acid) and the corresponding alcohol (glycerol). In GC-MS analysis ( Fig.11 ), 3-hydroxybutyric acid (~10%) and glycerol (~23%) were seen at 17.1 min and 20.2 min retention RT respectively. Two monoglyceride peaks were observed at 30.4 min and 30.9 min retention time (RT). The peak at 30.4 min (~16%) and the peak at 30.9 min (~50%) gave a total KM monoglyceride percentage of ~66%. There was no diglyceride peak and triglyceride peak (should be seen after ~30 min RT). In LC-MS analysis ( Fig.12), the major peak corresponds to the loss of water from the monoglycerides. The major peak at 1.4 min RT corresponds to the [M-H2O]+ peak at 161 - confirming the results from NMR and from GC-MS.

[0202] Circular Dichroism (CD): Chiral molecules are mirror images or enantiomers. The physical properties of enantiomers are identical in every respect except as to how the molecules interact with polarized light and how they interact with other chiral molecules. Enantiomers are special forms of isomerism. Circular dichroism (CD), measured as a function of wavelength, is the difference in absorbance of left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL). When a chiral chromophore is present, one state of circularly polarized light is absorbed to a greater or lesser extent than the other. Thus, at the corresponding wavelength, the circular dichroism signal can be positive or negative, depending on whether L-CPL is absorbed to a greater extent (positive CD signal, associated with R / D configuration) or to a lesser extent (negative CD signal, associated with S / L configuration) than R-CPL. KM was used for CD measurements. The concentration of KM in water was 7 mg / ml. The starting material (R)-3-hydroxybutyric acid, also known as D-β-hydroxybutyric acid, is chiral and introduces chirality into KM. The circular dichroism (CD) spectrum of KM is positive, indicating the R / D configuration in KM ( Fig.13 ).

[0203] KM Evaluation: Analysis of KM showed 80-70% monoglycerides based on NMR and GC-MS, respectively. No diglycerides and triglycerides were present based on GC-MS analysis. 10-20% glycerol and 10% 3-hydroxybutyric acid starting material were also seen in NMR and GC-MS, respectively. No other impurities were seen.

[0204] Quality Control Parameters: Based on the study of quality control parameters of ketone molecules (KM), GC-MS and LC-MS analysis are important quality control and characterization tools for the comprehensive characterization of KM encapsulation within plant proteins based on green nanotechnology.

[0205] Example 4a-NR Characterization:

[0206] Appearance: NR is a fine white powder.

[0207] Odor: The odor of NR is neutral.

[0208] Solubility: NR is water soluble and miscible with ethanol. The chloride salt of nicotinamide riboside has good solubility in water.

[0209] pH: NR shows a pH of 4.0 in water, indicating an acidic aqueous solution.

[0210] UV-visible absorption spectrum: Based on UV-visible absorption measurement, the electronic transition in NR belongs to the ultraviolet region (UV). NR shows a broad absorption up to ∼300 nm in the UV region, with peaks at 220 nm, 230 nm, and 265 nm ( Figure 2 ). There is no chromogenic characteristic absorption peak in the visible region. Therefore, UV-visible spectroscopy was not used for tracking protein nanoparticles encapsulating NR.

[0211] NMR spectra were recorded at 500 MHz on a Bruker 500 MHz spectrometer. 1 H nuclear magnetic resonance (NMR) spectra were recorded at 126 MHz on a Bruker 500 MHz spectrometer. 13 C NMR spectra. NMR spectra recorded in deuterated water (D2O). Reported relative to internal D2O 1 H chemical shift ( Figure 4 ). Reported relative to an external tetramethylsilane (TMS) standard 13 CNMR chemical shift ( Figure 8 ). Both proton and carbon NMR of NR showed clean spectra without impurities.

[0212] Mass Spectrometry: A sophisticated array of mass spectrometry techniques have been utilized to develop reliable quality control parameters for NR and its protein encapsulated products. Analysis of NR was performed using electrospray ionization mass spectrometry (ESI-MS) techniques. In addition to obtaining the elemental / isotopic signature of NR using MS, the [M+H]+ peak was also used for molecular weight estimation. The nicotinamide riboside anion [M] of 255.09 was detected. + The peak is the main peak ( Fig.10 ). The peak at 123.05 is consistent with the nicotinamide [M+H] + Fragment peak correlation.

[0213] Circular dichroism (CD): NR was evaluated by circular dichroism. The concentration of NR in water was 0.2 mg / ml. The circular dichroism spectrum of NR was negative, indicating S / L configuration (Figure 14). The anionic part of NR may also introduce chirality.

[0214] NR evaluation: Analysis of NR showed compounds containing nicotinamide riboside. NMR showed a clean spectrum with no starting material or solvent impurities. Mass spectrometry analysis showed peaks associated with nicotinamide riboside and nicotinamide units. The circular dichroism (CD) spectrum of NR was negative, indicating an S / L configuration.

[0215] Quality Control Parameters: Based on the study of quality control parameters of nicotinamide riboside (NR), the above analytical tools are important for the development of various NR related products through green nanotechnology based NR encapsulation.

[0216] Example 4b-CA Characterization

[0217] Appearance: CA is a fine white powder.

[0218] Odor: The odor of CA is neutral and the taste is bitter.

[0219] Solubility: CA is water soluble (25 mg / mL).

[0220] pH: CA shows a pH of 7.0 in water, indicating a neutral aqueous solution

[0221] Liquid chromatography-mass spectrometry (LC-MS): Liquid chromatography-mass spectrometry (LC-MS) is used for quality control confirmation of CA. LC-MS technology is an excellent analytical and quality control tool for detecting the purity, amount, and presence of other substances in CA. In LC-MS analysis ( Fig. 14B ). The main peak at 3.0 min RT corresponds to [M+H] of 195 + peak – confirms results from the caffeine literature.

[0222] CA evaluation: LC-MS analysis of CA showed pure material. CA is a well-recognized compound, and other detailed characterization data can be obtained from the National Institute of Standards and Technology (NIST) and the Spectral Database for Organic Compounds (SDBS) website.

[0223] Quality Control Parameters: Based on the study of quality control parameters of caffeine (CA), LC-MS analytical tools are important for the development of various CA related products through green nanotechnology based CA encapsulation.

[0224] Example 4b - TH Characterization

[0225] Appearance: TH is a fine white powder.

[0226] Odor: The odor of TH is neutral and tastes very bitter.

[0227] Solubility: CA is water soluble (25 mg / mL).

[0228] pH: CA shows a pH of 7.0 in water, indicating a neutral aqueous solution

[0229] Liquid chromatography-mass spectrometry (LC-MS): Liquid chromatography-mass spectrometry (LC-MS) is used for quality control confirmation of TH. LC-MS technology is an excellent analytical and quality control tool for detecting the purity, amount, and presence of other substances in TH. In LC-MS analysis ( Fig. 14C ). The main peak at 3.0 min RT corresponds to [M+H] + Peak - confirms the results from theophylline literature.

[0230] TH evaluation: LC-MS analysis of TH showed pure material. TH is a well-recognized compound and other detailed characterization data can be obtained from the National Institute of Standards and Technology (NIST) website.

[0231] Quality Control Parameters: Based on the study of quality control parameters of theophylline (TH), LC-MS analytical tools are important for the development of various TH-related products through TH encapsulation based on green nanotechnology.

[0232] Example 5 - Characterization of Proteins from Plant Sources

[0233] Table 2: Sources of protein from plant sources

[0234]

[0235] Pea protein

[0236] Water-soluble pea protein (PP) was purchased from the WaterSolubleProtein.com website. The plant source is pea (Pisum sativum), natural and non-GMO yellow pea, which has been hydrolyzed to be highly water-soluble (100 mg / ml). The protein solution is a clear yellow liquid with no precipitate or residue. It has a protein content of 80%. It is edible and dissolves immediately in water and forms a stable solution.

[0237] Pea protein UV-visible absorption spectrum: Based on UV-visible absorption measurement, the electronic transition in water-soluble pea protein (PP) belongs to the ultraviolet region (UV). PP shows a weak absorption peak at 265nm and a broad absorption in the 250nm-200nm UV region ( Fig.17A ). There is no characteristic absorption peak in the visible region.

[0238] Pea protein fluorescence spectrum: Phenylalanine, tyrosine and tryptophan amino acids in protein produce 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 at 352 nm was seen in the emission spectrum of PP ( Fig. 17B). Changes in the tryptophan microenvironment can be monitored by fluorescence spectroscopy. The shift of the maximum fluorescence peak and the change in fluorescence intensity can provide insight into the interaction between pea protein and ligand (references: Akbar, SM et al., Journal of Bioenergetics and Biomembranes, 2016, 48, 241–247 and Vivian, JT et al., Biophysical Journal 2001, 80, 2093–2109).

[0239] Matrix-Assisted Laser Desorption / Ionization Mass Spectrometry (MALDI-MS) of Pea Protein: Water-soluble pea protein (PP) was analyzed using matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) to examine the molecular weight of the protein components. A sinapinic acid matrix was used. The mass range of 2kDa-400kDa was investigated on a Bruker AutoFlex Speed ​​MALDI-MS, and no significant peaks were seen. The mass range was then reduced to 5kDa, and peaks at approximately 877 and 993 m / z were seen, indicating that the protein molecular weight was less than ~1kDa ( Fig.18 ).

[0240] Pea protein NMR. Fig.41 Shown is the 1H NMR of water-soluble pea protein (PP) in D2O.

