Protein particles, and methods for producing and using them.

Abstract

Particles comprising a protein and optionally an activator, for example, particles comprising whey protein hydrolysate and optionally tryptophan, are described herein. Methods for preparing and using particles comprising a protein and optionally an activator are also described herein.

Description

[Technical Field]

[0001] Statement on Electronic Submission of Sequence Listings An XML array list (4,495 bytes in size) with the filename 1213-9WO_ST26.xml, generated on April 2, 2024, and submitted herein, is incorporated herein by reference in its entirety.

[0002] [Technical field] The present invention relates to particles comprising a protein and optionally an activator, as well as to methods for producing and using such particles. [Background technology]

[0003] L-tryptophan (Trp) is a nonpolar aromatic essential amino acid and can be obtained from dietary proteins. It is a precursor to important biomolecules such as serotonin, melatonin, tryptamine, niacin, quinolinic acid, and kynurenic acid-nicotinamide adenine dinucleotide. Therefore, Trp plays a crucial role in regulating neurobehavioral processes, such as appetite, mood, sleep, cognition, pain perception, and behavior. Supplementation with this amino acid has been demonstrated to be effective in the medical treatment of various diseases, including depression, sleep disorders, cognitive impairment, anxiety disorders, and neurodegenerative diseases. In addition, Trp has demonstrated angiotensin-converting enzyme (ACE) inhibitory activity, antioxidant activity, antidiabetic activity, and satiety-promoting activity (Nongonierma & FitzGerald, 2015). Due to these properties, Trp is considered an essential dietary component. However, due to the pronounced bitterness of tryptophan, which is caused by aromatic and hydrophobic residues, its uses are limited. Of the free amino acids, Trp has the lowest bitterness threshold (BTT: 4 mmol / L) (Di Pizio & Nicoli, 2020 Molecules (Basel, Switzerland), 25(20), doi.org / 10.3390 / molecules25204623). The bitterness of Trp may lead to reduced intake of Trp-containing nutritional supplements and limit its use in food ingredients. [Overview of the project] [Means for solving the problem]

[0004] A first aspect of the present invention relates to a method for preparing particles of the present invention, comprising: preparing a protein in a composition (for example, dissolved and / or suspended in an aqueous composition); adjusting the pH of the composition to a basic pH, then adjusting the pH of the composition to an acidic pH; and then optionally heating the composition, thereby forming particles containing the protein.

[0005] A second aspect of the present invention relates to a method for preparing particles of the present invention, comprising: mixing a protein and an activator to form a mixture; adjusting the pH of the mixture to a basic pH; then adjusting the pH of the mixture to an acidic pH; and then optionally forming particles comprising the protein and the activator.

[0006] A further aspect of the present invention is a particle directed toward the above particle, the particle comprising a protein; and optionally an activator. In some embodiments, when the activator is present in the particle, the activator is present within the protein (e.g., within the tertiary structure of the protein). In some embodiments, when the activator is present in the particle, the activator is nonspecifically bound to the protein (e.g., via hydrophobic interactions, electrostatic interactions and / or hydrogen bonds, etc.).

[0007] An additional aspect of the present invention is directed towards the plurality of particles of the present invention.

[0008] A further aspect of the present invention is directed toward compositions comprising a carrier (e.g., water and / or oil) and particles of the present invention (e.g., particles prepared according to the method of the present invention). In some embodiments, the composition is a dispersion.

[0009] Further aspects of the present invention are directed toward articles comprising the particles and / or compositions of the present invention. In some embodiments, such articles are foods (e.g., infant formula, dairy products, etc.), nutritional supplements, therapeutic drinks, and / or cosmetics.

[0010] It should be noted that any aspect described in relation to one embodiment may be incorporated into a different embodiment, even if not specifically described in that embodiment. That is, all embodiments and / or features of any embodiment may be combined in any way and / or in any combination. The applicant reserves the right to modify the initially filed claims and / or to file new claims accordingly, including the right to modify the initially filed claims to depend on and / or incorporate features of any other claim or set of claims, even if not initially claimed so. These and other aspects and / or features of the present invention are described in detail in the specification below. Further features, advantages and details of the present invention will be understood by those skilled in the art by reading the accompanying drawings and the subsequent detailed description of preferred embodiments, but such description is merely illustrative of the present invention. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a graph showing the particle size distribution of WPI-Trp nanoparticles formed by heat treatment at 70°C for 60 minutes after a pH shift to either 11 to 6 or 11 to 7. [Figure 2] Figure 2 is a graph showing the effect of pH shift alone and heat treatment after pH shift on the particle size of the WPI-Trp mixture. Different letters indicate a statistically significant difference (p<0.05). [Figure 3] Figure 3 is a graph showing the intrinsic fluorescence spectra of the WPI-Trp mixture with only a pH shift applied, and the intrinsic fluorescence spectra of the WPI-Trp mixture after heat treatment following the pH shift. [Figure 4] Figure 4 is a schematic diagram of the proposed mechanism of Trp aggregation-induced fluorescence emission. [Figure 5]Figure 5 is a graph showing the particle size and polydispersity index (PDI) of suspensions containing native whey protein isolate (WPI) particles, WPI-Trp nanoparticles with a pH shift only from 11 to 6, and WPI-Trp nanoparticles heat-treated at 50 °C, �0 °C, 70 °C, or 80 °C for 60 minutes after a pH shift from 11 to 6. [Figure 6] Figure 6 is a graph showing the particle size of a pH-shifted (from pH 11 to pH 6) WPI-Trp mixture heated at various temperatures (50 / 60 / 70 / 80 °C). [Figure 7] Figure 7 is a graph showing the intrinsic fluorescence spectrum of a pH-shifted (from pH 11 to pH 6) WPI-Trp mixture heated at various temperatures (50 / 60 / 70 / 80 °C). [Figure 8] Figure 8 shows a graph based on the intensity percentage or volume percentage of the particle size distribution of a WPI-Trp sample that was pH-shifted from pH 11 to pH 6 and heat-treated at 70 °C for 0 to 60 minutes. [Figure 9] Figure 9 shows a graph based on the intensity percentage or volume percentage of the particle size distribution of a WPI sample that was pH-shifted from pH 11 to pH 6 and heat-treated at 70 °C for 0 to 60 minutes. [Figure 10] Figure 10 shows a graph based on the intensity percentage or volume percentage of the particle size distribution of a Trp sample that was pH-shifted from pH 11 to pH 6 and heat-treated at 70 °C for 0 to 60 minutes. [Figure 11] Figure 11 shows a graph based on the intensity percentage or volume percentage of the particle size distribution of a WPI-Trp sample at pH 6 heat-treated at 70 °C for 0 to 60 minutes without a pH shift. [Figure 12]Figure 12 is a graph showing the turbidity of samples of WPI-Trp, WPI, and Trp that were pH-shifted from pH 11 to pH 6 and heat-treated at 70 °C for 0 to 60 minutes. [Figure 13] Figure 13 is a graph showing the turbidity of samples of WPI-Trp, WPI, and Trp at pH 6 that were heat-treated at 70 °C for 0 to 60 minutes without a pH shift. [Figure 14] Figure 14 is a graph showing the particle size and polydispersity index (PDI) for both WPI-Trp nanoparticles and WPI nanoparticles after heat treatment at 70 °C for 0, 10, 20, 30, 40, 50, or 60 minutes following a pH shift from 11 to 6. [Figure 15] Figure 15 shows graphs of the intrinsic fluorescence spectra of samples of WPI-Trp (Figure 15, panel A), WPI (Figure 15, panel B), and Trp (Figure 15, panel C) that were pH-shifted from pH 11 to pH 6 and heat-treated at 70 °C for 0 to 60 minutes. [Figure 15-1] Figure 15 shows graphs of the intrinsic fluorescence spectra of samples of WPI-Trp (Figure 15, panel A), WPI (Figure 15, panel B), and Trp (Figure 15, panel C) that were pH-shifted from pH 11 to pH 6 and heat-treated at 70 °C for 0 to 60 minutes. [Figure 16] Figure 16 is a graph showing the particle size of a WPI-Trp mixture at pH 6, a WPI-Trp mixture with only heating, a WPI-Trp mixture with only a pH shift from pH 11 to pH 6, and WPI-Trp NPs that were pH-shifted from pH 11 to pH 6 in combination with heating. Different letters indicate significant differences (p < 0.05). [Figure 17] Figure 17 is a graph of the intrinsic fluorescence spectra of a WPI-Trp mixture at pH 6, a WPI-Trp mixture with only heating, a WPI-Trp mixture with only a pH shift from pH 11 to pH 6, and WPI-Trp NPs that were pH-shifted from pH 11 to pH 6 in combination with heating. [Figure 18]Figure 18 is a graph showing the particle size distribution of WPI-Trp nanoparticles formed by shifting the pH from 11 to 6, followed by heat treatment at 70°C for 20 minutes. [Figure 19] Figure 19 is a graph showing the particle size distribution of WPI-Trp nanoparticles formed by heat treatment at 70°C for 20 minutes without a pH shift from 11 to 6. [Figure 20] Figure 20 shows SEM images of the WPI-Trp mixture after different treatments, before and after freeze-drying. Figure 20, Panel A shows a freeze-dried WPI-Trp sample at pH 6 (FD, WPI-Trp mixture at pH 6: FD, WPI-Trp mixture, pH 6), Figure 20, Panel B shows a freeze-dried WPI-Trp sample after heat treatment (70°C, 20 mins) at pH 6 (FD, WPI-Trp mixture with heating: FD, WPI-Trp mixture with heating), Figure 20, Panel C shows a freeze-dried WPI-Trp sample after a pH shift from 11 to 6 (FD, WPI-Trp mixture with pH-shifting: FD, WPI-Trp mixture with pH shifting), Figure 20, Panel D shows a freeze-dried WPI-Trp sample obtained by a pH shift from 11 to 6 followed by heat treatment (70°C, 20 mins) (FD, WPI-Trp mixture with pH-shifting and heating: WPI-Trp mixture Figure 20, Panel E shows a WPI-Trp sample obtained by heat treatment at pH 6 (70°C, 20 min) before freeze-drying (WPI-Trp mixture with heating), and Figure 20, Panel F shows WPI-Trp NPs before freeze-drying: pH shift (from 11 to 6) followed by heat treatment (70°C, 20 min) (WPI-Trp Nps: WPI-Trp nanoparticles). [Figure 21] Figure 21 is a graph showing the surface hydrophobicity (H0) of WPI-Trp mixtures treated with different methods. Different letters indicate statistically significant differences (p<0.05). [Figure 22]Figure 22 is a graph showing the surface hydrophobicity (H0) of WPI treated with different methods. Different letters indicate a statistically significant difference (p<0.05). [Figure 23] Figure 23 is a graph showing the free sulfhydryl (SH) group content of WPI-Trp mixtures treated with different methods. Different letters indicate statistically significant differences (p<0.05). [Figure 24] Figure 24 is a graph showing the free sulfhydryl (SH) group content of WPI treated with different methods. Different letters indicate statistically significant differences (p<0.05). [Figure 25] Figure 25 is a graph of the circular dichroism (CD) spectra of WPI-Trp that have undergone different processing. [Figure 26] Figure 26 shows the circular dichroism (CD) spectra of WPI that have undergone different processing. [Figure 27] Figure 27 shows the FTIR spectra of WPI-Trp NP, natural WPI, and natural Trp. [Figure 28] Figure 28 is a graph showing the DPPH radical scavenging capacity of WPI-Trp at pH 6 after heating at 70°C for 20 minutes only, WPI-Trp at pH 6 after only a pH shift from pH 11 to pH 6, and WPI-Trp at pH 6 after a pH shift from pH 11 to pH 6 and heating at 70°C for 20 minutes. [Figure 29] Figure 29 is a graph showing the DPPH radical scavenging capacity of the WPI natural product at pH 6 after only heating at 70°C for 20 minutes, the WPI natural product at pH 6 after only a pH shift from pH 11 to pH 6, and the WPI natural product at pH 6 after a pH shift from pH 11 to pH 6 and heating at 70°C for 20 minutes. [Figure 30]Figure 30 is a graph showing the DPPH radical scavenging capacity of the Trp natural product at pH 6 after being heated at 70°C for 20 minutes only, the Trp natural product at pH 6 after being pH-shifted from pH 11 to pH 6 only, and the Trp natural product at pH 6 after being pH-shifted from pH 11 to pH 6 and then heated at 70°C for 20 minutes. [Figure 31] Figure 31 is a graph of the ABTS radical scavenging capacity of WPI-Trp at pH 6 and 0.05 mg / ml after heating at 70°C for 20 minutes only, WPI-Trp at pH 6 and 0.05 mg / ml after only a pH shift from pH 11 to pH 6, and WPI-Trp at pH 6 and 0.05 mg / ml after a pH shift from pH 11 to pH 6 and heating at 70°C for 20 minutes. Different letters indicate a statistically significant difference (p<0.05). [Figure 32] Figure 32 is a graph of the ABTS radical scavenging capacity of the WPI natural product at pH 6 and 0.05 mg / ml after heating at 70°C for 20 minutes only, after pH shifting from pH 11 to pH 6 only, and after pH shifting from pH 11 to pH 6 and heating at 70°C for 20 minutes. Different letters indicate statistically significant differences (p<0.05). [Figure 33] Figure 33 is a graph of the ABTS radical scavenging capacity for Trp at pH 6 and 0.05 mg / ml after heating at 70°C for 20 minutes only, Trp at pH 6 and 0.05 mg / ml after only a pH shift from pH 11 to pH 6, and Trp at pH 6 and 0.05 mg / ml after a pH shift from pH 11 to pH 6 and heating at 70°C for 20 minutes. Different letters indicate a statistically significant difference (p<0.05). [Figure 34]Figure 34 shows the molecular docking complexes of α-LA-Trp (Figure 34, Panel A), β-LG-Trp (Figure 34, Panel B), and BSA-Trp (Figure 34, Panel C), and also shows details of the binding sites of α-LA-Trp, β-LG-Trp, and BSA-Trp. The green dashed lines indicate hydrogen bonds, and the red-colored amino acid residues indicate hydrophobic interactions with Trp. [Figure 35] Figure 35 is a schematic diagram of the proposed mechanism for the formation of WPI-Trp nanoparticles. [Figure 36] Figure 36 is a flowchart illustrating an exemplary process for preparing whey protein isolate-tryptophan nanoparticles using ultrafiltration or nanofiltration. [Modes for carrying out the invention]