[0241] Soy Protein Isolate

[0242] Soy protein isolate was obtained from MP Biomedicals, catalog number ICN90545605. Soy protein is an abundant and inexpensive plant protein obtained from soybeans. Consumption of soybeans is beneficial for heart health and reduces inflammation. Therefore, soybeans have important applications for diabetes, atherosclerosis and cancer treatment. Soy protein isolate (SPI) is a soy food with the largest soy protein content (92%). SPI is water-soluble and is a useful drug delivery vehicle for water-soluble drugs. SPI is generally regarded as a safe drug, biodegradable, inexpensive and also available in large quantities. The anti-inflammatory properties of soy protein can also be utilized in nanotherapeutic formulations.

[0243] UV-visible absorption spectrum of soy protein isolate: Based on UV-visible absorption measurement, the electronic transition of SPI belongs to the ultraviolet region (UV). SPI shows a broad absorption in the 240nm–200nm UV region ( Fig.42 ). There is no characteristic absorption peak in the visible region.

[0244] Matrix-Assisted Laser Desorption / Ionization Mass Spectrometry (MALDI-MS) of Soy Protein Isolate: Water-soluble pea protein (PP) was analyzed using matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) to examine the molecular weight of the protein components. A sinapinic acid matrix was used. The mass range of 2 kDa-400 kDa was investigated on a Bruker AutoFlex Speed ​​MALDI-MS. A significant peak was seen at 8960 m / z, indicating a protein molecular weight of ~9 kDa ( Fig.34 ).

[0245] Okra Extract

[0246] Okra extract was obtained from Bulk Supplements, SKU OKR25kg. Okra is a flowering plant belonging to the Malvaceae 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 mucus or gel from the pods is a natural polysaccharide and includes D-galactose, L-rhamnose and L-galacturonic acid. Okra mucus is extracted in water and forms a gel-like viscous solution. Natural polysaccharides are biocompatible, non-toxic and have biosourced polymer applications in the form of microspheres, nanomatrices and nanoparticles. Okra extract (OE), a finely ground dry powder, was purchased from the BulkSupplements.com website. It is edible, water-soluble and can be stored at room temperature. OE produces a slightly turbid brown solution of okra mucus or gel in water. Natural polysaccharides such as okra gel can be used for encapsulation, stabilization and drug delivery applications.

[0247] Matrix Assisted Laser Desorption / Ionization Mass Spectrometry (MALDI-MS) of Okra Mucus: Okra mucus or gel from green seed pods was extracted in water. It is a gel-like viscous solution. Okra mucus was analyzed using matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) to check the molecular weight of protein components. Sinapic acid matrix was used. The mass range of 2 kDa-400 kDa was studied on a Bruker AutoFlex SpeedMALDI-MS. For okra polysaccharide mucus, a peak of about 8 kDa was seen ( Fig.29 ).

[0248] UV-visible absorption spectrum of okra mucus: Based on UV-visible absorption measurement, the electronic transitions in okra mucus mostly belong to the ultraviolet region (UV). Okra mucus shows a weak broad absorption between 400nm-300nm. In addition, in the UV region, it shows a stronger broad absorption in the region of 300nm-200nm ( Fig.30 ). There is no characteristic absorption peak in the visible region.

[0249] Example 6 - Phytochemical Characterization

[0250] Proton (1H) NMR spectra were recorded at 600 MHz on a Bruker 600 MHz spectrometer. Proton (13C) NMR spectra were recorded at 151 MHz on a Bruker 600 MHz spectrometer. NMR spectra recorded in deuterated water (D2O) or deuterated methanol (CD3OD) or a combination of the two. 1H chemical shifts are reported relative to internal D2O or CD3OD. 13C NMR chemical shifts are reported relative to external tetramethylsilane (TMS) standards. Carbon NMR shows corresponding changes.

[0251] Hydrogen bonding is a characteristic of KM, protein and phytochemical structures. Opportunities for both intermolecular and intramolecular hydrogen bonding are extensive. Hydrogen bonding can facilitate the interaction of KM with proteins and phytochemicals for nanoparticle synthesis and encapsulation purposes. The extensive hydrogen bonding feature facilitates KM nanoencapsulation and the interaction of KM with proteins and phytochemicals ( Fig.30 ). It also facilitates the interaction of proteins and phytochemicals.

[0252] Fig.42 It shows that mangiferin in CD3OD 1 H NMR.

[0253] Fig.45 The results show that epigallocatechin gallate (EGCG) in D2O 1 HNMR.

[0254] Fig.48 The results showed that quercetin in CD3OD 1 H NMR.

[0255] Fig.51 The results showed that resveratrol in CD3OD 1 H NMR.

[0256] Phytochemicals come from:

[0257] Table 3: Sources of phytochemicals

[0258]

[0259] Example 7- Cross-linking of pea protein with mangiferin and encapsulation of ketone molecules in pea protein cross-linked with mangiferin

[0260] Synthesis of KM-encapsulated pea protein nanoparticles (KM-PP-MP-NP) by cross-linking of mango peel: 15 g of water-soluble pea protein (PP), 15 g of mango peel (MP) powder and 6 g of ketone molecules (KM) were weighed and transferred to a 2L conical flask with a stirring bar. 1L of distilled water was added to the flask, and the contents of the reaction mixture were stirred overnight. The mixing was stopped and the insoluble residue was then allowed to settle. The insoluble residue was separated from the solution by first decanting and then filtering (75 micron size). 625 mL of the filtered solution was transferred to a 2L round-bottom flask with a stirring bar. 210 ml of anhydrous ethanol (one-third volume of the filtered solution) was added to the contents of the flask and stirred vigorously. The reaction mixture was then stirred overnight. The KM-encapsulated pea protein nanoparticles (KM-PP-MP-NP) obtained by cross-linking mango peel were then characterized by TEM, DLS size and ZP measurements. The KM content was estimated by GC-MS analysis and pure KMst.

[0261] Gas chromatography-mass spectrometry (GC-MS) procedure: 1 μL of the KM sample to be analyzed was diluted 1000 times by adding 1000 μL of pyridine. 25 μL of this solution was trimethylsilylated with 25 μL of MSTFA [N-methyl-N-(trimethyl-silyl)trifluoroacetamide] + 1% TMCS (trimethylchlorosilane) reagent by incubation at 50°C for 1 hour. The derivatized sample was then analyzed using an Agilent 6890GC (Agilent Technologies, Inc., Santa Clara, CA) coupled to a 5973N MSD mass spectrometer with a scan range of 50 to 650 m / z. 1 μL of the sample was injected into the GC column with a split ratio of 1:1 and a run time of 60 min. Separation was achieved on a 60m DB-5MS column (J&W Scientific, 0.25mm ID, 0.25um film thickness) and with a constant flow rate of helium at 1.0ml / min, at 80°C for 2min, then at 5°C / min to 315°C, and at a temperature program of 12min at 315°C. Standard alkane mixtures were used for GC-MS quality control and retention index calculations. AMDIS was used to deconvolute the chromatographic peaks in the sample and annotated by mass spectrometry and retention indices matched to an internally constructed mass spectral library and a commercial NIST17 mass spectral library. Pure KM standards with five different concentrations prepared in pyridine were similarly analyzed. Peak areas were calculated using Agilent Enhanced Data Analysis (EDA) software to generate calibration curves. The concentration of monoglycerides in the KM solution was accurately calculated using the calibration curve and the peak area of ​​the GC peak of the sample solution at ~30min RT.

[0262] The KM-PP-MP-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life. Blank pea protein nanoparticles (PP-MP-NPs) obtained by cross-linking mango peel were synthesized according to the same procedure without adding KM.

[0263] Fig.43 1H NMR (expanded) of the reaction mixture of mangiferin and water-soluble pea protein (PP) in CD3OD / D2O is shown (arrows indicate changes in protons).

[0264] Fig.44 The results show that mangiferin, water-soluble pea protein (PP) and KM reaction mixture in CD3OD / D2O 1 H NMR (extended).

[0265] The aromatic ring protons of phytochemicals show significant changes, indicating covalent interactions with proteins. These changes lead to cross-linking with the amino functional groups of proteins. Mangiferin proton NMR shows equivalent peaks at 7.5, 6.8 and 6.4 ppm. But after reaction with pea protein, there are significant changes in the 6.8 and 6.4 ppm peaks.

[0266] KM-encapsulated pea protein nanoparticles obtained by cross-linking mango peel (KM-PP-MP-NP): Naturally available plant-based materials were explored for cross-linking. Mangiferin (MGF), a phytochemical found in mango, was extracted from mango peel. The multiple hydroxyl groups in MGF are effective for protein cross-linking. Water-soluble pea protein nanoparticles (KM-PP-MP-NP) encapsulated with KM obtained by cross-linking mango peel were attempted using an ethanol desolvation method. Fig.15 ). After nanoparticle synthesis, any insoluble residues were removed by centrifugation / filtration. KM-PP-MP-NPs ranged in size from 336-422 nm with an average of 367 nm (using DLS) and exhibited an average zeta potential of -15 mV (Table 4), indicating reasonable stability. A polydispersity index (PDI) of 0.2 indicated uniformly sized nanoparticles (unimodal distribution). The nanoparticle core size ( Fig.23 ). It showed good particles with size in the range of -400 nm. KM-PP-MP-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial level production.

[0267] 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 uniformly sized nanoparticles (unimodal distribution). The core size of the nanoparticles ( Fig.19). It showed good particles with size in the range of ~400 nm. Blank nanoparticles were comparable to KM encapsulated nanoparticles with a slight increase in size. Blank pea protein nanoparticles obtained by cross-linking mango peel were synthesized without KM for comparison (Serial No. 5).

[0268] Table 4: Characterization of KM encapsulated pea protein nanoparticles (KM-PP-MP-NPs) obtained by ethanol desolvation method and mango peel cross-linking.

[0269]

[0270] a = Mango peel obtained from finely ground dried mango peel obtained from mango fruit (MP)

[0271] Fluorescence spectra of KM-PP-MP-NPs and PP-MP-NPs: The fluorescence emission spectra of KM-PP-MP-NPs and PP-MP-NPs were recorded by excitation at 285 nm corresponding to tryptophan amino acid. Fig.21 ) in the emission spectrum of 100 μg / mL pea protein nanoparticles. A broad emission peak with a fluorescence intensity of 40 at 351 nm was seen in the emission spectrum of 100 μg / mL pea protein nanoparticles. Changes in the tryptophan microenvironment can be monitored by fluorescence spectroscopy. The fluorescence maximum peak and intensity of KM-PP-MP-NP and PP-MP-NP are comparable, indicating that there is not much change in the tryptophan region of pea protein nanoparticles. Since KM is water-soluble and hydrophilic, it does not interact with the hydrophobic tryptophan microenvironment of pea protein. Fluorescence measurements give insights into the interaction of pea protein with KM.

[0272] GC-MS analysis of KM encapsulated pea protein nanoparticles (KM-PP-MP-NPs) obtained by cross-linking of mango peel: GC-MS analysis was used to determine the amount of KM in the nanoparticle formulation. It was found to be quantitative, indicating 100% KM encapsulation. High KM encapsulation efficiency may be important for biodelivery of KM.

[0273] Extensive hydrogen bonding is a feature of the KM, protein, and phytochemical structures. This is not surprising given the ester, hydroxyl, amine, and oxo functional groups in their structures. Opportunities for both intermolecular and intramolecular hydrogen bonding are extensive. Hydrogen bonding greatly facilitates the interaction of KM with proteins and phytochemicals for nanoparticle synthesis and encapsulation purposes. The extensive hydrogen bonding feature facilitates KM nanoencapsulation and the interaction of KM with proteins and phytochemicals ( Fig.39 ). It also facilitates the interaction of proteins and phytochemicals.

[0274] Example 8a- Cross-linking of pea protein with mangiferin and encapsulation of nicotinamide riboside molecules in pea protein cross-linked with mangiferin

[0275] Synthesis of NR-encapsulated pea protein nanoparticles (NR-PP-MP-NP) by mango peel cross-linking: 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 2L conical flask with a stirring bar. 1L of distilled water was added to the flask and the contents of the reaction mixture were stirred overnight. The mixing was stopped and the insoluble residue was then allowed to settle. 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 2L round-bottom flask with a stirring bar. 234 ml of anhydrous ethanol (one-third volume of the filtered solution) was added to the flask and the contents were stirred vigorously. The reaction mixture was then stirred overnight and any insoluble residue was removed by centrifugation / filtration (75 micron size). The NR-encapsulated pea protein nanoparticles (NR-PP-MP-NP) obtained by mango peel cross-linking were then characterized by TEM, DLS size and ZP measurements (Table 5). The NR-PP-MP-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life.

[0276] Blank pea protein nanoparticles (PP-MP-NPs) obtained by cross-linking mango peel were synthesized following the same procedure without adding NR.

[0277] NR encapsulated pea protein nanoparticles (NR-PP-MP-NP) obtained by cross-linking mango peel: Naturally available plant-based materials were explored for cross-linking. Mangiferin (MGF), a phytochemical found in mango, can be extracted from mango peel. The multiple hydroxyl groups in MGF are effective for protein cross-linking. As mentioned above, water-soluble pea protein nanoparticles (NR-PP-MP-NP) encapsulated NR obtained by cross-linking mango peel were attempted by ethanol desolvation method ( Fig.16 ). After nanoparticle synthesis, any insoluble residues larger than 75 microns in size were removed by centrifugation / filtration. The NR-PP-MP-NPs ranged in size from 286-369 nm with an average of 297 nm (using DLS) and exhibited an average zeta potential of -18 mV (Table 5a), indicating reasonable stability. A polydispersity index (PDI) of 0.2 indicated uniformly sized nanoparticles (unimodal distribution). The nanoparticle core size was explored by TEM using a negative staining technique (Figure 20). It showed particles with a size in the range of ~300 nm. NP-PP-MP-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial level production. Blank nanoparticles were comparable to those encapsulated with NR, with a slightly increased size.

[0278] Table 5a: Characterization of NR encapsulated pea protein nanoparticles (NR-PP-MP-NPs) obtained by ethanol desolvation method and mango peel cross-linking.

[0279]

[0280] a = Mango peel obtained from finely ground dried mango peel (MP)

[0281] Example 8b- Cross-linking of pea protein with mangiferin and encapsulation of caffeine molecules in pea protein cross-linked with mangiferin

[0282] Synthesis of CA-encapsulated pea protein nanoparticles (CA-PP-MP-NPs) by cross-linking of mango peel: 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 conical flask with a stirring 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 any insoluble residues were removed by centrifugation / filtration (75 micron size). The CA-encapsulated pea protein nanoparticles (CA-PP-MP-NPs) obtained by cross-linking of mango peel were then characterized by TEM, DLS size, and ZP measurements (Table 5b). The CA-PP-MP-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life.

[0283] CA encapsulated pea protein nanoparticles (CA-PP-MP-NPs) obtained by cross-linking mango peel: Naturally available plant-based materials were explored for cross-linking. Mangiferin (MGF), a phytochemical found in mango, can be extracted from mango peel. The multiple hydroxyl groups in MGF are effective for protein cross-linking. CA-PP-MP-NPs had an average size of 652 nm (using DLS) and showed an average zeta potential of -16 mV (Table 5b), indicating reasonable stability. The polydispersity index (PDI) of ~0.3 indicated mostly uniformly sized nanoparticles (unimodal distribution). The nanoparticle core size ( Fig. 20B ). It showed longitudinal particles with sizes in the range of ~200 nm. CA-PP-MP-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial level production.

[0284] Table 5b: Characterization of CA encapsulated pea protein nanoparticles (CA-PP-MP-NPs) obtained by cross-linking of mango peel.

[0285]

[0286] a = Mango peel obtained from finely ground dried mango peel (MP)

[0287] Example 8c- Cross-linking of pea protein with mangiferin and encapsulation of theophylline molecules in the cross-linked pea protein with theophylline

[0288] Synthesis of TH-encapsulated pea protein nanoparticles (TH-PP-MP-NPs) by cross-linking of mango peel: 25 g of water-soluble pea protein (PP), 8 g of mango peel (MP) powder, and 13 g of theophylline (TH) were weighed and transferred to a 1 L conical flask with a stirring 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 any insoluble residues were removed by centrifugation / filtration (75 micron size). The TH-encapsulated pea protein nanoparticles (TH-PP-MP-NPs) obtained by cross-linking of mango peel were then characterized by TEM, DLS size, and ZP measurements (Table 5c). The TH-PP-MP-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life.

[0289] TH encapsulated pea protein nanoparticles (TH-PP-MP-NPs) obtained by cross-linking mango peel: Naturally available plant-based materials were explored for cross-linking. Mangiferin (MGF), a phytochemical found in mango, can be extracted from mango peel. The multiple hydroxyl groups in MGF are effective for protein cross-linking. TH-PP-MP-NPs had an average size of 563 nm (using DLS) and showed an average zeta potential of -17 mV (Table 5c), indicating reasonable stability. The polydispersity index (PDI) of ~0.5 indicated a slightly broad size distribution of nanoparticles. The nanoparticle core size ( Fig. 20C ). It showed particles with sizes in the range of ~100 nm. TH-PP-MP-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial level production.

[0290] Table 5c: Characterization of TH-encapsulated pea protein nanoparticles (TH-PP-MP-NPs) obtained by cross-linking of mango peel.

[0291]

[0292] a = Mango peel obtained from finely ground dried mango peel (MP)

[0293] Example 9 - Cross-linking of pea protein with EGCG and encapsulation of ketone molecules in pea protein cross-linked with EGCG

[0294] Synthesis of KM-encapsulated pea protein nanoparticles (KM-PP-TE-NP) by cross-linking with tea extract: A tea extract (TE) solution was prepared for nanoparticle synthesis. 6 g of Darjeeling loose leaf tea (Table 3) was weighed and transferred to a 1 L conical flask. 300 mL of distilled water was added to the flask, and the contents were boiled at 100 ° C for 10 minutes. The heating was then stopped and the tea extract solution was cooled to room temperature. Insoluble tea residues were separated from the solution 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 conical flask with a stirring 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 another 30 minutes. 100 ml of TE solution was added, and the reaction mixture was stirred for another 2 hours. Then, 170 mL of anhydrous ethanol (one-third volume of 500 mL of water) was added to the contents of the flask while stirring vigorously. The reaction mixture was stirred overnight. The pea protein nanoparticles (KM-PP-TE-NPs) encapsulated with KM obtained by cross-linking with tea extract were then characterized by TEM, DLS size and ZP measurements. The KM content was estimated by the above-mentioned GC-MS analysis and calibration with pure KM standards. The KM-PP-TE-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life. Blank pea protein nanoparticles (PP-TE-NPs) obtained by cross-linking with tea extract were synthesized according to the same procedure without the addition of KM.