[0012] Herein, the present invention is described herein with reference to the accompanying drawings and examples illustrating embodiments of the invention. This description is not intended to be a detailed catalog of all different ways in which the invention can be carried out or all features that may be added to the invention. For example, a feature illustrated in relation to one embodiment may be incorporated into another embodiment, and a feature illustrated in relation to a particular embodiment may be omitted from that embodiment. Accordingly, the present invention is intended to show that in some embodiments of the invention, any one or combination of features described herein may be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, but they do not depart from the invention. Accordingly, the following description is intended to illustrate some specific embodiments of the invention and not to exhaustively specify all permutations, combinations, and variations thereof.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Terms used in the description of the invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention.

[0014] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety with respect to the teachings relating to the sentences and / or paragraphs in which they are referenced.

[0015] Unless the context indicates otherwise, it is particularly intended that the various features of the present invention described herein may be used in any combination. Furthermore, in some embodiments of the present invention, it is also intended that any feature or combination of features described herein may be excluded or omitted. For example, when this specification states that a composition comprises component A, component B, and component C, it is particularly intended that A, B, or C, or any combination thereof, may be omitted and excluded.

[0016] When used in the detailed description of the present invention and the appended claims, the singular forms "a, an" and "the" are intended to also encompass the plural forms unless the context clearly indicates otherwise.

[0017] Furthermore, as used herein, “and / or” means and includes any and all possible combinations of one or more related items listed, and, where interpreted as an alternative ("or"), the absence of any combination.

[0018] As used herein, the term "approximately" when referring to a measurable value, such as quantity or concentration, means to include not only the specified value but also variations of ±10%, ±5%, ±1%, ±0.5%, and even ±0.1% of the specified value. For example, "approximately X" means that if X is a measurable value, it includes X, as well as variations of X of ±10%, ±5%, ±1%, ±0.5%, or ±0.1%. The range of measurable values ​​provided herein may include any other range and / or individual values ​​within that range.

[0019] When used herein, phrases such as "between X and Y" and "about X and Y" should be interpreted as including X and Y. When used herein, phrases such as "about X and Y" mean "about X and about Y," and phrases such as "about X to Y" mean "about X to about Y."

[0020] The ranges of values ​​described herein are intended to function simply as abbreviations for individually referring to each individual value that falls within the range, unless otherwise stated herein, and each individual value is incorporated herein as if it were individually described herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13 and 14 are also disclosed.

[0021] As used herein, the terms “comprises” and “comprising” identify the presence of the described features, integers, processes, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof.

[0022] When used herein, the transitional phrase "essentially" applied to the compositions of the present invention should be interpreted as encompassing the specific materials or processes described in the claim and one or more essential and novel features of the claimed invention that do not substantially affect them. Accordingly, the word "essentially" as used in the claims of the present invention is not intended to be interpreted as equivalent to "includes".

[0023] As used herein, the words “increase,” “grow,” “boost,” “enhance,” “improve,” and “improve” (and their grammatical variations) represent an increase of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more, compared to another measurable characteristic or quantity (e.g., a control value).

[0024] As used herein, the words “reduction,” “reduced,” “to reduce,” “to lessen,” and “decrease” (and their grammatical variations) represent, for example, a reduction of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% compared to another measurable characteristic or quantity (e.g., a control value). In some embodiments, the reduction may result in no detectable activity or quantity at all, or essentially no activity at all (i.e., an insignificant amount, e.g., less than about 10%, or even less than 5%).

[0025] A "portion" or "fragment" of a nucleotide sequence or polypeptide (including a domain) is a nucleotide sequence or polypeptide (e.g., one or more nucleotides or one or more peptides) of a length reduced from that of the respective reference nucleotide sequence or reference polypeptide (e.g., a reduction of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues), and the reference nucleotide sequence or reference polypeptide Each of these should be understood to mean a sequence of nucleotides or polypeptides consisting of consecutive residues that are identical or nearly identical (for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical), essentially comprising and / or consisting of the above nucleotide sequences or polypeptides.

[0026] As used herein, “sequence identity” means the degree to which two optimally aligned polynucleotide sequences or polypeptide sequences remain invariant across an alignment window of their constituent elements, such as nucleotides or amino acids. "Identity" can be readily calculated by known methods, including but not limited to those described in the following literature: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0027] As used herein, the terms “percent sequence identity” or “percent identity” mean the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule compared to a test ("subject") polynucleotide molecule (or its complementary chain) when the two sequences are optimally aligned. In some embodiments, “percent identity” may mean the percentage of identical amino acids in the amino acid sequence compared to a reference polypeptide.

[0028] When used herein, in the context of two nucleic acid molecules, nucleotide sequences, or protein sequences, the phrase "substantially identical" or "substantial identity" is used. Identity refers to two or more sequences or subsequences that, when compared and aligned to obtain the greatest possible correspondence, have at least approximately 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identity of nucleotides or amino acid residues, as measured using one of the sequence comparison algorithms below or by visual inspection. In some embodiments of the present invention, the substantial identity exists over a region of consecutive nucleotides in the nucleotide sequence of the present invention, in terms of length, ranging from about 10 to about 20 nucleotides, about 10 to about 25 nucleotides, about 10 to about 30 nucleotides, about 15 to about 25 nucleotides, about 30 to about 40 nucleotides, about 50 to about 60 nucleotides, about 70 to about 80 nucleotides, about 90 to about 100 nucleotides, or more nucleotides, and any range up to the full length of the sequence. In some embodiments, the nucleotide sequence can be substantially identical over at least about 20 nucleotides (for example, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40 nucleotides).In some embodiments, substantially identical nucleotides or protein sequences perform substantially the same function as the nucleotide (or encoded protein sequence) that is substantially identical to them.

[0029] In sequence comparison, typically one sequence acts as a reference sequence, which is compared to a test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, and, if necessary, the subset coordinates and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage for one or more test sequences relative to the reference sequence, based on the specified program parameters.

[0030] The optimal alignment of sequences for aligning the comparison window is well known to those skilled in the art and may be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally these algorithms, e.g., GCG 登録商標 Wisconsin Package 登録商標This may be performed by computerized implementations of GAP, BESTFIT, FASTA, and TFASTA, available as part of Accelrys Inc., San Diego, CA. The "identity fraction" of the aligned segments of the test sequence and the reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (e.g., the entire reference sequence or a smaller defined portion thereof). The sequence identity percentage is expressed as the identity fraction multiplied by 100. Comparison of one or more polynucleotide sequences may be performed against the full-length polynucleotide sequence or a portion thereof, or against a longer polynucleotide sequence. For the purposes of this invention, the "identity percentage" may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0031] Particles comprising a protein and optionally an activator are provided according to embodiments of the present invention. When the particles of the present invention comprise a protein and an activator, the activator may be associated with the protein. In some embodiments, the particles of the present invention comprise a protein and an activator associated with the protein. In some embodiments, the particles of the present invention comprise a protein but not an activator. In some embodiments, when an activator is present in the particles of the present invention, the activator is present within the protein present in the particles of the present invention, and / or the activator is present on the surface of the protein present in the particles of the present invention. In some embodiments, the activator is present within the tertiary structure of the protein. In some embodiments, the activator is nonspecifically bound to the protein, for example, via hydrophobic interactions, electrostatic interactions, hydrogen bonds, etc. One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) protein molecules (e.g., one or more protein monomers) may be present in the particles of the present invention. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) activators are present between two or more protein molecules that are associated with each other (e.g., via nonspecific interactions).

[0032] The particles of the present invention may contain one or more (e.g., 1, 5, 10, 20, 30, 40, 50, or more) activators, which may be identical or different from each other, and / or one or more (e.g., 1, 5, 10, 20, 30, 40, 50, or more) protein molecules, which may be identical or different from each other. In some embodiments, the particles of the present invention contain an activator located within a region of the tertiary structure of the protein (e.g., within a tertiary fold). In some embodiments, the activator is located within a region of the tertiary structure of the protein (e.g., within a tertiary fold), which includes at least one nonspecific hydrophobic interaction between two or more amino acid residues. In some embodiments, the activator is located within a hydrophobic pocket of the protein. In some embodiments, the activator is located within the protein core of the protein. In some embodiments, the activator is located within a folded region of the protein, which may have zero solvent accessibility. In some embodiments, when the particles of the present invention contain a plurality of activators (wherein the plurality of activators may be the same or different from one another), at least one of the plurality of activators may be present within the protein, and one or more of the plurality of activators may be present on the surface of the protein.

[0033] The exemplary proteins of the present invention include, but are not limited to, dairy proteins (e.g., milk proteins), plant proteins, and / or animal (e.g., meat) proteins. As used herein, “dairy proteins,” “milk proteins,” “plant proteins,” “animal proteins,” and “meat proteins” refer to proteins found naturally in dairy products, milk, plants, animals, and meat, and / or proteins derived from such naturally occurring proteins having an amino acid sequence that has at least 70% sequence identity with the amino acid sequence of the naturally occurring protein. For example, in some embodiments, dairy proteins, milk proteins, plant proteins, animal or meat proteins are found in dairy products, milk, plants, animals, or meat, respectively, and / or the proteins are isolated from dairy products, milk, plants, animals, or meat, respectively, or the proteins are synthetically prepared to have an amino acid sequence that has at least 70% sequence identity with the amino acid sequence of the naturally occurring protein. In some embodiments, the protein comprises milk proteins, for example, but not limited to, α-lactalbumin, β-lactoglobulin, and / or lactoferrin. In some embodiments, whey protein isolate (WPI), which may contain α-lactalbumin and β-lactoglobulin and lactoferrin, is used to prepare the particles of the present invention. In some embodiments, the whey protein isolate (WPI) contains about 50%, about 55%, about 60%, about 65%, about 70%, about 80%, or about 90% β-lactoglobulin and / or about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% α-lactalbumin. In some embodiments, the particles of the present invention may contain whey protein isolate (WPI) and may not contain activators. In some embodiments, the particles of the present invention contain one or more α-lactalbumins and one or more β-lactoglobulins.

[0034] The protein used to prepare the particles of the present invention may have a molten globule state and / or a bilobal structure. The protein may contain two or more (e.g., 2, 3, 4, 5, or more) domains and / or, at pH from about 5 to about 9, the protein may contain one or more (e.g., 1, 2, 3, 4, or more) intramolecular disulfide bonds. In some embodiments, the protein contains at least two domains and the protein contains at least one disulfide bridge connecting the two domains of the protein. In some embodiments, the protein is monomeric. In some embodiments, the protein has about 100 amino acids to about 200, about 300, about 400, or about 500 amino acids and / or a molecular weight from about 10 kDa to about 20, about 30, about 40, or about 50 kDa. The protein may have about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acids. In some embodiments, the protein has a molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, or about 50 kDa. The protein may have an isoelectric point (pI) from about 4, about 4.1, about 4.2, about 4.3, or about 4.4 to about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5. In some embodiments, the protein has a pI ranging from about 4.2 to about 4.5 or about 5.2.The protein comprises α-helices in amounts of approximately 10% or 15% to 20%, 25%, or 30% of the total tertiary structure and / or total secondary structure (which may be arbitrarily calculated by the percentage of amino acids present in the α-helices relative to the total number of amino acids in the protein), β-sheets in amounts of approximately 1% or 5% to 10%, 15%, 20%, or 25% of the total tertiary structure and / or total secondary structure (which may be arbitrarily calculated by the percentage of amino acids present in the β-sheets relative to the total number of amino acids in the protein), and approximately 1% or 5% to 10%, 15% of the total tertiary structure and / or total secondary structure. The structure may have a β-turn in an amount of %, approximately 20%, or approximately 25% (which may be arbitrarily calculated by the percentage of amino acids present in the β-turn relative to the total number of amino acids in the protein), and / or an unordered tertiary structure and / or secondary structure in an amount ranging from approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, or approximately 60% to approximately 65%, approximately 70%, or approximately 75% of the total tertiary structure and / or total secondary structure (which may be arbitrarily calculated by the percentage of amino acids present in the unordered tertiary structure and / or secondary structure relative to the total number of amino acids in the protein).