[0295] Fig.46 The results show that the reaction mixture of epigallocatechin gallate (EGCG) and water-soluble pea protein (PP) in D2O 1 H NMR (expanded) (arrows indicate proton changes).

[0296] Fig.47 The results show the reaction mixture of epigallocatechin gallate (EGCG), water-soluble pea protein (PP) and KM in D2O. 1 H NMR (extended).

[0297] The aromatic ring protons of phytochemicals show distinct changes, indicating covalent interactions with proteins. These changes lead to cross-linking with the amino functional groups of proteins. In the EGCG proton NMR, the doublet at 6.1 ppm disappears after reaction with pea protein.

[0298] Tea extract contains various polyphenols, including flavonoids, epigallocatechin gallate (EGCG), and other catechins. Therefore, tea extract can effectively crosslink protein nanoparticles. Water-soluble pea protein nanoparticles encapsulating KM (KM-PP-TE-NPs) obtained by crosslinking using tea extract were implemented by ethanol desolvation method. After nanoparticle synthesis, any insoluble residues were removed by centrifugation / filtration. Ethanol was removed from the formulation by rotary evaporation at 37°C. KM-PP-TE-NPs ranged in size from 316-373nm, with an average size of 338nm (using DLS), and showed an average zeta potential of -13mV (Table 6), indicating stability. The polydispersity index (PDI) of ~0.2 indicated uniformly sized nanoparticles (unimodal distribution). The nanoparticle core size ( Fig.23 ). It showed stable particles with sizes in the range of ~300nm. KM-PP-TE-NPs were formulated and characterized to define quality control parameters for scale-up and commercial level production. Small scale (~10mL and 50mL) nanoparticles were initially formulated. After establishing characterization and quality control parameters, they were scaled up to ~1L. KM from repeated batches were used for large-scale synthesis. Large-scale batches showed reliable scale-up and characterization parameters.

[0299] GC-MS analysis of KM encapsulated pea protein nanoparticles (KM-PP-TE-NP) obtained by cross-linking of tea extract: The amount of KM in the nanoparticle formulation was determined using GC-MS analysis. It was found to be quantitative, indicating 100% KM encapsulation. High KM encapsulation efficiency may be important for biodelivery of KM. The GC-MS column provides good separation of the starting material and various glycerides. Quantification of KM can be performed by using neat KM to obtain a calibration curve. The curve has good linearity and the analyzed concentrations fall within the calibration range. GC-MS can be used for quality control of the KM encapsulation process.

[0300] Blank protein nanoparticles obtained by crosslinking tea extract (PP-TE-NP): Blank pea protein nanoparticles obtained by crosslinking tea extract were synthesized in the absence of KM for comparison. They 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 indicated uniformly sized nanoparticles (unimodal distribution). The core size of the nanoparticles ( Fig.23 ). It shows good particles with size in the range of ~400 nm. Blank nanoparticles are comparable to KM encapsulated nanoparticles with a slight increase in size and potential.

[0301] Table 6: Characterization of KM-encapsulated pea protein nanoparticles (KM-PP-TE-NPs) obtained by ethanol desolvation method and tea extract cross-linking

[0302]

[0303] a = Tea leaves boiled at 100°C for tea extract (TE) (20 mg / mL)

[0304] Fluorescence spectra of KM-PP-TE-NPs and PP-TE-NPs: The fluorescence emission spectra of KM-PP-TE-NPs and PP-TE-NPs were recorded by excitation at 285 nm corresponding to tryptophan amino acid. Fig.25 ) in the emission spectrum of 100 μg / ml pea protein nanoparticles. A broad emission peak with a fluorescence intensity of 30 at 353 nm was seen in the emission spectrum of 100 μg / ml pea protein nanoparticles. Changes in the tryptophan microenvironment can be monitored by fluorescence spectroscopy. The fluorescence maximum peak and intensity of KM-PP-TE-NP and PP-TE-NP are comparable, indicating that there is not much change in the tryptophan region of pea protein nanoparticles. Since KM is water-soluble and hydrophilic, it does not interact with the hydrophobic tryptophan microenvironment of pea protein. Fluorescence measurements give insights into the interaction of pea protein with KM.

[0305] Example 10 - Cross-linking of pea protein with EGCG and encapsulation of NR molecules in pea protein cross-linked with EGCG

[0306] Synthesis of NR-encapsulated pea protein nanoparticles (NR-PP-TE-NP) by cross-linking with tea extract: A tea extract (TE) solution was prepared for nanoparticle synthesis by weighing 5 g of Darjeeling loose leaf tea and transferring it to a 1 L conical flask. 250 mL of distilled water was added to the flask, and the contents were boiled at 100 ° C for 10 minutes. The heating was stopped and the tea extract solution was cooled to room temperature. Insoluble tea residues were separated from the solution 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 conical flask with a stirring 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 another 30 minutes. Further, 100 ml of TE solution was added, and the mixture was stirred for another 2 hours. Then, 170 mL of anhydrous ethanol (one-third volume of 500 mL of water) was added to the contents of the flask while stirring vigorously. The reaction mixture was then stirred overnight. The NR-encapsulated pea protein nanoparticles (NR-PP-TE-NPs) obtained by cross-linking with tea extract were then characterized by TEM, DLS size and ZP measurements. The NR content was estimated by the above-mentioned GC-MS analysis and calibration with pure KM standards. The NR-PP-TE-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life. Blank pea protein nanoparticles (PP-TE-NPs) obtained by cross-linking with tea extract were synthesized according to the same procedure without the addition of NR.

[0307] Tea extract contains various polyphenols, such as flavonoids, epigallocatechin gallate (EGCG), and other catechins. Therefore, tea extract can effectively crosslink protein nanoparticles. Water-soluble pea protein nanoparticles (NR-PP-TE-NPs) encapsulating NR obtained by crosslinking tea extracts were implemented by an ethanol desolvation method. After the synthesis of the nanoparticles, any insoluble residues larger than 75 microns were removed by centrifugation / filtration. The NR-PP-TE-NPs were between 370-429 nm in size, with an average size of 415 nm (using DLS), and showed an average ζ potential of -14 mV (Table 7), indicating stability. The polydispersity index (PDI) of ~0.2 indicates nanoparticles of uniform size (unimodal distribution). Using negative staining techniques, the nanoparticle core size was estimated by TEM (Figure 24). It shows stable particles with a size range of ~400 nm. NR-PP-TE-NPs were formulated and characterized to define quality control parameters for scale-up and commercial-level production. Nanoparticles were initially formulated on a small scale (~10 mL). After characterization and quality control parameters were established, they were scaled up to ~1 L. NRs from duplicate batches were used for large-scale synthesis. Large-scale batches showed reliable scale-up and characterization parameters.

[0308] Blank protein nanoparticles obtained by crosslinking tea extract (PP-TE-NP): Blank pea protein nanoparticles obtained by crosslinking tea extract were synthesized in the absence of NR for comparison. They 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 uniformly sized nanoparticles (unimodal distribution). The core size of the nanoparticles ( Fig.16 ). It shows good particles with size in the range of ~300 nm. The blank nanoparticles are comparable to the NR encapsulated nanoparticles with a slight increase in size and potential.

[0309] Table 7a: Characterization of NR-encapsulated pea protein nanoparticles (NR-PP-TE-NPs) obtained by ethanol desolvation method and tea extract crosslinking

[0310]

[0311] a = Tea leaves boiled at 100°C for tea extract (TE) (20 mg / mL)

[0312] Example 11 - Cross-linking of pea protein with EGCG and encapsulation of caffeine molecules in pea protein cross-linked with EGCG

[0313] Synthesis of CA-encapsulated pea protein nanoparticles (CA-PP-TE-NP) by cross-linking of tea extract: A tea extract (TE) solution was prepared for nanoparticle synthesis by weighing 5 g of Darjeeling loose leaf tea and transferring it to a 1 L conical flask. 250 mL of distilled water was added to the flask, and the contents were boiled at 100 °C for 10 minutes. The heating was stopped and the tea extract solution was cooled to room temperature. Insoluble tea residues were separated from the solution 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 conical flask with a stirring 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 another 30 minutes. Further, 100 ml of TE solution was added, and the mixture was stirred overnight. The CA encapsulated pea protein nanoparticles (CA-PP-TE-NPs) obtained by tea extract cross-linking were then characterized by TEM, DLS size and ZP measurements. The CA-PP-TE-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life.

[0314] Tea extract contains various polyphenols such as flavonoids, epigallocatechin gallate (EGCG) and other catechins. Therefore, tea extract can effectively crosslink protein nanoparticles. Water-soluble pea protein nanoparticles encapsulated with CA (CA-PP-TE-NPs) were crosslinked using tea extract. After the synthesis of the nanoparticles, any insoluble residues larger than 75 microns in size were removed by centrifugation / filtration. CA-PP-TE-NPs had an average size of 330 nm (using DLS) and a zeta potential of -13 mV (Table 7b). The polydispersity index (PDI) of ~0.4 indicated fairly uniformly sized nanoparticles (unimodal distribution). The nanoparticle core size ( Fig. 24B ). It showed longitudinal particles with sizes in the range of ~500nm. CA-PP-TE-NPs were formulated and characterized to define quality control parameters for scale-up and commercial level production. Initially small scale (~10mL) nanoparticles were formulated. After establishing characterization and quality control parameters, they were scaled up to ~500mL. Large scale batches showed reliable scale-up and characterization parameters.

[0315] Table 7b: Characterization of CA-encapsulated pea protein nanoparticles (CA-PP-TE-NPs) obtained by cross-linking of tea extract

[0316]

[0317] a = Tea leaves boiled at 100°C for tea extract (TE) (20 mg / mL)

[0318] Example 12 - Cross-linking of pea protein with EGCG and encapsulation of theophylline molecules in pea protein cross-linked with EGCG

[0319] Synthesis of TH-encapsulated pea protein nanoparticles (TH-PP-TE-NP) by cross-linking of tea extract: A tea extract (TE) solution was prepared for nanoparticle synthesis by weighing 5 g of Darjeeling loose leaf tea and transferring it to a 1 L conical flask. 250 mL of distilled water was added to the flask, and the contents were boiled at 100 ° C for 10 minutes. The heating was stopped and the tea extract solution was cooled to room temperature. Insoluble tea residues were separated from the solution 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 conical flask with a stirring bar. 400 mL of distilled water was added to the flask, and the pea protein solution was stirred for 30 minutes. 10 g of TH was weighed and transferred to the flask. The solution was stirred for another 30 minutes. Further, 100 ml of TE solution was added, and the mixture was stirred overnight. The TH-encapsulated pea protein nanoparticles (TH-PP-TE-NPs) obtained by cross-linking of tea extract were then characterized by TEM, DLS size and ZP measurements.The TH-PP-TE-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life.

[0320] Tea extract contains various polyphenols such as flavonoids, epigallocatechin gallate (EGCG) and other catechins. Therefore, tea extract can effectively crosslink protein nanoparticles. Water-soluble pea protein nanoparticles encapsulating TH (TH-PP-TE-NPs) obtained by crosslinking using tea extract were implemented. After the synthesis of nanoparticles, any insoluble residues larger than 75 microns in size were removed by centrifugation / filtration. TH-PP-TE-NPs had an average size of 322 nm (using DLS) and a zeta potential of -12 mV (Table 7c). The polydispersity index (PDI) of ~0.4 indicated fairly uniformly sized nanoparticles (unimodal distribution). The nanoparticle core size ( Fig.24C ). It showed particles with sizes in the range of ~100 nm. TH-PP-TE-NPs were formulated and characterized to define quality control parameters for scale-up and commercial level production. Initially small scale (~50 mL) nanoparticles were formulated. After characterization and quality control parameters were established, they were scaled up to ~500 mL. Large scale batches showed reliable scale-up and characterization parameters.

[0321] Table 7c: Characterization of TH-encapsulated pea protein nanoparticles (TH-PP-TE-NPs) obtained by cross-linking of tea extract

[0322]

[0323] a = Tea leaves boiled at 100°C for tea extract (TE) (20 mg / mL)

[0324] Example 13 - Cross-linking of pea protein with quercetin and encapsulation of ketone molecules in pea protein cross-linked with quercetin

[0325] Synthesis of KM-encapsulated pea protein nanoparticles (KM-PP-BE-NP) by cross-linking of berry extract: An aqueous solution of blackberry extract (BE) was prepared for nanoparticle synthesis. 100 g of dried purple berry powder was weighed and transferred to a 1 L conical flask. 1000 mL of distilled water was added to the flask and the contents were boiled at 100°C for 15 minutes. The heating was then stopped and the solution was cooled to room temperature to allow any remaining insoluble berry powder residue to settle. The insoluble residue was separated from the purple solution by decanting and then filtering (using a filter with a 75 micron mesh or centrifuging). For nanoparticle synthesis, the filtered clear purple BE aqueous solution was used. 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 conical flask with a stirring bar. 1000 mL of BE extract aqueous solution was added to the flask and the contents were boiled at 100°C for 15 minutes. Then, 333 mL of anhydrous ethanol was added to the flask while stirring, and the contents of the reaction mixture were stirred overnight. The pea protein nanoparticles encapsulated with KM (KM-PP-BE-NPs) obtained by cross-linking with berry extract were characterized by TEM, DLS size and ZP measurements. The KM-PP-BE-NP solution was refrigerated (5±3°C) to extend the shelf life. Blank pea protein nanoparticles (PP-BE-NPs) obtained by cross-linking with berry extract were synthesized according to the same procedure without the addition of KM.

[0326] Quercetin proton NMR showed equivalent peaks at 7.7, 7.6, 6.9, 6.4 and 6.2 ppm. After reaction with pea protein, the double peak at 6.4 ppm was significantly reduced ( Fig.48 and Fig.49 ). NMR analysis clearly showed significant changes in quercetin, indicating that covalent interactions with proteins open up cross-linking pathways. The aromatic ring protons of the phytochemicals showed significant changes, indicating covalent interactions with proteins. These changes lead to cross-linking with protein amino functional groups. Quercetin proton NMR showed equivalent peaks at 7.7, 7.6, 6.9, 6.4, and 6.2 ppm. But after reaction with pea protein, the double peak at 6.4 ppm was significantly reduced.

[0327] Blackberry extract containing quercetin is a mixture of phytochemicals that contains various other active ingredients that can also play a role in the cross-linking and encapsulation process while adding nutritional value to the formulation.

[0328] Fig.491H NMR (expanded) of a reaction mixture of quercetin and water-soluble pea protein (PP) in CD3OD / D2O is shown (arrows indicate changes in protons).

[0329] Fig.50 The results show the reaction mixture of quercetin, water-soluble pea protein (PP) and KM in CD3OD / D2O. 1 H NMR (extended).

[0330] A naturally occurring plant-based material from blackberry was used to study cross-linking. Quercetin (QE), a phytochemical present in berries, can be extracted from berry extracts. The hydroxyl groups in QUE are effective in cross-linking proteins. Water-soluble pea protein nanoparticles (KM-PP-BE-NPs) encapsulated with KM with berry extract cross-linking were prepared using an ethanol desolvation method. Centrifugation / filtration was used to eliminate any insoluble residue after nanoparticle production. The KM-PP-BE-NPs ranged in size from 392-474 nm with an average of 433 nm (determined by DLS) and had an average zeta potential of -9 mV (Table 8a), indicating reasonable stability. A polydispersity index (PDI) of 0.3 indicated mostly uniformly sized nanoparticles (unimodal distribution). TEM was used to study the size of the nanoparticles using a negative staining technique ( Fig.27A ). It showed particle sizes in the range of ~100-500nm. KM-PP-BE-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial manufacturing. Initially, small-scale (~10mL) nanoparticles were formulated. After establishing characterization and quality control parameters, they were successfully scaled up to ~1L to test large-scale production. Large-scale batches showed reproducible scale-up and characterization parameters. These experiments helped define quality control parameters for scale-up and commercial-level production.

[0331] Table 8a: Characterization of KM encapsulated pea protein nanoparticles (KM-PP-BE-NPs) obtained by ethanol desolvation method and berry extract cross-linking.

[0332]

[0333] a = Powdered berry extract boiled at 100°C (25 mg / ml)

[0334] b = berry powdered extract boiled at 100°C (50 mg / ml)

[0335] Example 14 - Cross-linking of pea protein with quercetin and encapsulation of caffeine molecules in pea protein cross-linked with quercetin

[0336] Synthesis of CA-encapsulated pea protein nanoparticles (CA-PP-BE-NP) by cross-linking of berry extract: An aqueous solution of blackberry extract (BE) was prepared for nanoparticle synthesis. 25 g of dried purple berry powder was weighed and transferred to a 1 L conical flask. 500 mL of distilled water was added to the flask and the contents were boiled at 100°C for 15 minutes. The heating was then stopped and the solution was allowed to cool to room temperature to allow any remaining insoluble berry powder residue to settle. The insoluble residue was separated from the purple solution by decanting and then filtering (using a filter with a 75 micron mesh or centrifuging). For nanoparticle synthesis, a filtered clear purple BE aqueous solution was used. 25 g of water-soluble pea protein (PP) and 13 g of caffeine (CA) were weighed and transferred to a 1 L conical flask with a stirring 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-NPs) obtained by cross-linking of berry extracts were characterized by TEM, DLS size and ZP measurements. The CA-PP-BE-NP solutions were refrigerated (5±3°C) to extend the shelf life.

[0337] A naturally occurring plant-based material from blackberry was used to study cross-linking. Quercetin (QE), a phytochemical present in berries, can be extracted from berry extracts. The hydroxyl groups in QUE are effective in cross-linking proteins. Water-soluble pea protein nanoparticles (CA-PP-BE-NPs) encapsulated CA with berry extract cross-linking were prepared. Centrifugation / filtration was used to eliminate any insoluble residue after nanoparticle production. The CA-PP-BE-NPs had an average size of 386 nm (determined by DLS) and an average zeta potential of -14 mV (Table 8b), indicating reasonable stability. A polydispersity index (PDI) of 0.4 indicated fairly uniformly sized nanoparticles (unimodal distribution). TEM was used to study the size of the nanoparticles using a negative staining technique ( Fig.27C ). It showed particle sizes in the range of ~200nm. CA-PP-BE-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial manufacturing. Initially, small-scale (~10mL) nanoparticles were formulated. After establishing characterization and quality control parameters, they were successfully scaled up to ~500mL to test large-scale production. Large-scale batches showed reproducible scale-up and characterization parameters. These experiments helped define quality control parameters for scale-up and commercial-level production.

[0338] Table 8b: Characterization of CA encapsulated pea protein nanoparticles (CA-PP-BE-NPs) obtained by cross-linking of berry extracts.