[0035] The particles of the present invention may contain multiple proteins. In some embodiments, the particles of the present invention contain about 5, about 10 or about 20 to about 25, about 30, about 40 or about 50 protein molecules (e.g., protein monomers). In some embodiments, the particles of the present invention contain about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 protein molecules (e.g., protein monomers). The particles of the present invention may contain protein in a total amount (e.g., one or more protein molecules) of about 75% by weight, about 80% by weight or about 85% to about 90% by weight, about 95% by weight, about 99% by weight or about 100% by weight of the particles. In some embodiments, protein may be present in the particles in an amount of about 100% by weight of the particles. In some embodiments, the activator may be present in the particles in a total amount (e.g., one or more activators) ranging from about 0% by weight, about 0.1% by weight, about 0.5% by weight, about 1% by weight, or about 5% by weight to about 10% by weight, about 15% by weight, about 20% by weight, or about 25% by weight. In some embodiments, the activator may be present in the particles in an amount ranging from about 10% by weight, about 11% by weight, about 12% by weight, about 13% by weight, or about 14% by weight to about 15% by weight, about 16% by weight, about 17% by weight, about 18% by weight, about 19% by weight, or about 20% by weight. In some embodiments, the particles of the present invention comprise a total amount of protein (e.g., one or more protein molecules) of about 75% by weight, about 80% by weight, or about 85% to about 90% by weight, about 95% by weight, about 99% by weight, or about 100% by weight of the particles, and an activator (e.g., one or more activators) of a total amount of about 0% by weight, about 0.1% by weight, about 0.5% by weight, about 1% by weight, or about 5% to about 10% by weight, about 15% by weight, about 20% by weight, or about 25% by weight of the particles.

[0036] Furthermore, exemplary proteins that may be present in the particles of the present invention include, but are not limited to, whey protein isolate, α-lactalbumin, lysozyme, cytochrome c, apomyoglobin, staphylococcal nuclease, β-lactoglobulin, lactoferrin, and any combination thereof. In some embodiments, the protein that may be present in the particles of the present invention is whey protein isolate. The protein of the present invention may be from any source (e.g., plants, animals, etc.). In some embodiments, the protein is obtained from and / or derived from an animal source, e.g., mammals (e.g., cattle or humans). In some embodiments, the particles of the present invention contain about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 whey protein isolate molecules. In some embodiments, the proteins present in the particles of the present invention have amino acid sequences with approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% sequence identity with one or more of the sequence ID numbers: 1 to 3. In some embodiments, the proteins present in the particles of the present invention have amino acid sequences with at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity with one or more of the sequence ID numbers 1 to 3. In some embodiments, the proteins present in the particles of the present invention have amino acid sequences with about 100% sequence identity with one or more of the sequence ID numbers 1 to 3.

[0037] The activator used to prepare the particles of the present invention may be an organic compound, such as, but not limited to, an amino acid. In some embodiments, the activator has a molecular weight ranging from about 70 g / mol, about 100 g / mol, about 150 g / mol or about 200 g / mol to about 250 g / mol, about 300 g / mol, about 400 g / mol or about 500 g / mol. The activator may have a solubility in water at 25°C of about 15 mg / mL or less, for example, about 15 mg / mL, about 14 mg / mL, about 13 mg / mL, about 12 mg / mL, about 11 mg / mL, about 10 mg / mL, about 9 mg / mL, about 8 mg / mL, about 7 mg / mL, about 6 mg / mL or about 5 mg / mL, or less. In some embodiments, the activator may have solubility in water at 25°C at concentrations of approximately 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL to approximately 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / L. In some embodiments, the activator has a pKa of approximately 1.5, 2, 2.5, 2.6, 2.7, or 2.8 to approximately 2.9, 3, 3.2, or 3.5 and / or a pI of approximately 5, 5.5, 5.6, 5.7, or 5.8 to approximately 5.9, 6, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the activator has a solubility of about 10 mg / mL in water at 25°C or below, and / or a pKa of about 2.7 or about 2.8 to about 2.9 or about 3, and / or a pI of about 5.7 or about 5.8 to about 5.9, about 6 or about 6.1. Exemplary activators include, but are not limited to, amino acids (e.g., tryptophan, leucine, phenylalanine, cysteine ​​and / or tyrosine), vitamin E, and any combination thereof. In some embodiments, the activator present in the particles of the present invention is tryptophan.

[0038] The particles of the present invention may have a size (e.g., diameter) in at least one dimension ranging from about 50 nm, about 75 nm, or about 100 nm to about 125 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm, when optionally measured using microscopy (e.g., scanning electron microscopy (SEM) and / or transmission electron microscopy (TEM)) and / or dynamic light scattering (DLS). In some embodiments, the particles have a size (e.g., diameter) in at least one dimension of about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm. In some embodiments, the particles have a size (e.g., diameter) in at least one dimension of about 125 nm. In some embodiments, the particles have a size (e.g., diameter) in at least one dimension of about 110 nm. In some embodiments, the particles are nanoparticles. In some embodiments, the plurality of particles of the present invention, particles prepared according to the method of the present invention, and / or particles present in the composition of the present invention have a Dv(50) ranging from about 50, about 75, or about 100 nm to about 125, about 150, or about 200 nm when optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS. In some embodiments, the plurality of particles of the present invention have a Dv(50) of about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, or about 200 nm when optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS. In some embodiments, the plurality of particles of the present invention have a polydispersity index (PDI) of less than about 0.5, optionally less than about 0.3.In some embodiments, the plurality of particles of the present invention have a polydispersity index (PDI) of about 0.2.

[0039] In some embodiments, the particles of the present invention have a spherical structure. In some embodiments, the particles of the present invention have a rod-like structure. In some embodiments, the particles of the present invention have a cube-like structure. In some embodiments, the particles of the present invention have a loose matrix structure. In some embodiments, each of the plurality of particles of the present invention, when present in a composition (e.g., a liquid, e.g., water, a buffer, milk (e.g., skim milk), and / or an acidic whey beverage), has the same or different structure, selected from a spherical structure, a rod-like structure, a cube-like structure, and / or a loose matrix structure. In some embodiments, the particles of the present invention consist of α-helices in amounts of about 10% or about 15% to about 20%, about 25%, or about 30% of the total tertiary structure and / or total secondary structure (arbitrarily calculated by the percentage of amino acids present in the α-helices relative to the total number of amino acids in the protein), β-sheets in amounts of about 1% or about 5% to about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% of the total tertiary structure and / or total secondary structure (arbitrarily calculated by the percentage of amino acids present in the β-sheets relative to the total number of amino acids in the protein), and the total tertiary structure and / or total secondary structure The structure may have a β-turn in an amount of approximately 1% or 5% to approximately 10%, 15%, 20%, or 25% of the total structure (arbitrarily calculated by the percentage of amino acids present in the β-turn relative to the total number of amino acids in the protein), and / or a disordered tertiary structure and / or secondary structure in an amount of approximately 30%, 35%, 40%, 45%, 50%, 55%, or 60% to approximately 65%, 70%, or 75% of the total tertiary structure and / or total secondary structure (arbitrarily calculated by the percentage of amino acids present in the disordered tertiary structure and / or secondary structure relative to the total number of amino acids in the protein).In some embodiments, exposure to a temperature of about 70°C for about 20 minutes maintains that the amount of one or more of the α-helices, β-sheets, β-turns, and disordered tertiary structures and / or secondary structures present in the particles of the present invention remains within about ±5%, about ±10%, about ±15%, about ±20%, about ±25%, about ±30%, or about ±35% of the respective amounts of the α-helices, β-sheets, β-turns, and disordered tertiary structures and / or secondary structures present in the particles before exposure (for example, the respective amounts of the α-helices, β-sheets, β-turns, and disordered tertiary structures and / or secondary structures present in the particles at the time of initial formation and / or immediately before exposure).

[0040] In some embodiments, the particles of the present invention may have a free sulfhydryl (SH) group content of about 5 μmol SH / g, about 6 μmol SH / g, about 7 μmol SH / g, about 8 μmol SH / g, about 9 μmol SH / g or about 10 μmol SH / g to about 11 μmol SH / g, about 12 μmol SH / g, about 13 μmol SH / g, about 14 μmol SH / g or about 15 μmol SH / g, when optionally measured using Elman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) i.e., DTNB) and UV-Vis spectroscopy. In some embodiments, the particles of the present invention may have a free sulfhydryl (SH) group content of about 5 μmol SH / g, about 6 μmol SH / g, about 7 μmol SH / g, about 8 μmol SH / g, about 9 μmol SH / g, about 10 μmol SH / g, about 11 μmol SH / g, about 12 μmol SH / g, about 13 μmol SH / g, about 14 μmol SH / g, or about 15 μmol SH / g when optionally measured using Elman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) i.e., DTNB) and UV-Vis spectroscopy. In some embodiments, the particles of the present invention may have a free sulfhydryl (SH) group content of about 4 × 10⁻¹⁶ when optionally measured using 8-anilino-1-naphthalene sulfonate (ANS) as a fluorescent probe. 7 ~Approx. 7×10 7It may have surface hydrophobicity. In some embodiments, particles of the present invention comprising a protein and an activator may have increased intrinsic fluorescence intensity compared to the intrinsic fluorescence intensity of the protein and / or activator alone (for example, when the protein and / or activator are not present in the particles of the present invention).

[0041] In some embodiments, the protein and / or activator used to prepare the particles of the present invention is soluble in water at a temperature of about 25°C and a pH of about 11. In some embodiments, the protein and / or activator is soluble in water at a temperature of about 25°C and a pH of about 11 in amounts ranging from about 25 mg / mL, about 30 mg / mL, about 40 mg / mL or about 45 mg / mL to about 50 mg / mL, about 100 mg / mL, about 150 mg / mL, about 200 mg / mL, about 250 mg / mL, about 300 mg / mL, about 350 mg / mL or about 400 mg / L. The protein and / or activator may have a negative charge in water at a pH of about 11.

[0042] Particles of the present invention comprising a protein and optionally an activator may have improved (e.g., increased) storage, stability, activity, and / or function compared to the protein alone (e.g., the protein is not present in the particles of the present invention and is not associated with the optional activator). Particles of the present invention comprising a protein and an activator may have improved (e.g., increased) storage, stability, activity, and / or function compared to the protein alone (e.g., the protein is not present in the particles of the present invention and is not associated with the activator). In some embodiments, when stored in a closed container at about 4°C to about 10°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months, the size of the particles in at least one dimension (e.g., diameter) remains within about ±20% of its original size (e.g., size at initial formation of the particles and / or size on day 1 of storage). For example, at an initial point (e.g., the start of day 1 of the storage period), the particles may have a diameter of about 125 nm, and after storage in a sealed container at about 4°C to about 10°C for about 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months from the start of day 1 of the storage period, the particles may have an increased or decreased size of about 20% or less. Thus, particles having a starting size of about 125 nm may have a size in the range of about 100 nm to about 150 nm at the end of the storage period. In some embodiments, after storage in a sealed container at about 4°C to about 10°C for about 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, the particles of the present invention have a size (e.g., diameter) in at least one dimension that has increased by less than about 20% compared to their original size. In some embodiments, after heating (for example, heating at a temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, or about 85°C for a period of about 10 minutes, about 20 minutes, or about 30 minutes to about 60 minutes, about 90 minutes, or about 120 minutes), the size of the particle in at least one dimension (e.g., diameter) is maintained within about ±20% of its original size (e.g., the size of the particle during its initial formation and / or the size immediately before heating).In some embodiments, the particles may have a diameter of about 125 nm in the initial state before heating, and after heating at a temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C or 90°C for a period of about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes or about 30 minutes to about 60 minutes, about 90 minutes or about 120 minutes, the particles may have an increased or decreased size of about 20% or less. In some embodiments, dried particles (e.g., freeze-dried and / or spray-dried particles and / or particles containing about 0% to about 5% by weight of water) are stored in a sealed container at about 4°C to about 10°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months, and optionally, at the end of the storage period, the size (e.g., diameter) of the dried particles is measured, and / or the dried particles are reconstituted (e.g., dissolved and / or dispersed) in a composition (e.g., water and / or buffer), and the size (e.g., diameter) of the particles in the composition is measured. In some embodiments, particles present in a composition (e.g., water and / or buffer) are stored in a sealed container at approximately 4°C to approximately 10°C for approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, or approximately 6 months, and optionally, the size (e.g., diameter) of the particles in the composition is measured at the end of the storage period.