[0339]

[0340] a = berry powder boiled at 100°C (50 mg / mL) for berry extract (BE)

[0341] Example 15 - Cross-linking of pea protein with quercetin and encapsulation of theophylline molecules in pea protein cross-linked with quercetin

[0342] Synthesis of TH-encapsulated pea protein nanoparticles (TH-PP-BE-NP) by cross-linking of berry extract: An aqueous solution of blackberry extract (BE) was prepared for nanoparticle synthesis. 25 g of dried purple berry powder was weighed and transferred to a 1 L conical flask. 500 mL of distilled water was added to the flask and the contents were boiled at 100 ° C for 15 minutes. The heating was then stopped and the solution was cooled to room temperature to allow any remaining insoluble berry powder residues to settle. The insoluble residues were separated from the purple solution by decanting and then filtering (using a filter with a 75 micron mesh or centrifuging). For nanoparticle synthesis, a filtered clear purple BE aqueous solution was used. 25 g of water-soluble pea protein (PP) and 13 g of theophylline (TH) were weighed and transferred to a 1 L conical flask with a stirring bar. 230 mL of BE extract aqueous solution and 230 mL of distilled water were added to the flask and the mixture was stirred overnight. TH-encapsulated pea protein nanoparticles (TH-PP-BE-NPs) obtained by cross-linking of berry extracts were characterized by TEM, DLS size and ZP measurements.TH-PP-BE-NP solutions were refrigerated (5±3°C) to extend the shelf life.

[0343] A naturally occurring plant-based material from blackberry was used to study cross-linking. Quercetin (QE), a phytochemical present in berries, can be extracted from berry extracts. The hydroxyl groups in QUE are effective in cross-linking proteins. Water-soluble pea protein nanoparticles (TH-PP-BE-NPs) encapsulated with TH with berry extract cross-linking were prepared. Centrifugation / filtration was used to eliminate any insoluble residue after nanoparticle production. The TH-PP-BE-NPs had an average size of 360 nm (determined by DLS) and an average zeta potential of -15 mV (Table 8c), indicating reasonable stability. A polydispersity index (PDI) of 0.3 indicated uniformly sized nanoparticles (unimodal distribution). TEM was used to study the size of the nanoparticles using a negative staining technique ( Fig.27D). It showed particle sizes in the range of ~100 nm. TH-PP-BE-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial manufacturing. Initially, small-scale (~10 mL) nanoparticles were formulated. After establishing characterization and quality control parameters, they were successfully scaled up to ~500 mL to test large-scale production. Large-scale batches showed reproducible scale-up and characterization parameters. These experiments helped define quality control parameters for scale-up and commercial-level production.

[0344] Table 8c: Characterization of TH encapsulated pea protein nanoparticles (TH-PP-BE-NPs) obtained by cross-linking of berry extracts.

[0345]

[0346] a = berry powder boiled at 100°C (50 mg / mL) for berry extract (BE)

[0347] Example 16 - Cross-linking of pea protein with resveratrol and encapsulation of ketone molecules in pea protein cross-linked with resveratrol

[0348] 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 together and the contents were boiled at 100° C. for 15 minutes. The heating was then stopped and the solution was cooled to room temperature to allow any remaining insoluble grape residues to settle. The insoluble residues were separated from the clarified solution by decantation and then filtration (using a filter with a 75 micron mesh or centrifugation). The filtered clarified grape extract aqueous solution was used to synthesize nanoparticles. 50.0 g of water-soluble pea protein (PP) and 300.0 g of ketone molecules (KM) were weighed and transferred to a 2L conical flask with a stirring bar. 1000 mL of GE extract aqueous solution was added to the flask and the contents were boiled at 100° C. for 15 minutes. Then, while stirring, 333 mL of anhydrous ethanol was added to the flask. The reaction mixture contents were stirred overnight. Pea protein nanoparticles encapsulating KM obtained by grape extract cross-linking (KM-PP-GE-NPs) were characterized by TEM, DLS size and ZP measurements. The KM-PP-GE-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life. Blank pea protein nanoparticles obtained by grape extract cross-linking (PP-BE-NPs) were synthesized following the same procedure without the addition of KM.

[0349] Fig.52 The results show that the reaction mixture of resveratrol and water-soluble pea protein (PP) in CD3OD / D2O 1 H NMR (expanded) (arrows indicate proton changes).

[0350] Fig.53 The results show the reaction mixture of resveratrol, water-soluble pea protein (PP) and KM in CD3OD / D2O. 1 HNMR (extended).

[0351] In the resveratrol proton NMR, after reaction with pea protein, 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 ( Fig.51 and Fig.52 ). NMR analysis clearly shows significant changes in resveratrol, indicating that covalent interactions with proteins open up crosslinking pathways. The aromatic ring protons of the phytochemicals show significant changes, indicating covalent interactions with proteins. These changes lead to crosslinking with protein amino functional groups. In the resveratrol proton NMR, the 6.8-6.7ppm region peaks show a significant change in the peak splitting pattern and a slight decrease in the peak at 6.2ppm after reaction with pea protein, indicating covalent interactions with proteins. Carbon NMR shows corresponding changes.

[0352] Resveratrol is a well-known antioxidant with an electroactive hydroxyl group facilitated by a planar structure due to double bonds. Moreover, grape extracts containing resveratrol are a mixture of phytochemicals containing various other active ingredients that can also play a role in the cross-linking and encapsulation process while adding nutritional value to the formulation.

[0353] The framework structure of grape extract (GE) was studied for nanoencapsulation of KM. Grape extract contains flavonoids and resveratrol as well as other polyphenols. Pea protein grape extract cross-linked nanoparticles (KM-PP-GE-NPs) encapsulating KM were produced by combining an aqueous solution of GE, pea protein and KM and stirring overnight. KM was absorbed or incorporated into the grape framework, which formed a nanomatrix. The size of KM-PP-GE-NPs ranged from 499-607nm, with an average size of 553nm (using DLS), and the average zeta potential was determined to be -10mV (Table 9), showing reasonable stability. The polydispersity index (PDI) of 0.2 indicated that the size of the nanoparticles was uniform and exhibited a unimodal distribution. TEM was used to study the size and morphology of the nanoparticles using a negative staining technique ( Fig.27B). It showed good particle size in the range of ~200-600nm. KM-PP-GE-NPs were formulated and fully characterized to establish quality control standards for scale-up and commercial production. Initially small-scale (~10mL) nanoparticles were formulated, and then production was scaled up to ~1L after establishing characterization and quality control parameters. For large-scale synthesis, KM from several batches was used for large-scale synthesis. Large-scale batches showed reliable scale-up and characterization parameters. These studies helped define quality control parameters for scale-up and commercial level production.

[0354] Table 9: Characterization of KM-encapsulated pea protein nanoparticles (KM-PP-GE-NPs) obtained by grape extract cross-linking

[0355]

[0356] a = Grape extract boiled at 100°C (25 mg / mL)

[0357] b = Grape extract boiled at 100°C (50 mg / mL)

[0358] Example 17 - Encapsulation of Ketone Molecules in Soy Protein Isolate

[0359] Synthesis and characterization of soy protein isolate nanoparticles encapsulating KM: Soy protein isolate (SPI) backbone was explored for nanoencapsulation of KM by desolvation method. Since soy protein was explored at the beginning, glutaraldehyde was used for crosslinking to check nanoparticle formation before utilizing plant-based phytochemicals. But mango peel, tea, berry and grape extracts can also be used for crosslinking nanoparticles. Soy protein isolate nanoparticles loaded with KM (KM-SPI-NP) were prepared by ethanol desolvation method. KM-SPI-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial level production. Various KM concentrations were explored for nanoparticle synthesis. KM-SPI-NP sizes were between 151-230nm (using DLS). A polydispersity index (PDI) of ~0.2 indicated uniformly sized nanoparticles (unimodal distribution). They showed zeta potentials between 35-53mV, indicating very good stability (Table 10). Using negative staining techniques, the nanoparticle core size ( Fig.35 ).

[0360] Table 10: Characterization of KM-SPI-NPs encapsulated with soy protein isolate obtained by ethanol desolvation method

[0361]

[0362] a = Add 100 μL of 5M NaOH to change pH 5 to pH 7 and dissolve SPI

[0363] b = Add 200 μL of 5M NaOH to change pH 4 to pH 7 and dissolve the SPI

[0364] c = 20 uL of 25% glutaraldehyde in water for cross-linking

[0365] Example 18 - Encapsulation of Ketone Molecules in Okra Extract Cross-linked with Okra Extract

[0366] Synthesis of KM encapsulated okra extract nanoparticles (KM-OE-NPs): 10 g of okra extract (OE) powder and 25 g of ketone molecule (KM) were weighed and transferred to a 1 L conical flask with a stirring 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-NPs) were characterized by TEM, DLS size and ZP measurements. The KM content was estimated by GC-MS analysis and calibration with pure KM standards. The KM-OE-NPP solution was refrigerated (5°C ± 3°C) to extend the shelf life. Blank okra extract (OE) without the addition of KM was used for comparison with KM-OE-NPs.

[0367] Example 19 - Encapsulation of Ketone Molecules in Okra Extract

[0368] Okra extract (OE) skeleton was explored for nanoencapsulation of KM. KM okra extract nanoparticles were prepared by mixing an aqueous solution of OE and KM and stirring overnight. KM was absorbed or embedded into okra mucus or gel (a form of nanomatrix). The KM-OE-NP size was 158nm (using DLS) and showed a zeta potential of -22mV (Table 11), indicating reasonable stability. The polydispersity index (PDI) of 0.6 indicated that the nanoparticles were not very uniformly sized (departing from a unimodal distribution). The nanoparticle core size ( Fig.31). It showed good particles with sizes in the range of ~100-500nm. KM-OE-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial level production. Initially, small-scale (~10mL) nanoparticles were formulated. After establishing characterization and quality control parameters, they were scaled up to ~1L. KM from various batches were used for large-scale synthesis. Ascorbic acid was also added as a preservative. Therefore, the size and zeta potential changed. The size was 280nm (using DLS) and showed a zeta potential of -9mV (Table 11). Large-scale batches showed reliable scale-up and characterization parameters. These experiments help define quality control parameters for scale-up and commercial level production.

[0369] Blank okra extract: A blank aqueous okra extract without KM was also characterized for comparison. It 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 indicated heterogeneous size of the okra gel nanomatrix (multimodal distribution). The core size of the okra extract ( Fig.31 ). It showed sparse particles with size in the range of ~100 nm. KM encapsulated okra extract nanoparticles (KM-OE-NPs) showed more particles with better polydispersity value compared to blank okra extract.

[0370] Table 11: Characterization of KM okra gel nanoparticles (KM-OE-NPs)

[0371]

[0372] a = Ascorbic acid added as a preservative for 500 ml scale

[0373] GC-MS Analysis of KM Encapsulated Okra Extract Nanoparticles (KM-OE-NP): GC-MS analysis was used to determine the amount of KM in the nanoparticle formulation. It was found to be quantitative indicating 100% KM encapsulation. High KM encapsulation efficiency may be important for biodelivery of KM. The GC-MS column provided good separation of the starting material and various glycerides. Quantification of KM can be done by using tributyrin (GTB) standards and obtaining a calibration curve. The curve should have good linearity and the analyzed concentration should fall within the calibration range. GC-MS can be used for quality control of the KM encapsulation process.

[0374] Example 20 - Encapsulation of NR molecules in okra extract cross-linked with okra extract.

[0375] Synthesis of NR encapsulated okra extract nanoparticles (NR-OE-NPs): 10 g of okra extract (OE) powder (Table 3) and 25 g of NR were weighed and transferred to a 1 L conical flask with a stirring bar. 500 mL of distilled water was added to the flask and the contents were stirred for 1 hour. 2 g of ascorbic acid (preservative) was weighed and transferred to the flask and stirred overnight. The resulting NR encapsulated okra extract (NR-OE-NPs) were characterized by TEM, DLS size and ZP measurements. The NR-OE-NP solution was refrigerated (5°C ± 3°C) to extend the shelf life. Blank okra extract (OE) without NR added was used for comparison with NR-OE-NPs.

[0376] Okra extract (OE) skeleton was explored for nanoencapsulation 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 into okra mucus or gel (a form of nanomatrix). NR-OE-NP size was 149nm (using DLS) and showed a zeta potential of -17mV (Table 12), indicating reasonable stability. The polydispersity index (PDI) of 0.5 indicated that the nanoparticles were not very uniformly sized (departing from a unimodal distribution). The nanoparticle core size ( Fig.32 ). It showed good particles with sizes in the range of ~200nm. NR-OE-NPs were formulated and fully characterized to define quality control parameters for scale-up and commercial level production. Initially small-scale (~10mL) nanoparticles were formulated. After establishing characterization and quality control parameters, they were scaled up to ~1L. NR from various batches were used for large-scale synthesis. Ascorbic acid was also added as a preservative. However, the size and zeta potential did not change much. The size was 162nm (using DLS) and showed a zeta potential of -9mV (Table 8). Large-scale batches showed reliable scale-up and characterization parameters. These experiments help define quality control parameters for scale-up and commercial level production.

[0377] Blank okra extract: A blank aqueous okra extract without NR was also characterized for comparison. It 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 heterogeneous size of the okra gel nanomatrix (multimodal distribution). The core size of the okra extract ( Fig.32 ). It showed sparse particles with size in the range of ~100 nm. NR encapsulated okra extract nanoparticles (NR-OE-NPs) showed more particles with better polydispersity value compared to blank okra extract.

[0378] Table 12: Characterization of NR okra gel nanoparticles (NR-OE-NPs)

[0379]

[0380] a = Ascorbic acid added as a preservative for 500 ml scale

[0381] Example 21 - Folin-Ciocalteu analysis of total phenolics of pea protein nanoparticles encapsulating KM obtained by cross-linking of plant-based (mango peel, tea, berry and grape extracts)

[0382] Total phenolic content can be an important component of beverages, supplements, functional foods, food additives and nutraceuticals industries, providing flavor, color and organoleptic qualities such as bitterness and astringency, odor and haze formation during storage. Phenols can exhibit antioxidant activity due to their extensive conjugated π-electron system, which promotes the donation of electrons or hydrogen atoms from hydroxyl moieties to free radicals. The oxygen radical absorption capacity (ORAC) of whole foods, juices and food additives has previously been used as an industrial 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 / polyphenols that exhibit physiological functions in the human diet is also associated with a reduced risk of developing chronic diseases induced by oxidative stress. Therefore, they limit the growth of carcinogenic tumors, reduce the risk of cardiovascular disease, and have antibacterial, anti-inflammatory, anti-obesity, anti-diabetic, anti-aging, anti-spasmodic and anti-diarrhea properties. In addition, the energy-enhancing KM within the nanoencapsulation platform contains many health-promoting ingredients, including antioxidant polyphenols from mango peel, tea, berries and grape extracts. Products rich in antioxidants are important because they protect the body's cells from the destructive effects of free radicals (which can damage proteins, lipids and DNA), thereby reducing and preventing the development of various degenerative diseases such as cancer and coronary heart disease. Phenols provide health benefits through several mechanisms, including the elimination of free radicals and the protection and regeneration of other dietary antioxidants (such as vitamin E), acting as a group of protective and valuable plant ingredients due to their high antioxidant activity and healing properties. The total phenolic content was determined using the Folin–Ciocalteu method, which involves the reduction of the Folin–Ciocalteu reagent with the polyphenols present in the nanoformulation. The total phenolic content was quantified using total gallic acid equivalents (GAE), the most commonly used reference molecule. The total phenolic content of KM-PP-MP-NP was 236 mg GAE / g, the total phenolic content of KM-PP-TE-NP was 127 mg GAE / g, the total phenolic content of KM-OE-NP was 205 mg GAE / g, the total phenolic content of KM-PP-BE-NP was 324 mg GAE / g, the total phenolic content of KM-PP-GE-NP was 275 mg GAE / g, and the total phenolic content of KM-PP-NP was 12 mg GAE / g. Fig.28 shown.

[0383] Example 22: Cellular toxicity study of KM and KM-encapsulated pea protein nanoparticles obtained by cross-linking tea extract (KM-PP-TE-NP)

[0384] Cellular toxicity studies of KM and KM-encapsulated pea protein nanoparticles obtained by cross-linking tea extract (KM-PP-TE-NPs) were performed using human aortic endothelial cells (HAECs). The cell proliferation assay kit uses 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a tetrazolium dye used to measure cell viability. Using the MTT assay, the cell viability of KM and KM-encapsulated nanoparticles was measured. HAECs after 24, 48, and 72 hours (hr) of incubation with KM showed no toxicity in the concentration range of 81-1300 μg / mL. This Fig.26A Pea protein isolate nanoparticles encapsulating KM cross-linked with tea extract (KM-PP-TE-NP) were also synthesized. HAECs incubated with KM-PPI-NPs for 24, 48, and 72 hours showed no toxicity in the concentration range of 81-650 μg / mL. Fig.26B The toxic concentration of KM encapsulated nanoparticles was slightly lower compared to free KM. This observation inferred enhanced bioavailability of KM in KM-PP-TE-NPs. The KM concentration in KM-PP-MP-NPs was used to plot the concentration values.

[0385] Example 23: Cellular toxicity study of KM and KM-encapsulated pea protein nanoparticles obtained by cross-linking mango peel (KM-PP-MP-NP)

[0386] Cellular assay toxicity studies of KM and KM-encapsulated pea protein nanoparticles obtained by crosslinking mango peel (KM-PP-MP-NP) were performed using human aortic endothelial cells (HAEC). The cell proliferation assay kit uses 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a tetrazolium dye used to measure cell viability. Using the MTT assay, the cell viability of KM and KM-encapsulated nanoparticles was measured. HAECs after 24, 48, and 72 hours (hr) of incubation with KM showed no toxicity in the concentration range of 9-142 μg / mL. This was Fig.22A Pea protein isolate nanoparticles encapsulating KM cross-linked with mango peel (KM-PP-MP-NP) were also synthesized. HAEC incubated with KM-PPI-NPs for 24, 48, and 72 hours showed no toxicity in the concentration range of 9-71 μg / mL. Fig. 22B The toxic concentration of nanoparticles encapsulating KM was slightly lower compared to free KM. This observation inferred enhanced bioavailability of KM in KM-PP-MP-NPs. The KM concentration in KM-PP-MP-NPs was used to plot the concentration values.

[0387] Although preferred embodiments of the present invention have been shown and described herein, 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, those skilled in the art will now appreciate that many variations, changes and replacements are possible. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention. The following claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for encapsulating a plurality of small molecules, the method comprising: a) obtaining protein and one or more phytochemicals from plant sources; and b) 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 comprising encapsulated small molecules.