[0043] In some embodiments, the particles and / or multiple particles of the present invention are stable in that they have a single peak particle size distribution in a composition at pH about 3, a composition at pH about 7, and / or a composition at pH about 11, when optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS. In some embodiments, a composition containing multiple particles (e.g., a composition having pH values ​​of about 3, about 7, and / or 11) has two or more peak particle size distributions when optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS, indicating that the multiple particles are unstable. In some embodiments, the particles of the present invention are stable in that they do not detach (e.g., precipitate) and / or aggregate from the composition of the present invention. In some embodiments, the particles of the present invention are stable in that they do not detach (e.g., precipitate) and / or aggregate from the composition of the present invention when the pH of the composition is adjusted (e.g., from pH value of about 11 to pH value of about 3).

[0044] In some embodiments, the particles of the present invention and / or the proteins therein have increased activity and / or function (e.g., increased antioxidant activity) compared to the activity and / or function of proteins not provided in the particles of the present invention. In some embodiments, the particles of the present invention have improved function. In some embodiments, the particles of the present invention enhance the properties of the proteins and / or activity present in the particles. For example, in some embodiments, the particles of the present invention have increased activity and / or function (e.g., increased antioxidant activity) when optionally measured by Di Pizio & Nicoli, 2020 Molecules (Basel, Switzerland), 25 (20), doi.org / 10.3390 / molecules25204623 (which is incorporated herein) for a method of measuring bitterness and / or BTT. The bitterness of an activator (e.g., tryptophan) can be reduced by increasing the threshold. For example, the particles of the present invention can provide a BTT of about 4 mmol / L, about 5 mmol / L, or about 6 mmol / L to about 7 mmol / L or about 8 mmol / L with respect to the activator.

[0045] According to several embodiments, compositions comprising particles of the present invention are provided. In some embodiments, the composition comprises a plurality of particles of the present invention. In some embodiments, the composition comprises particles of the present invention and a carrier. The carrier may be a liquid, such as, but not limited to, water and / or oil. In some embodiments, the carrier is a food-grade ingredient, such as, but not limited to, alcohol (e.g., ethanol, e.g., in amounts of about 5% to about 20%). One or more additives may be present in the composition of the present invention. Exemplary additives are, but not limited to, pectin and / or gum. In some embodiments, the composition of the present invention does not contain masking agents, flavorings, cyclodextrins (e.g., β-cyclodextrin), and / or physical barriers optionally configured to mask or reduce the taste of activators that may be present in the particles of the present invention. In some embodiments, the composition is not a gel (e.g., hydrogel, e.g., protein hydrogel) and / or is not an emulsion. In some embodiments, the particles of the present invention are not present in a gel (e.g., hydrogel, e.g., protein hydrogel) or emulsion. In some embodiments, the composition of the present invention does not contain any agents configured and / or designed to reduce the bitterness and / or off-flavor of any activators that may be present in the composition, rather than agents configured and / or designed to provide a desired flavor or taste of the composition.

[0046] In some embodiments, the compositions of the present invention are foods, nutritional supplements, therapeutic drinks and / or cosmetics. In some embodiments, the particles of the present invention may be present in foods. In some embodiments, the foods are dairy products (e.g., milk, yogurt, etc.). The particles may be present in the compositions and / or articles of the present invention in an amount of about 1% by weight, about 2% by weight, about 3% by weight, about 4% by weight, about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, about 25% by weight or about 30% by weight of the compositions and / or articles.

[0047] When the activator is present in the particles of the present invention, when it is present in the carrier and / or composition of the present invention, it may remain associated with the particles and / or proteins present in the particles (e.g., by forming complexes or being present within them). In some embodiments, about 30% or less of the total amount of activator added to the carrier and / or composition is present in the carrier and / or composition free (i.e., not associated with the particles and / or proteins). For example, about 0%, about 1%, about 2%, about 5%, about 10%, or about 15% to about 20%, about 25%, or about 30% of the activator present in the particles of the present invention, which is provided in the carrier and / or composition and / or used to prepare the particles of the present invention, may be present in the carrier and / or composition as free activator (i.e., activator not associated with the particles and / or proteins). Therefore, in the case of particles containing an activator in a given amount, when the particles are added to a carrier and / or composition, the amount of free activator present in the carrier and / or composition may be about 30% or less of the given amount of activator present in the particles. In some embodiments, a composition comprising water and the particles of the present invention (the particles present in an amount of about 100 mg per 1 mL of water) contains the free activator in an amount ranging from about 0% by weight, about 1% by weight, about 2% by weight, about 5% by weight, about 10% by weight or about 15% by weight to about 20% by weight, about 25% by weight or about 30% by weight of the total amount of activator present in the particles. The composition of the present invention may be a dispersion (e.g., a colloidal dispersion). In some embodiments, the composition of the present invention does not have visible aggregates in the composition (e.g., it does not contain visible clumps, aggregates, or particulates). In some embodiments, the composition is transparent, not cloudy or opaque. In some embodiments, the composition may appear cloudy, but sediments and / or aggregates are not present.

[0048] Methods for preparing particles of the present invention are provided according to several embodiments of the present invention. In some embodiments, the method of the present invention includes providing a protein in a composition (e.g., a protein dissolved and / or suspended in an aqueous composition (e.g., a solution)); adjusting the pH of the composition to a basic pH, then adjusting the pH of the composition to an acidic pH; and then optionally heating the composition to form particles containing the protein. In some embodiments, the particles do not contain an activator. In some embodiments, the particles contain the protein and an activator. In some embodiments, the method of the present invention includes putting the protein and the activator together to form a mixture; adjusting the pH of the mixture to a basic pH, then adjusting the pH of the mixture to an acidic pH; and then optionally heating the mixture to form particles containing the protein and the activator.

[0049] In some embodiments, the composition containing the protein is an aqueous composition. In some embodiments, the mixture containing the protein and the activator is an aqueous composition. In some embodiments, the protein and the optional activator may be dissolved in the aqueous composition used in the method of the present invention. In some embodiments, the protein and / or the activator may be dissolved in the aqueous composition. In some embodiments, the mixture used in the method of the present invention contains protein and activator in a weight ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1 or about 15:1 to about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1 or about 30:1 (protein:activator). In some embodiments, the mixture contains the protein and the activator in a weight ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, or about 30:1 (protein:activator). In some embodiments, the mixture contains the protein and the activator in a weight ratio of about 5:1 to about 20:1 (protein:activator). In some embodiments, the mixture contains the protein and the activator in a weight ratio of about 5:1 (protein:activator). In some embodiments, the mixture used in the method of the present invention has a total solids content ranging from about 1 w / v%, about 2 w / v%, about 3 w / v%, about 4 w / v%, or about 5 w / v% to about 6 w / v%, about 7 w / v%, about 8 w / v%, about 9 w / v%, or about 10 w / v%. In some embodiments, the mixture has a total solids content of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the mixture has a total solids content of about 2.5 w / v%.

[0050] In some embodiments, the activator may be present in the composition (e.g., mixture) used in the method of the present invention in amounts of 0 mg / mL, about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL or from about 50 mg / mL to about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL or about 100 mg / mL, and / or the protein may be present in the composition in amounts of about 1 mg / mL, about 5 mg / mL It may be present in amounts of mL, approximately 10 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 75 mg / mL, approximately 80 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, approximately 95 mg / mL, or from approximately 100 mg / mL to approximately 110 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, approximately 140 mg / mL, approximately 150 mg / mL, approximately 160 mg / mL, approximately 170 mg / mL, approximately 180 mg / mL, approximately 190 mg / mL, or approximately 200 mg / mL. For example, in some embodiments, the activator (e.g., tryptophan) may be dissolved in the mixture of the present invention in an amount of about 30 mg / mL to about 50 mg / mL, and the protein (e.g., WP) may be dissolved in the same mixture in an amount of about 50 mg / mL or about 75 mg / mL to about 100 mg / mL, about 150 mg / mL, or about 200 mg / L. In some embodiments, WPI is dissolved in the mixture of the present invention in an amount of about 200 mg / mL or less, for example, in an amount of about 50 mg / mL to about 100 mg / mL. In some embodiments, WPI is dissolved in the mixture of the present invention in an amount of about 50 mg / mL or less, for example, in an amount of about 10 mg / mL to about 50 mg / mL.

[0051] In some embodiments, adjusting the pH of a composition (e.g., a mixture) used in the method of the present invention to a basic pH includes adjusting the pH of the composition from about 8, about 9, or about 10 to about 11, about 12, or about 13. In some embodiments, adjusting the pH of the composition to a basic pH includes adjusting the pH of the composition to about 11. In some embodiments, adjusting the pH of a composition used in the method of the present invention to an acidic pH includes adjusting the pH of the composition to less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, or less than about 2. In some embodiments, adjusting the pH of the composition to an acidic pH includes adjusting the pH of the composition from about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5 to about 5.5, about 6, or about 6.5. In some embodiments, adjusting the pH of the composition to an acidic pH includes adjusting the pH of the composition to about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, or about 6.5. In some embodiments, adjusting the pH of the composition to an acidic pH includes adjusting the pH of the composition to about 6. In some embodiments, the method of the present invention includes adjusting the pH of the composition of the present invention to about 11, and then adjusting the pH of the composition to about 6.

[0052] Acids and / or bases (for example, organic or inorganic acids and / or organic or inorganic bases, respectively) may be used to adjust the pH of the composition of the present invention. In some embodiments, adjusting the pH of the composition to a basic pH may involve adding a base to the composition. In some embodiments, the base is sodium hydroxide (NaOH) and / or a NaOH solution. In some embodiments, adjusting the pH of the composition to an acidic pH involves adding an acid to the composition. In some embodiments, the acid is hydrochloric acid (HCl) and / or an HCl solution.

[0053] In some embodiments, adjusting the composition to an acidic pH optionally includes adding the acid dropwise to the composition while mixing it. In some embodiments, adjusting the composition to an acidic pH optionally includes adding the acid to the composition in a manner in which less than the total volume of the acid is added to the composition in a single addition, rather than adding the entire volume of the acid in a single addition, such that the entire volume of the acid is added in two or more additions over a period of time. In some embodiments, the acid is added to the composition in two or more additions (e.g., two, four, six, eight, ten, or more) (each addition being less than the total volume of the acid added to the composition during the adjustment and / or the method) while mixing the composition. In some embodiments, adjusting the composition to an acidic pH optionally includes adding the composition to the acid, by adding the entire volume of the acid to the composition in a single addition. In some embodiments, adjusting the composition to an acidic pH while optionally mixing the composition may include adding hydrochloric acid (HCl) and / or an HCl solution to the composition dropwise. In some embodiments, adjusting the composition to an acidic pH while optionally mixing the composition may include adding HCl and / or an HCl solution to the composition by two or more additions of HCl and / or an HCl solution (each addition being less than the total volume of HCl and / or an HCl solution added to the composition during the adjustment and / or the method). In some embodiments, adjusting the composition to an acidic pH may include adding HCl and / or an HCl solution to the composition by directly mixing the total volume of HCl and / or an HCl solution into the composition. In some embodiments, the method of the present invention does not involve heating the composition (e.g., a mixture) of the present invention (i.e., lacking heating), but includes adjusting the composition to an acidic pH (the adjustment involves adding an acid to the composition via two or more additions of the acid (e.g., two, four, six, eight, ten, or more) (each addition being less than the total volume of the acid added to the composition during the adjustment and / or the method)).In some embodiments, the method of the present invention does not involve heating the composition of the present invention, but includes adjusting the composition to an acidic pH (such adjustment includes adding the entire volume of the acid by directly mixing it into the composition).

[0054] In some embodiments, the pH of the composition is adjusted to an acidic pH, from about 15 minutes, 20 minutes, or 25 minutes after the pH of the composition has been adjusted to a basic pH, to about 30 minutes, 35 minutes, or 40 minutes. In some embodiments, the pH of the composition is adjusted to an acidic pH, from about 30 minutes after the pH of the composition has been adjusted to a basic pH. In some embodiments, depending on the adjustment of the pH of the composition to a basic pH, the composition may be mixed at the basic pH from about 15 minutes, 20 minutes, or 25 minutes, to about 30 minutes, 35 minutes, or 40 minutes. In some embodiments, depending on the adjustment of the pH of the composition to a basic pH, the composition may be mixed at the basic pH for about 30 minutes. In some embodiments, depending on adjusting the pH of the composition to an acidic pH, the composition may be mixed at the acidic pH for about 15 minutes, about 20 minutes, or about 25 minutes to about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, depending on adjusting the pH of the composition to an acidic pH, the composition may be mixed for about 30 minutes.

[0055] In some embodiments, the method of the present invention includes heating the composition (e.g., a mixture) of the present invention. In some embodiments, heating the composition (e.g., a mixture) in the method of the present invention includes heating the composition for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes to about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, the method of the present invention includes heating the composition for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, the method of the present invention includes heating the composition for about 20 minutes. In some embodiments, the method of the present invention includes heating the composition at a temperature ranging from about 50, about 55, or about 60°C to about 65, about 70, about 75, or about 80°C. In some embodiments, the method of the present invention includes heating the composition to a temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or about 80°C. In some embodiments, the method of the present invention includes heating the composition to a temperature of about 70°C. In some embodiments, the method of the present invention includes heating the composition to a temperature ranging from about 50°C, about 55°C, or about 60°C to about 65°C, about 70°C, about 75°C, or about 80°C, and maintaining the temperature for about 10 minutes, about 15 minutes, about 20 minutes, about 25 or about 30 minutes to about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, the method of the present invention includes heating the composition to a temperature of about 70°C and maintaining the temperature for about 20 minutes. In some embodiments, the method of the present invention does not include heating the composition of the present invention (i.e., it lacks heating). In some embodiments, the method of the present invention does not involve heating the composition of the present invention to a temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C or about 80°C (i.e., lacks heating), and / or involves maintaining such temperature for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes or about 30 minutes to about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes or about 60 minutes.

[0056] In some embodiments, the method of the present invention comprises: combining a protein (e.g., whey protein isolate) and an activator (e.g., tryptophan) to form a mixture; adjusting the pH of the mixture to about 11; mixing the mixture for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 60 minutes (e.g., shaking, stirring); and then adjusting the pH of the mixture to about 6; and heating the mixture at a temperature of about 50°C, 60°C, 70°C or 80°C for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 60 minutes. In some embodiments, the mixture contains the protein and the activator in a ratio of about 5:1 w / w (protein:activator, e.g., whey protein isolate:tryptophan). In some embodiments, the mixture has a total solids content of about 2.5 w / v%. In some embodiments, the protein is whey protein isolate. In some embodiments, the activator is tryptophan. In some embodiments, adjusting the pH of the mixture to about 11 involves adding sodium hydroxide (NaOH) and / or a NaOH solution to the mixture. In some embodiments, mixing the mixture (e.g., shaking, stirring) is carried out for about 20 minutes. In some embodiments, adjusting the pH of the mixture to about 6 involves adding hydrochloric acid (HCl) and / or an HCl solution to the mixture. In some embodiments, heating the mixture is carried out at a temperature of about 70°C for about 1 minute.

[0057] As shown in Figure 36, in some embodiments, the method of the present invention optionally involves adding water to a mixer and optionally mixing and / or heating the water to a certain temperature; optionally, adding an antifoaming agent (e.g., a food-grade antifoaming agent, e.g., MAGRABAR) 登録商標The process includes: adding PD-602, a silicone defoamer, and / or a vegetable oil-based defoamer to a mixer; adding a protein (e.g., whey protein isolate) to the mixer to provide a composition; adjusting the pH of the composition to about 11; optionally adding an activator (e.g., tryptophan) dissolved and / or suspended in an aqueous composition (e.g., a solution) to the composition in the mixer; optionally adding water to the composition to its final weight; mixing the composition for a certain period of time, optionally checking the pH of the composition and adjusting it as necessary (e.g., to a pH of about 11); and adjusting the pH of the composition to about 6; heating the composition to a temperature of about 70°C and maintaining that temperature for a certain period of time, then cooling the composition to a temperature of about 10°C or lower; optionally checking the particle size of the particles in the composition and / or the viscosity of the composition; optionally concentrating the composition using ultrafiltration or nanofiltration; and optionally spray-drying the composition. In some embodiments, the mixer is an in-line mixer. In some embodiments, the method includes adding water to the mixer and mixing the water while heating it to a temperature of about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, or about 25°C, optionally to a temperature of about 21°C. In some embodiments, the method includes adding an antifoaming agent to the mixer in an amount of about 30 ppm, about 31 ppm, about 32 ppm, about 33 ppm, about 34 ppm, about 35 ppm, about 36 ppm, about 37 ppm, about 38 ppm, about 39 ppm, or about 40 ppm, optionally in an amount of about 35 ppm. In some embodiments, the method includes adding a protein (e.g., whey protein isolate) to the mixer in amounts of about 1 w / v%, about 2 w / v%, about 3 w / v%, about 4 w / v%, or about 5 w / v%, optionally in amounts of about 4 w / v%. In some embodiments, the protein is whey protein isolate.In some embodiments, the method includes adjusting the pH of the composition to about 11 by adding sodium hydroxide (NaOH) and / or a NaOH solution to the composition, where optionally, NaOH is 1N NaOH. In some embodiments, the method includes adding an activator (e.g., tryptophan) in an amount of about 0.5 w / v%, about 0.6 w / v%, about 0.7 w / v%, about 0.8 w / v%, about 0.9 w / v%, or about 1.0 w / v%, optionally in an amount of about 0.8 w / v%. In some embodiments, the activator is tryptophan. In some embodiments, mixing the composition for a certain period of time includes mixing the composition for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes. In some embodiments, adjusting the pH to about 6 includes adding hydrochloric acid (HCl) and / or an HCl solution to the composition (where the HCl is 1N HCl), and optionally mixing the composition for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes, and optionally mixing the composition at a pH of about 6 for about 2 minutes. In some embodiments, the method includes heating the composition at a temperature of about 70°C for about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, or about 50 seconds to about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes, and optionally about 1 minute. In some embodiments, the method includes checking the particle size of the particles in the composition and / or checking the viscosity of the composition. In some embodiments, checking the particle size involves checking that the average particle size (e.g., average particle diameter) of the particles in the composition is about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, or about 450 nm, optionally about 400 nm. In some embodiments, the method involves concentrating the composition using ultrafiltration or nanofiltration. In some embodiments, the composition is concentrated by an ultrafiltration method, optionally using a 4-inch 10 kDa membrane. In some embodiments, the composition is concentrated by a nanofiltration method, optionally using a 4-inch 300 Dalton membrane.In some embodiments, the method of the present invention includes removing particles having less than 10 kDa (e.g., by filtration). In some embodiments, the method of the present invention includes removing salts and / or free amino acids (e.g., free tryptophan) from the composition of the present invention. The method of the present invention can produce particles of the present invention having a particle size distribution from about 50 nm, about 100 nm, about 200 nm or about 300 nm to about 400 nm, about 500 nm, about 600 nm or about 700 nm. In some embodiments, the particles may have a polydispersity index of less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25 or less than about 0.2. In some embodiments, the particles may have a polydispersity index of about 0.2. In some embodiments, the particles may have an average particle size (e.g., average particle diameter) from about 50, about 75 or about 100 nm to about 125 nm, about 150 nm or about 200 nm. In some embodiments, the particles may have an average particle size (e.g., average particle diameter) of about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, or about 200 nm. In some embodiments, the particles may have an average particle size (e.g., average particle diameter) of about 125 nm. In some embodiments, the particles may have an average particle size (e.g., average particle diameter) of about 110 nm. In some embodiments, the particles have a free sulfhydryl (SH) group content ranging from about 5 μmol SH / g, about 6 μmol SH / g, about 7 μmol SH / g, about 8 μmol SH / g, about 9 μmol SH / g or about 10 μmol SH / g to about 11 μmol SH / g, about 12 μmol SH / g, about 13 μmol SH / g, about 14 μmol SH / g or about 15 μmol SH / g, when optionally measured using Elman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) i.e., DTNB) and UV-Vis spectroscopy.In some embodiments, the method of the present invention can produce particles of the present invention having an increased sulfhydryl (SH) group content compared to particles not produced according to the method of the present invention, when optionally measured using Elman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) i.e., DTNB) and UV-Vis spectroscopy. In some embodiments, the particles have a sulfhydryl (SH) group content of about 4 × 10¹⁵ when optionally measured using 8-anilino-1-naphthalene sulfonate (ANS) as a fluorescent probe. 7 ~Approx. 7×10 7 The particles may have surface hydrophobicity. In some embodiments, the method of the present invention can produce particles of the present invention having increased surface hydrophobicity compared to particles not produced according to the method of the present invention, when optionally measured using 8-anilino-1-naphthalene sulfonate (ANS) as a fluorescent probe. In some embodiments, the method of the present invention can produce particles of the present invention having increased fluorescence intensity compared to particles not produced according to the method of the present invention, when optionally measured by fluorescence spectroscopy.

[0058] In some embodiments, the activator is present in the particles in an amount ranging from about 0% by weight, about 5% by weight, or about 10% by weight to about 15% by weight, about 20% by weight, or about 25% by weight of the particles. In some embodiments, the activator is present in the particles in an amount ranging from about 0% by weight, about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, or about 25% by weight of the particles. In some embodiments, the protein is present in the particles in an amount ranging from about 75% by weight, about 80% by weight, or about 85% by weight to about 90% by weight, about 95% by weight, or about 100% by weight of the particles. In some embodiments, the protein is present in the particles in an amount ranging from about 75% by weight, about 80% by weight, about 85% by weight, about 90% by weight, about 95% by weight, or about 100% of the particles. In some embodiments, the activator is present in the particles in an amount ranging from about 0% by weight, about 5% by weight, or about 10% by weight to about 15% by weight, about 20% by weight, or about 25% by weight of the particles, and the protein is present in the particles in an amount ranging from about 75% by weight, about 80% by weight, or about 85% by weight to about 90% by weight, about 95% by weight, or about 100% by weight of the particles.

[0059] In some embodiments, the method of the present invention includes dehydrating the particles of the present invention and / or a composition containing the particles. Dehydrating the particles of the present invention and / or a composition containing the particles of the present invention can be done using methods and / or devices known in the art. In some embodiments, dehydrating the particles of the present invention and / or a composition containing the particles of the present invention includes freeze-drying and / or spray-drying the particles and / or the composition. In some embodiments, dehydrating the particles of the present invention includes freeze-drying and / or spray-drying the composition after any heating.

[0060] The methods of the present invention may include reducing the size (e.g., diameter) of the particles of the present invention. For example, in some embodiments, the size (e.g., diameter) of the particles of the present invention may be reduced in accordance with adjusting the pH of the composition in which the particles are present. In some embodiments, the methods of the present invention include reducing the size (e.g., diameter, optionally average diameter) of the particles of the present invention from about 5%, about 10%, about 15%, or about 20% to about 25%, about 30%, about 35%, or about 40% compared to the size of the particles in the composition at a pH of about 11. In some embodiments, the size (e.g., diameter, optionally average diameter) of the particles of the present invention present in the composition may be reduced from about 5%, about 10%, about 15%, or about 20% to about 25%, about 30%, about 35%, or about 40% after the pH of the composition has been adjusted from a pH of about 11 to a pH of about 6 or 3.

[0061] In some embodiments, the methods of the present invention involve administering a therapeutically effective amount of the particles and / or compositions of the present invention to a subject. Where used herein, the term “therapeutably effective amount” means the amount of the particles and / or compositions of the present invention that elicits a therapeutically beneficial response in the subject. Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as some benefit is provided to the subject.

[0062] As used herein, “to treat,” “treat,” or “treatment of” (and their grammatical variations) means any type of treatment that provides a benefit to a subject, and may mean that the severity of the subject’s condition is reduced, at least partially improved, or improved, and / or some relief, reduction or decrease is achieved in at least one clinical symptom associated with the subject’s condition, and / or a delay occurs in the progression of the symptom. In some embodiments, the severity of symptoms associated with sleep quality and / or mental health may be reduced in the subject compared to the severity of symptoms in the absence of the method of the present invention. In some embodiments, the particles and / or compositions of the present invention are administered to a subject to improve sleep quality (e.g., increase the length of sleep and / or rapid eye movement (REM) sleep duration, reduce sleep interruptions, etc.), to improve mental health, and / or to treat those diseases and / or their symptoms.

[0063] In some embodiments, the particles and / or compositions of the present invention may be administered in a treatment-effective amount. As used herein, a “treatment-effective” amount is an amount sufficient to treat a subject (as defined herein). Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as it provides some benefit to the subject. In some embodiments, a treatment-effective amount may be achieved by administering a composition of the present invention.

[0064] The words “prevent,” “preventing,” and “prevention” (and their grammatical variations) mean avoidance, reduction, and / or delay of the onset of symptoms associated with a disease, disorder, or condition, and / or reduction of the severity of the onset of symptoms associated with the disease, disorder, or condition compared to what would occur in the absence of the method of the present invention. Prevention may be complete, for example, the complete absence of symptoms. Such prevention may also be partial, such that the occurrence and / or severity of the onset of symptoms in the subject is less than what would occur in the absence of the method of the present invention. In some embodiments, the particles and / or compositions of the present invention are administered to a subject to prevent a disease, disorder, or condition.

[0065] In some embodiments, the particles and / or compositions of the present invention may be administered in a prevention effective amount. As used herein, a “prevention effective” amount is sufficient to prevent (as defined herein) the symptoms associated with a disease, disorder, or condition in a subject. Those skilled in the art will understand that the level of prevention does not need to be complete, as long as some benefit is provided to the subject. In some embodiments, a prevention effective amount may be achieved by administering a composition of the present invention.