2. The method of claim 1, wherein the plurality of small molecules comprises a plurality of ketone molecules.

3. The method of claim 1, wherein the plurality of small molecules comprises a plurality of nicotinamide riboside molecules or nicotinamide riboside analogs. The method of claim 1 , wherein the plurality of small molecules comprises a plurality of caffeine molecules. The method of claim 1 , wherein the plurality of small molecules comprises a plurality of theophylline molecules.

6. The method of any one of the preceding claims, wherein the plant-derived protein is from a first source and the one or more phytochemicals are from a second source different from the first source.

7. The method of claim 6, wherein the first source is from a first plant species and the second source is from a second plant species.

8. The method according to any one of the preceding claims, wherein the protein of vegetable origin is pea protein.

9. The method according to any one of the preceding claims, wherein the protein of vegetable origin is banana protein, okra protein or soy protein.

10. The method of claim 9, wherein the vegetable-derived protein is soy protein, wherein the soy protein is soy protein.

11. The method of any one of the preceding claims, wherein the one or more phytochemicals are derived from mango, tea, okra, berries or grapes.

12. The method of any one of the preceding claims, wherein the one or more phytochemicals are derived from mango peel powder, dried tea leaves, okra extract powder, berry extract, or grape extract.

13. The method according to any one of the above claims, wherein the one or more phytochemicals comprises mangiferin, catechin or quercetin.

14. The method of claim 13, wherein the one or more phytochemicals comprises quercetin, and the weight ratio of the one or more phytochemicals to the plant-derived protein is between 1:10,000 and 1:40,000.

15. The method of claim 13, wherein the one or more phytochemicals comprises mangiferin, and the weight ratio of the one or more phytochemicals to the plant-derived protein is between 1:30 and 1:

50.

16. The method of claim 13, wherein the one or more phytochemicals comprises a catechin, wherein the catechin is epigallocatechin gallate.

17. The method of claim 16, wherein the weight ratio of the one or more phytochemicals to the plant-derived protein is between 1:200 and 1:

400.

18. The method of any one of the above claims, wherein the one or more phytochemicals comprise polyphenols.

19. The method of claim 18, wherein the polyphenols comprise one or more of flavonoids and resveratrol.

20. The method of claim 19, wherein the weight ratio of the one or more phytochemicals to the plant-derived protein is between 1:300 and 1:

500.

21. The method of any of the above claims, wherein the mixture comprises a dry weight ratio of the plurality of small molecules to plant-derived protein of between 1 :1 and 1 :

5.

22. A method according to any one of the preceding claims, wherein the mixture comprises a weight ratio of protein of vegetable origin to water of between 1:50 and 1:100 or between 1:10 and 1:

100.

23. The method of any of the above claims, further comprising (c) mixing the mixture with ethanol.

24. The method according to claim 23, wherein (c) is performed by adding the ethanol to the mixture at a volume ratio of the ethanol to the mixture of 1:10 to 1:

1.

25. The method according to claim 23, wherein (c) is performed by adding ethanol to the mixture at a volume ratio of the ethanol to the mixture of 1:10 to 1:

5.

26. The method of claim 23, wherein (c) is performed by adding ethanol to the mixture at a volume ratio of the ethanol to the mixture of 1:4 to 1:

2.

27. The method of any one of claims 23-26, further comprising (d) removing the ethanol via evaporation of the ethanol (eg, at 25°C to 40°C).

28. The method of any one of the preceding claims, wherein the particles have an average diameter greater than 80 nm.

29. A method according to any one of the preceding claims, wherein the particles have an average diameter of less than 700 nm.

30. A method according to any one of the preceding claims, wherein the particles have an average diameter of between 100 nm and 500 nm or between 300 nm and 500 nm.

31. The method of any of the above claims, further comprising cross-linking the phytochemical to a plant protein.

32. The method of claim 31 , wherein the phytochemical is cross-linked to the water-soluble plant protein via an imine linkage.

33. The method of claim 31 , wherein the cross-linking is performed without the addition of an aldehyde.

34. The method of claim 33, wherein the aldehyde is glutaraldehyde.

35. The method of claim 31, wherein the cross-linking occurs at one or more functional amino acid groups of the plant protein.

36. The method of claim 33, wherein the functional amino acid group comprises lysine.

37. The method of any of the above claims, wherein the mixture comprises a soluble portion and an insoluble portion.

38. The method of claim 37, further comprising separating the soluble portion from the insoluble portion.

39. The method of claim 38, wherein separating the soluble portion from the insoluble portion comprises filtering the insoluble portion from the soluble portion.

40. The method of any one of claims 1-2 or 6-39, wherein the plurality of small molecules comprises (R)-1,3 dihydroxypropan-2-yl 3-hydroxybutyrate.

41. The method of any one of claims 1-2 or 6-40, wherein at least 50% of the small molecules are (R)-1,3 dihydroxypropan-2-yl 3-hydroxybutyrate.

42. The method of any one of the preceding claims, wherein at least 50% of the small molecules comprise a glyceryl backbone conjugated to 3-hydroxybutyrate.

43. The method of any one of claims 1 or 3 or 6-39, wherein the plurality of small molecules is nicotinamide riboside.

44. The method of any one of claims 1 or 3 or 6-39 or 43, wherein at least 50% of the small molecules are nicotinamide riboside.

45. The method of any one of claims 1 or 4 or 6-39, wherein the plurality of small molecules is caffeine.

46. ​​The method of any one of claims 1 or 4 or 6-39 or 45, wherein at least 50% of the small molecules are caffeine.

47. The method of any one of claims 1 or 5-39, wherein the plurality of small molecules is theophylline.

48. The method of any one of claims 1 or 5-39 or 47, wherein at least 50% of the small molecules are theophylline.

49. The process of any one of the preceding claims, wherein (b) is performed at 20°C to 30°C.

50. The method of any one of the preceding claims, wherein (b) is performed at a pH of 3.5 to 11.

0.

51. The method of any one of the preceding claims, wherein (b) is performed at a pH of 3.5 to 7.

0.

52. The method of any one of the preceding claims, wherein (b) is performed at a pH of 5.0 to 9.

0.

53. The method of any of the above claims, wherein (b) is performed at a pH of 7.0 to 11.

0.

54. The method of any of the above claims, wherein the plant-derived protein is water-soluble.

55. The method of claim 54, wherein the vegetable-derived protein is at least 95% water soluble.

56. The method of claim 54, wherein the vegetable-derived protein is at least 98% water soluble.

57. The method of claim 54, wherein the vegetable-derived protein is at least 99% water soluble.

58. A nanoencapsulation composition formed by the method of any of the preceding claims.

59. A composition comprising small molecules encapsulated within a plant protein cross-linked with one or more phytochemicals.

60. The composition of claim 59, wherein the plant protein is pea protein.

61. The composition of any one of claims 59-60, wherein the plant protein is banana protein, okra protein or soy protein.

62. The composition of claim 61, wherein the plant protein is soy protein, wherein the soy protein is soy protein.

63. The composition of any one of claims 59-62, wherein the one or more phytochemicals are derived from mango, tea leaves, okra, berries, or grapes.

64. The composition of any one of claims 59-63, wherein the one or more phytochemicals are derived from mango peel powder, dried tea leaves, okra extract powder, berry extract, or grape extract.

65. The composition of any one of claims 59-64, wherein the one or more phytochemicals comprise mangiferin, catechin, or quercetin.

66. The composition of claim 65, wherein the one or more phytochemicals is a catechin, wherein the catechin is epigallocatechin gallate.

67. The composition of any one of claims 59-62, wherein the one or more phytochemicals comprise polyphenols.

68. The composition of claim 67, wherein the polyphenol comprises one or more of flavonoids and resveratrol.

69. The composition of any one of claims 59-68, wherein the one or more phytochemicals are cross-linked to the plant protein via an imine linkage.

70. The composition of any one of claims 59-69, wherein the cross-linking occurs at the functional amino acid groups of the plant protein.

71. The composition of claim 70, wherein the functional amino acid group comprises lysine.

72. The composition of any one of claims 59-71, wherein the small molecule comprises a ketone molecule.

73. The composition of claim 72, wherein the ketone molecule comprises (R)-1,3 dihydroxypropan-2-yl 3-hydroxybutyrate.

74. The composition of any one of claims 59-71, wherein the small molecule comprises a nicotinamide riboside molecule or an analog thereof.

75. The composition of any one of claims 59-71, wherein the small molecule comprises a caffeine molecule.

76. The composition of any one of claims 59-71, wherein the small molecule comprises a theophylline molecule.

77. A composition according to any one of claims 59-74, wherein the particles have an average diameter greater than 80 nm.

78. The composition of claims 59-77, wherein the particles have an average diameter of less than 700 nm.

79. A composition according to any one of claims 59-78, wherein the particles have an average diameter between 100 nm and 500 nm or between 300 nm and 500 nm.

80. The composition of any one of claims 59-79, wherein the composition has a phenolic content greater than 10 mg GAE / g.

81. The composition of any one of claims 59-80, wherein the composition has a phenolic content less than or equal to 350 mg GAE / g.

82. The composition of any one of claims 59-81, wherein the composition has a phenolic content between 10 and 500 mgGAE / g.

83. A composition according to any one of claims 59-82, wherein the composition has a phenolic content between 100 and 400 mgGAE / g.

84. A composition according to any one of claims 59-83, wherein the composition has a phenolic content between 200 and 300 mgGAE / g.

85. The composition of any one of claims 59-84, wherein the composition does not comprise glutaraldehyde.

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