[0066] The present invention finds use in both veterinary and medical applications. Suitable subjects to be treated by the method of the present invention include, but are not limited to, mammalian subjects. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates (e.g., apes and humans), non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas), etc., and intrauterine mammals. Any mammal that needs to be treated according to the present invention is suitable. Human subjects of both sexes and at any developmental stage (i.e., neonates, infants, young children, adolescents, and adults) can be treated according to the present invention. In some embodiments of the present invention, the subject is a mammal, and in some embodiments, the subject is a human. The human subject includes both males and females of all ages, including fetuses, neonates, infants, young children, adolescents, adults, and elderly individuals, as well as pregnant individuals. In certain embodiments of the present invention, the subject is a human adolescent and / or adult.

[0067] The method of the present invention may be carried out on animals, particularly mammals, such as mice, rats, dogs, cats, livestock, and horses, for veterinary purposes and / or for drug screening and drug development purposes.

[0068] In some embodiments, the subject "needs" or "has a need" for the method of the present invention, the subject is suspected of having a disease, disorder or condition, has findings typically associated with a disease, disorder or condition, and / or the subject has a disease, disorder or condition.

[0069] Herein, the present invention will be described with reference to the following embodiments. It should be understood that these embodiments are not intended to limit the scope of the claims of the present invention, but rather to illustrate specific embodiments. Any modifications of the illustrated methods that may arise for those skilled in the art are intended to fall within the scope of the present invention.

[0070] Examples

[0071] Example 1

[0072] 1. Introduction

[0073] Tryptophan (Trp) was encapsulated in whey protein nanoparticles using a combination of pH shift and heat treatment. The effect of this combination of pH shift and heat treatment on whey protein isolate-tryptophan nanoparticles (WPI-Trp-NPs) was investigated. The WPI-Trp particles had an average particle size of 110.1 nm and a low average PDI of 0.20. Fluorescence spectroscopy confirmed Trp encapsulation by WPI, indicating an increase in fluorescence intensity when Trp is encapsulated by WPI. Surface hydrophobicity, circular dichroism, particle size, free sulfhydryl, and antioxidant activity were used to characterize WPI-Trp-NPs. The driving forces for the formation of WPI-Trp-NPs were non-covalent bonds, such as hydrophobic interactions and hydrogen bonds. Molecular docking results showed that the formation of WPI-Trp nanocomplexes with alpha-lactalbumin (α-LA), bovine serum albumin, and β-lactoglobulin (β-LG) was inherently hydrophobic, demonstrating hydrogen bonding affinity between Trp and these proteins, where the strength of the interaction was ranked as α-LA > BSA > β-LG. The combination of pH shift and heating was an important method for improving the functionality of WPI and for effectively producing WPI-Trp nanoparticles.

[0074] 2. Materials and Methods

[0075] 2.1.Materials

[0076] Whey protein isolate powder (Provon 190) was provided by Glanbia Nationals, Inc. (Fitchburg, WI, USA). Tryptophan (Trp, reagent grade >98%), 2,2-diphenyl-1-picrylhydrazyl (DPPH, reagent grade >97%), 2,2-azinobis(3-ethylbenzothiazoline-6-s-sulfonic acid (ABTS, reagent grade >98%), and 8-anilino-1-naphthalenesulfonic acid (ANS, reagent grade >98%) were purchased from Sigma-Aldrich (10417, St Louis, MO, USA). 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was purchased from ThermoFisher (02451, Waltham, MA, USA). Hydrochloric acid (reagent grade >37%) and sodium hydroxide (reagent grade >97%) were purchased from Fisher Scientific (Hampton, NH, USA). Milli-Q water was purchased from the Millipore water purification system (Millipore Prepared from Sigma (Burlington, MA, USA). All other chemicals used in this study were analytical grade.

[0077] 2.2 Preparation of whey protein-tryptophan nanoparticles (WPI-Trp NPs)

[0078] 2.2.1 pH Shift Treatment

[0079] The WPI-Trp solution was prepared by dissolving a powder sample of WPI and a powder sample of Trp in a 5:1 ratio (w / w) in Milli-Q water and stirring with a magnetic stirrer (800 rpm) for 2 hours. The WPI-Trp solution was stored in a refrigerator (4°C) for 12 hours to allow for complete hydration. Next, the WPI-Trp solution was adjusted to pH 11 with 1 mol / L NaOH and stirred at 800 rpm for 1 hour. Then, the WPI-Trp solution at pH 11 was divided into three parts and further adjusted to pH 5, 6, and 7 with 1 mol / L HCl, and stirred at 800 rpm for another hour. The resulting WPI-Trp solution was adjusted to a final concentration of 2.5 w / v% with Milli-Q water to prepare a pH-shifted WPI-Trp stock solution. The un-pH-shifted WPI-Trp solution was divided into three parts, and each was adjusted to pH 5, 6, and 7 with 1 mol / L HCl, with a final concentration of 2.5 w / v%.

[0080] Control samples WPI and TRP with and without pH shift were prepared using the same protocol.

[0081] 2.2.2 Heat treatment of pH-shifted samples

[0082] In a sample preparation method combining pH shifting and heat treatment, 2 mL each of pH-shifted WPI-Trp, WPI, and Trp sample solutions (2.5 w / v%) were separately filled into glass tubes and placed in a constant temperature (50°C, 60°C, 70°C, 80°C) water bath (Model WB20, VWR International). Each tube was then heated for 10, 20, 30, 40, 50, and 60 minutes, respectively, to induce heat-induced nanoparticles. The heat treatment was terminated by immersing these glass tubes in an ice bath for 4 minutes to allow them to reach room temperature.

[0083] 2.3 Characterization of WPI-Trp NPs

[0084] 2.3.1 Particle Size Analysis

[0085] Particle size measurements were performed to investigate the effects of pH shift and heat treatment on protein aggregation. WPI-Trp, WPI, and Trp nanoparticles were analyzed for their average diameter, particle size distribution, and polydispersity index (PDI) using a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern, UK). The analysis was performed at 25°C in a 1 cm path cuvette. The measurements were performed at least six times, three times for each analysis.

[0086] 2.3.2 Circular Dichroism (CD) Spectroscopy

[0087] CD spectroscopy was performed using an AVIV-202-01 spectropolarimeter (Lakewood, NJ, USA) to investigate the secondary structure changes of WPI and WPI-Trp samples after heat treatment and pH shift. CD spectra were measured at 25°C and wavelengths of 190–260 nm. Samples with a concentration of 2 mg / mL were measured in a 1 mm pass quartz cell. A web-based DichroWeb was used to calculate the secondary structure of the samples.

[0088] 2.3.3 Intrinsic fluorescence

[0089] Intrinsic fluorescence spectroscopy was performed to study the aggregation-induced effects of WPI and Trp, and the dynamic structural changes of WPI. The intrinsic fluorescence of the prepared WPI-Trp samples, WPI samples, and Trp samples was recorded at room temperature using a fluorescence spectrophotometer (Hitachi, F-7000, Japan). The fluorescence emission spectra of the samples were excited at 295 nm and recorded at 310–500 nm (Zhan, F., et al, (2020). Food Hydrocolloids, 105, 105767).

[0090] 2.3.4 Surface hydrophobicity

[0091] Surface hydrophobicity measurements were carried out to investigate protein structural changes between pH shift and heating. Surface hydrophobicity was detected using ANS as a fluorescent probe (Jiang, H., et al., (2022). Food Chemistry, 393, 133358). An ANS stock solution (8.0 mM) was prepared with phosphate buffer (10 mM, pH 7.4). Samples of six concentrations were prepared from 0.01 mg / mL to 0.25 mg / mL using the above buffer. Next, 2 μL of ANS was added separately to 200 μL of WPI alone and WPI-Trp mixture (with or without treatment: 5:1, w / w) in a 96-well plate. Then, the plate was held in the dark for 20 min and read on a microplate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The surface hydrophobicity (H0) of the samples was calculated as the slope of the fluorescence intensity curve against the sample concentration.

[0092] 2.3.5 Turbidity

[0093] Turbidity was measured to analyze the progress of protein aggregation during pH shift and heating. The turbidity of WPI, Trp, and WPI-Trp was measured according to a previously reported method (Lin, T., et al. (2022). Food Hydrocolloids, 131, 107736).

[0094] 2.3.6 Fourier transform infrared spectroscopy (FTIR)

[0095] FTIR spectra were carried out to analyze the driving force for the formation of WPI-Trp nanoparticles. The FTIR spectra of WPI, WPI-Trp, and Trp were recorded from 4000 cm -1 ~400 cm -1The measurements were recorded using an FTIR spectrometer (Shimadzu Instrument, Kyoto, Japan) within the wavenumber range of 4 cm. -1 This was performed using an average of 32 scans at a resolution of [resolution value].

[0096] 2.3.7 Scanning electron microscopy (SEM)

[0097] The microstructure of lyophilized and solution samples was imaged using a scanning electron microscope (SEM) (Zeiss Gemini 500, Jena, Germany). Samples were vacuum-dried, then scanned and imaged using the SEM. SEM analysis was performed according to a previously reported method (Lin et al., 2022).

[0098] 2.4 Antioxidant activity

[0099] 2.4.1 DPPH radical scavenging capacity

[0100] The DPPH radical scavenging activity of the prepared WPI samples, Trp samples, and WPI-Trp samples was measured using a previously published method (Dong, H., et al., (2016). International Journal of Biological Macromolecules, 93, 179~185) with some modifications. The modifications are as follows: The samples were diluted with Milli-Q water to the following concentrations: 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL. Next, 200 μl of the sample was mixed with 100 μL of DPPH solution (0.2 mmol / mL), and the mixture was stored in the dark at room temperature (approximately 25°C) for 30 minutes.

[0101] 2.4.2 ABTS Radical Eradication Ability

[0102] The radical scavenging ability of ABTS was measured with some modifications to a previously published protocol (Dong et al., 2016). The modifications are as follows: The sample was dissolved in phosphate buffer (pH 7.4) to a concentration of 0.05 mg / mL. Next, 200 μL of the sample was mixed with 20 μL of ABTS working solution, and the mixture was stored in the dark at room temperature (approximately 25°C) for 20 minutes.

[0103] 2.5 Sulfhydryl group content (SH)

[0104] The free sulfhydryl group content of WPI and WPI-Trp samples was measured according to a previously published method (Jiang, Z., et al., (2022). International Dairy Journal, 127, 105211). A mixture of samples containing DTNB was considered a sample group, and samples without DTNB were considered a sample blank. A mixture of Tris-Gly buffer and DTNB was considered a reagent blank. The absorbance of the mixtures was recorded at 412 nm using a UV-Vis spectrophotometer (UV-2600, SHIMADZU Co., Japan). The free sulfhydryl group content was calculated according to the following formula.

number

[0105] Here, A 412 θ is the absorbance at 412 nm, C is the concentration of the sample, and D is the dilution factor.

[0106] 2.6 Molecular docking

[0107] Molecular docking was performed to further understand the interaction mechanism between WPI and Trp. Auto Dock Vina 1.2.0 software (Trott & Olson, 2009) was used to predict the interaction between whey protein and Trp. 3D crystal structures of α-LA (PDB: 1F6R), β-LG (PDB: 3NPO), and BSA (PDB: 4F5S) were obtained from the Web-based Protein Data Bank, which functions as acceptor molecules that merge nonpolar hydrogens and incorporate charge. Blind docking was performed to predict the lowest energy binding sites for Trp to α-LA, β-LG, and BSA. PyMOL ver.2.5.2 software was used to visualize the docking results, and the ligand-receptor interactions were analyzed using Ligplot (Wallace, AC, Laskowski, RA, & Thornton, JM (1995). LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions. Protein Engineering, Design and Selection, 8(2), 127~134).

[0108] 2.7 Data and Statistical Analysis

[0109] All data were analyzed and plotted using two software programs: JMP (version Pro15, SAS, USA) and GraphPad Prism9 (GraphPad Software Inc., USA). All experiments were performed three times, and the mean and standard deviation are shown in the graphs. Analysis of variance (ANOVA) and Tukey HSD comparative test (p<0.05) were used to analyze differences between samples.

[0110] 3. Results and Discussion

[0111] 3.1 Effect of pH shift on WPI-Trp nanoparticle formation

[0112] To determine the effect of pH shift on the formation of WPI-Trp nanoparticles, 2.5 w / v% solutions of WPI and Trp were mixed in a 5:1 (w / w) ratio and dissolved in an alkaline solution at pH 11. These solutions were then adjusted back to pH 5, pH 6, or pH 7, respectively, and subsequently heat-treated at 70°C for 60 minutes. Samples with a pH shift from 11 to 5 showed undesirable precipitation (e.g., large aggregates and sediments), as evidenced by the high turbidity of the mixture, and therefore did not exhibit proper WPI-Trp nanoparticle formation. Aggregation can occur even at room temperature at pH 5 because pH 5 is close to the isoelectric point of WPI (pI 5.1), where the protein carries a net-zero charge. Aggregation is further promoted by heat treatment. However, samples with a pH shift from 11 to 7 or 11 to 6 showed colloidal dispersion without visible aggregates and demonstrated nanoparticle formation. This result was confirmed by the particle size distribution results, as shown in Figure 1. As shown in Figure 1, the sample with a pH shift from 11 to 7 showed a smaller particle size compared to the sample with a pH shift from 11 to 6, indicating the formation of WPI-Trp nanoparticles. The particle sizes of WPI-Trp NPs obtained under pH shifts (from 11 to 7 and from 11 to 6) and heat treatment combined with pH shifts (pH shift from 11 to 7 and 6 followed by heating at 70°C for 1 hour) ranged from less than 50.0 nm to a maximum particle size of 156.0 ± 3.0 nm when the pH was shifted from 11 to 6 and then heated (Figure 2). While not bound by any specific theory, this may be due to the pH being close to the isoelectric point of WPI, and therefore to low electrostatic repulsion. Shifting the pH from 11 to 7 and then heating did not significantly increase the particle size of WPI-Trp (p>0.05), and the particles were smaller than those in the sample where the pH was shifted from 11 to 6 and then heated. This difference in size is thought to be due to the fact that the further the pH is from the isoelectric point, the much weaker the effect of heating-induced aggregation becomes.

[0113] Intrinsic fluorescence has been considered an effective approach for studying protein conformational changes. Tryptophan is one of the main protein fluorophores, and its binding properties are studied by recording fluorescence spectral emission at specific excitation wavelengths. Aggregation-induced emission is observed when a fluorescent molecule fluoresces when trapped in a protein aggregate, because the rotation of the fluorescent molecule is restricted. When it is freely dispersed in a buffer, the emission may be strongly quenched or non-fluorescent. Therefore, the intrinsic fluorescence intensity of Trp is positively correlated with the encapsulated Trp content in the protein aggregate and can be used to characterize WPI-Trp nanoparticles. Although not bound by any particular theory, a schematic diagram of the proposed mechanism of Trp aggregation-induced fluorescence emission is shown in Figure 4.

[0114] The fluorescence spectra of WPI-Trp samples with a pH shift from 11 to 6 showed higher intrinsic fluorescence intensity than samples with a pH shift from 11 to 7 (Figure 3). This suggests that a higher Trp content was captured in the samples obtained by shifting the pH from 11 to 6. While not bound by any specific theory, the theory of aggregation-induced luminescence indicates that a higher content of captured fluorescent Trp is positively correlated with a higher intrinsic fluorescence intensity of Trp.

[0115] Samples that were shifted from pH 11 to pH 6 and then heated at 70°C showed not only higher intrinsic intensity compared to heated or unheated samples shifted from pH 11 to pH 7, and unheated samples shifted from pH 11 to pH 6, but also exhibited red-shifted bands. This further supports the idea that shifting the pH from 11 to 6 and then heating can promote protein unfolding, resulting in an increase in the number of binding sites where Trp interacts with and encapsulates nanoparticles. The inventors found that the WPI-Trp mixture obtained by pH shifting and heating further promoted protein unfolding and showed further Trp complexation.

[0116] 3.2 Effect of heating temperature on WPI-Trp nanoparticle formation

[0117] Heating temperature is one of the major factors that can affect protein particle size. Previous studies have shown that the denaturation temperature of WPI is 65°C to 70°C (L. Zhang, et al., (2021) International Dairy Journal, 123, 105175). Heating temperature can affect the degree of protein denaturation. Denatured proteins with hydrophobic residues and highly reactive sulfhydryl groups exposed result in hydrophobic interactions, making it easier for disulfide bonds to form between protein particles.

[0118] In the inventors' samples, higher temperatures resulted in the formation of larger particles between WPI and Trp, ranging from 38.9±0.6 nm at 50°C to 341.1±5.1 nm at 80°C. This was evident from the higher turbidity of the higher-temperature samples, with the 80°C sample exhibiting the highest turbidity. The effect of heating temperature on the particle size of WPI-Trp nanoparticles was investigated by shifting the pH from 11 to 6, followed by heat treatment at different temperatures for 1 hour. Samples containing 2.5% (w / v) WPI-Trp in a 5:1 w / w ratio (WPI:Trp) were adjusted to pH 11, shaken for 30 minutes, adjusted to pH 6, and then heat-treated at 50°C, 60°C, 70°C, or 80°C for 1 hour. After incubation at these different temperatures, clear differences were observed in the appearance of the WPI-Trp mixtures. WPI-Trp nanoparticles formed at 60°C and 70°C exhibited colloidal suspensions with no visible aggregates. When the particle size of the colloidal suspension was measured by DLS (Figure 5), the samples produced by pH shift and heat treatment at 60°C and 70°C showed narrow particle size distributions (PDI < 0.3) with average particle sizes of approximately 70 nm and 150 nm, respectively. WPI-Trp that was pH shifted from pH 11 to pH 6 and subsequently heated at 80°C for 1 hour produced the largest particle size among the three temperature groups, which is thought to be due to heat-induced aggregation. The increase in temperature resulted in a redshift in the fluorescence spectrum, indicating that the protein was further unfolded and more internal amino acid groups were exposed as the temperature increased. Although not bound by any specific theory, these exposed hydrophobic groups are thought to interact with additional Trp molecules, thereby contributing to particle aggregation.

[0119] In addition, as shown in Figure 7, the fluorescence intensity of Trp increased at temperatures of 50°C to 70°C, and then decreased at 80°C. This is thought to be because the content of unfolded proteins increased, followed by stronger protein self-assembly, which limited Trp incorporation. In contrast, at 50°C and 60°C, there was less protein unfolding and aggregation, and therefore fewer exposed hydrophobic residues, which is thought to have limited Trp encapsulation. This is consistent with the smaller particle size (Figure 6).

[0120] 3.3 Effect of heating time on WPI-Trp nanoparticle formation

[0121] In addition to the heat treatment temperature, heating time also affects protein aggregation. One of the main indicators of protein aggregation is turbidity. The effect of heating time on the particle size of WPI-Trp nanoparticles was investigated with and without a pH shift from 11 to 6, followed by different heating times from 0 to 60 minutes, and heat treatment at 70°C. Clear differences were observed in the appearance of the suspensions formed after heat treatment at each time. With longer heating times, the WPI-Trp samples showed more turbid suspensions. Homogeneous nanoparticles were formed after heating for 10 minutes (PDI < 0.03). Both turbidity and particle size increased with increasing heating time in both the unshifted and pH-shifted groups (Figures 8-13). The unshifted WPI-Trp samples demonstrated significantly higher turbidity (Figure 13) and larger particle size (Figure 11) compared to the pH-shifted samples (p < 0.05, Figures 8-10 and 12), and precipitates were observed.

[0122] The differences in turbidity and particle size upon heating between pH-shifted and unshifted WPI-Trp samples (Figures 8-13) can be explained by changes in the WPI structure during the pH shift. Upon heating, WPI-Trp exhibited increased particle size and turbidity compared to WPI alone, indicating that WPI aggregation was promoted in the presence of Trp (Figure 14; Figures 8-13). While not bound by any specific theory, this is thought to be due to increased interaction between exposed hydrophobic residues on WPI and Trp.

[0123] The fluorescence intensity of the WPI-Trp mixture reached its peak after 20 minutes of heat treatment (Figure 15, Panel A). Heat treatment longer than 20 minutes decreased the fluorescence intensity and resulted in a slight red shift. This is thought to be due to restricted access of Trp to the protein interior because the longer heating time strengthened the protein-protein interactions. Upon heating, the intrinsic fluorescence intensity of the pH-shifted WPI sample increased during 30 minutes of heating due to protein aggregation in a nonpolar environment and the presence of Trp residues (Figure 15, Panel B). After 30 minutes of heating, the fluorescence intensity began to decrease. Fluorescence quenching of Trp fluorescence may be due to changes in proximal amino acids and disulfides that occurred during aggregation. The intrinsic fluorescence intensity of Trp remained much lower than that of the WPI and WPI-Trp samples (Figure 15, Panel C). Based on aggregation-induced luminescence, it is thought that fluorescence emission is reduced due to a strong quenching effect when Trp molecules are freely dispersed in a hydrophilic environment.

[0124] At pH 6, natural WPI-Trp had a particle size of 68.2 ± 2.1 nm, which decreased to 36.8 ± 0.6 nm after pH shift treatment, and showed a slight increase in intrinsic intensity with a red shift (Figure 16). This suggests that the structure of the whey protein becomes more flexible in the molten-globule state, exposing more hydrophobic residues, thereby resulting in a smaller particle size, more Trp, and therefore higher fluorescence intensity.

[0125] WPI-Trp that was not pH-shifted but heat-treated resulted in a large particle size of 1784.0 ± 85.4 nm, indicating a significant heat-induced protein aggregation effect. On the other hand, WPI-Trp solutions that were pH-shifted and then heated showed a significantly smaller particle size of 110.1 ± 0.8 nm (p<0.05), indicating that the pH shift reduced the heat sensitivity of WPI-Trp particles. Furthermore, WPI-Trp treated with both pH shifting and heating exhibited the highest fluorescence intensity, while samples heated only had slightly lower peak values ​​(Figure 17). It was considered that both heating and pH-shifting treatments functionalized the whey protein and contributed to Trp complexation.

[0126] 3.4 Effects of HCl addition method and concentration on WPI-Trp nanoparticle formation with pH shift and without heating

[0127] The effect of HCl addition method and concentration on the formation of WPI-Trp nanoparticles without heat treatment was investigated by either dropping or directly mixing a fixed amount of 1-5 M HCl solution into a basic solution of the WPI mixture. This experiment demonstrated that when the pH of a composition containing WPI and Trp in a WPI:Trp ratio of 5:1 (final total solids content (WPI and Trp) concentration of 4.8 w / v%) was adjusted from pH 11 to pH 6 using 1 M HCl, a clear difference was observed in the appearance of the suspensions formed by different addition methods. When 1 M HCl was dropped into the basic WPI-Trp solution after a pH shift from pH 11 to pH 6 without heat treatment, the suspension remained clear, indicating that no nanoparticles were formed. However, when the entire amount of 1 M HCl was added at once and directly mixed into the mixture, a colloidal dispersion was obtained instantaneously without heat treatment after adjusting the pH from pH 11 to pH 6 without heat treatment, indicating that nanoparticles were formed without heat treatment. Furthermore, when the pH was adjusted from pH 11 to pH 6 using 5M HCl, nanoparticles (colloidal dispersions) were formed without heat treatment by both dropwise and direct mixing. This indicates that the rate of HCl addition and / or acid concentration significantly affect the formation of WPI-Trp nanoparticles. Therefore, the protocol for WPI-Trp nanoparticle formation can be varied by the method and concentration of HCl addition during the pH shift. While we do not wish to be bound by any particular theory, heat treatment may induce intermolecular association, and nanoparticle formation may be possible when adjusting the pH from pH 11 to pH 6 by slowly adding low concentrations of HCl. In addition, while we do not wish to be bound by any particular theory, in the case of the direct mixing method, the fast reaction rate may induce the formation of molecularly self-assembled nanoparticles, and therefore, heat treatment may not have been necessary in the case of WPI. This unique observation was not observed in the case of alpha-lactalbumin.

[0128] 3.5 WPI-Trp Nanoparticle Size Distribution

[0129] WPI-Trp nanoparticles were prepared by pH shifting from 11 to 6, followed by heat treatment at 70°C for 20 minutes. As shown in Figure 18, compared to a sample without pH shifting under the same conditions (Figure 19), the pH-shifted WPI-Trp nanoparticles had a narrower particle size distribution and a particle size of approximately 125 nm. The pH-shifted samples also showed lower turbidity of the suspension compared to the samples without pH shifting.

[0130] 3.6 Thermally induced interactions of WPI nanoparticles in skim milk dispersions

[0131] Dispersions containing non-fat dry milk powder (NFDM) alone, dispersions containing NFDM and whey protein isolate (WPI), dispersions containing NFDM and WPI nanoparticles, and dispersions containing NFDM and WPI-tryptophan (Trp) nanoparticles were prepared by reconstituting the powder in deionized water so that the final protein content was 4 w / w% and the casein-to-protein ratio was 80:20 for dispersions containing NFDM alone, and so that the ratio was 60:40 for dispersions containing NFDM and WPI, dispersions containing NFDM and WPI nanoparticles, and dispersions containing NFDM and WPI-Trp nanoparticles. These powders were mixed at 30°C for 30 minutes. The compositions of these dispersions are given in Table 1.

[0132] After overnight refrigeration, the dispersion was heated to 30°C in a water bath, followed by heating to 90°C for 2 minutes. The dispersion was then immediately cooled to room temperature in an ice bath. To confirm any interaction between casein and whey protein induced by heating, the particle size of the dispersion was measured after heat treatment.

[0133] As shown in Table 1, the particle size of the NFDM dispersion decreased slightly after heating. However, the particle size of the NFDM / WPI dispersion (casein to whey protein 60:40 dispersion) increased from approximately 310 nm to approximately 410 nm upon heating. While we do not wish to be bound by any specific theory, this is thought to be due to heat-induced aggregation of casein-whey protein interactions or whey protein-whey protein interactions. The particle size of dispersions containing NFDM / WPINP or NFDM / WPITrpNP did not significantly increase after heating. These results demonstrate that WPI nanoparticles and WPI-Trp nanoparticles prepared using pH shift and heating methods can be used to produce functional WPI that does not interact in response to heating. These nanoparticles may be beneficial in applications where heat-induced interactions between proteins need to be limited, primarily to prevent heat-induced textural changes, such as dairy products. For example, WPI nanoparticle components can be used in high-protein yogurt formulations where the interaction between casein and whey protein needs to be minimized to control viscosity and texture.

[0134] [Table 1]

[0135] 3.7 Characterization of WPI-Trp NPs generated under selected conditions

[0136] Based on the data, WPI-Trp nanoparticles were prepared by pH shifting from pH 11 to pH 6, followed by heating at 70°C for 20 minutes. Under these conditions, the fluorescence intensity was maximized, and the nanoparticle size was 110.1 ± 0.8 nm, with a low PDI of 0.20 ± 0.02, meeting the requirements for being considered nanoparticles. The particles were obtained by pH shifting from pH 11 to pH 6 and / or heating at 70°C for 20 minutes, and optionally by lyophilization as further described below.

[0137] 3.7.1 SEM imaging

[0138] The microstructure of WPI-Trp subjected to different processing methods was analyzed using SEM (Figure 20). WPI-Trp samples at pH 6 after freeze-drying (FD) demonstrated large flake formation (Figure 20, Panel A). Several samples were freeze-dried after particle formation. Compared to untreated samples, WPI-Trp samples heated without pH shift showed higher levels of aggregation and conjugation (Figure 20, Panel B). WPI-Trp samples that were pH-shifted and freeze-dried (Figure 20, Panel C) showed a rod-like shape. For freeze-dried WPI-Trp NPs samples obtained by combining pH shift and heating (Figure 20, Panel D), square-like aggregates were formed and uniformly distributed at similar sizes, suggesting that the pH-shifted samples self-aggregated into more compact whey protein structures after heating (Figure 20, Panel D). However, the size of the freeze-dried WPI-Trp NPs was larger than that of the unfreeze-dried WPI-Trp NPs (Figure 20, Panel F), suggesting that freeze-drying may have caused further aggregation. The WPI-Trp NPs solution sample prepared by combining pH shift and heating (before freeze-drying) (Figure 20, Panel F) showed uniformly dispersed spherical nanoparticles, while the solution sample prepared by heat treatment only without pH shifting (Figure 20, Panel E) showed high-density and large aggregates. This result was consistent with the particle size distribution in which the heat-only WPI-Trp NPs sample showed significantly larger particle sizes. Based on differences in microstructure, the inventors confirmed that the pH-shifted sample showed lower heat sensitivity compared to the unshifted sample, thus supporting the formation of WPI-Trp NPs by a combination of pH shift (from pH 11 to pH 6) and heat treatment (70°C, 20 minutes).

[0139] 3.7.2 Surface hydrophobicity

[0140] The surface hydrophobicity (H0) of a protein indicates the number of hydrophobic groups exposed on the protein's surface. pH shift treatment significantly increased the H0 of WPI and WPI-Trp (Figures 21-22), indicating that the initial internal hydrophobic groups of the native protein were exposed on the protein surface after the pH shift. This is consistent with the redshift of WPI and the fluorescence spectrum of pH-shifted WPI-Trp. Heat-treated WPI and WPI-Trp samples also showed increased surface hydrophobicity (Figures 21-22). While not bound by any specific theory, the redshift in the fluorescence spectrum may be attributable to changes in the protein's spatial structure leading to unfolding and exposure of hydrophobic groups on the protein surface. This increased surface hydrophobicity may enhance non-covalent interactions between the WPI-Trp complex.

[0141] However, the surface hydrophobicity of nanoparticles obtained by pH shift combined with heating was lower than that of samples subjected to pH shift alone. This may be because, after the pH shift, the protein is in an unstable molten-globular state, exposing certain hydrophobic groups and giving them priority for hydrophobic interactions. Therefore, after heating, these exposed hydrophobic groups become buried again, reducing surface hydrophobicity. Heat-induced aggregation may contribute to the formation of WPI-Trp NPs complexes. Overall, the increase in H0 induced by the pH shift suggests unfolding and exposure of hydrophobic groups on WPI, thereby promoting hydrophobic interactions between WPI and Trp during the subsequent heating process.

[0142] 3.7.3 Free sulfhydryl group content

[0143] Oxidation and exchange between sulfhydryl (SH) groups and disulfide (SS) bonds are thought to play a crucial role in protein polymerization. The protein species in WPI, β-LG, α-La, and BSA contain 2, 4, and 17 intramolecular SS bonds, respectively. Each of β-LG and BSA contains one free thiol (-SH) group. Furthermore, SH groups can accept radicals and be oxidized to convert them into SS bonds. After heat treatment of our WPI-Trp sample, the sample showed an increased SH group content (Figure 23), which is thought to be due to protein denaturation exposing free sulfhydryl groups. More specifically, when β-LG is heated, it separates into monomers, thereby denaturing at high temperatures and exposing highly reactive sulfhydryl groups, resulting in an increased SH group content. However, this increase was not significant (p>0.05) because the oxidation of SH groups, i.e., the SH / SS exchange reaction, was limited by the antioxidant properties of Trp. In contrast, the total SH group content of heated WPI was significantly lower compared to natural WPI (p<0.05) (Figure 24), which is thought to be due to the oxidation of SH groups and the exchange between them and SS bond formation induced by heating. Oxidation of SH, or conversion of SH to SS bonds, may promote the polymerization of WPI.

[0144] The SH group content of WPI significantly increased after the pH shift (p<0.05) (Figure 24), which is thought to be related to protein unfolding, suggesting that SH groups originally embedded within the protein were exposed or that disulfide bonds in the protein were cleaved. The SH content of pH-shifted WPI-Trp did not show a significant difference compared to the untreated WPI-Trp mixture (p>0.05) (Figure 23), which is also thought to be related to a decrease in the oxidation of SH groups in the presence of Trp. WPI-Trp NPs obtained by combining pH shift and heating showed the highest SH group content. Although not bound by any specific theory, this is thought to be because the pH shift and heating synergistically promoted the exposure of SH groups, while suppressing the exchange between SH groups and SS bond formation due to the presence of Trp. The SH group content of WPI NPs obtained by combining pH shift and heating was slightly lower than that of WPI nanoparticles obtained by pH shift alone, which may also be due to the oxidation of reactive SH groups induced by heating. Overall, due to the presence of Trp, the formation of SS bonds by oxidation of SH groups may be limited during the formation of WPI-Trp NPs.

[0145] 3.7.4 Circular Dichroism (CD)

[0146] CD spectroscopy was used to identify the secondary structures of WPI and WPI-Trp and to analyze the effects of pH shift and heating on these secondary structures (Figures 25-26; Table 2). Untreated WPI showed a positive peak at 196 nm for the β-sheet structure and a negative peak at 208 nm for the α-helix structure. In the case of WPI and WPI-Trp, both pH shift treatment alone and heat treatment alone resulted in the smallest blue-shift of ellipticity (Figures 25-26), and therefore a decrease in α-helix content (Table 2), indicating that the WPI protein was unfolded and short peptides were released. This loss of α-helix structure suggests that both heating and pH shift may cause the protein to reach a molten-globule state with greater flexibility. Specifically, in the case of WPI, both pH shifting alone and heating alone resulted in a significant decrease in α-helix content (p<0.05) and a significant increase in β-sheet and β-turn content (p<0.05). In contrast, WPI-Trp showed negligible changes in α-helix, β-sheet, and β-turn content (p>0.05). While not bound by any particular theory, this difference may be due to an interaction between WPI and Trp that promotes a more stable secondary structure. The combination of pH shifting and heat treatment further decreased α-helix content and resulted in a significant increase in random coils in both WPI-Trp NPs and WPI NPs (p<0.05), thereby suggesting a conversion from α-helix to random coils. This conversion demonstrated that the modified whey protein structure becomes more disordered and has greater flexibility. Previous studies have confirmed that protein structure becomes less orderly as the random coil content increases. This is consistent with the increase in surface hydrophobicity of WPI-Trp NPs and WPI NPs resulting from a combination of pH changes and heat treatment (Figures 21-22), because small hydrophobic patches are exposed after the pH changes and heat treatment.In conclusion, both heating and pH shift may contribute to the disorder of WPI, which may be explained by the molten-globule state of the denatured protein, and the presence of Trp may help stabilize the protein's secondary structure.

[0147] [Table 2]

[0148] 3.7.5 FTIR

[0149] FTIR measurements were performed to analyze the interaction between Trp and WPI (Figure 27). 1600–1700 cm -1 The amide I band that appeared represented the C=O stretching vibration of the peptide bond. The amide II band was at 1500 cm. -1 ~1600cm -1It was closely related to the stretching vibrations of CN and the bending vibrations of NH (Dai, L., Sun, C., Li, R., Mao, L., Liu, F., & Gao, Y. (2017). Structural characterization, formation mechanism and stability of curcumin in zein-lecithin composite nanoparticles fabricated by antisolvent co-precipitation. Food Chemistry, 237, 1163~1171; Zhong, M., Sun, Y., Sun, Y., Fang, L., Wang, Q., Qi, B., & Li, Y. (2022). Soy lipophilic protein self-assembled by pH-shift combined with heat treatment: Structure, hydrophobic resveratrol encapsulation, emulsification, and digestion. Food Chemistry, 394, 133514). Typical amide I and amide II absorption bands of WPI are 1629 cm², respectively. -1 and 1521cm -1 It is located at [location]. Compared to WPI, the band of WPI-Trp nanoparticles was shifted to a lower wavenumber: namely, 1624 cm⁻¹ for amide I. -1 , and in the case of Amido II, 1517cm -1 The changes in amide I and amide II suggest that hydrophobic interactions can occur between WPI and Trp. 3200–3500 cm -1 The bands in the region are associated with the -OH stretching vibration in the protein and represent hydrogen bonds. The spectrum of WPI-Trp NPs is 3265 cm⁻¹. -1 It had a band at 3200–3500 cm. No significant shift was observed compared to WPI, but WPI-Trp NPs showed a difference of 3200–3500 cm. -1The broader and sharper bands suggested the formation of hydrogen bonds. In the case of Trp, 3394 cm⁻¹ -1 , 3010cm -1 and 742cm -1 The sharp bands corresponded to the NH stretching vibration of the indole ring, the aromatic CH stretching vibration, and the CH bending vibration of the aromatic ring, respectively.

[0150] After complexation with WPI, Trp's 3394 cm⁻¹ -1 and 3010cm -1 The distinctive band has disappeared, and the length is 742cm. -1 The band intensity decreased, indicating an interaction between the indole ring and WPI, which was likely related to hydrogen bonding and hydrophobic interactions between the indole ring of Trp and WPI. In the FTIR spectra of WPI-Trp NPs, no new bands were detected compared to WPI, suggesting that the interaction between WPI and Trp was primarily due to non-covalent bonding.

[0151] 3.7.6 DPPH and ABTS radical scavenging activity of WPI-Trp NPs

[0152] The relative ABTS and DPPH radical scavenging abilities of Trp, WPI, and WPI-Trp were measured to evaluate the effects of pH shift and heat treatment on antioxidant activity (Figures 28-33). DPPH is a stable, oil-soluble free radical and can be used to measure radical scavenging activity. Only heat-treated WPI-Trp demonstrated low DPPH radical scavenging ability and showed the lowest DPPH radical scavenging ability at 5 mg / ml (Figure 28). This is thought to be because, after heat treatment, hydrophobic interactions between exposed hydrophobic residues and WPI-Trp particles resulted in aggregation, which in turn resulted in low solubility in DPPH solution. At 5 mg / ml, the DPPH radical scavenging ability was lowest due to the low solubility of heated WPI-Trp and WPI samples in DPPH solution. The DPPH radical scavenging ability of WPI-Trp was not significantly affected by pH shift treatment (p>0.05), while the DPPH scavenging ability of WPI alone significantly increased (p<0.05) (Figure 29). This is thought to be because the internal amino acids of WPI were exposed after pH shift treatment, thereby enhancing its ability to scavenge free DPPH radicals. In the case of WPI-Trp NPs obtained by pH shift combined with heating, the DPPH scavenging ability of WPI-Trp decreased despite the encapsulation of Trp being maximized under these conditions (Figure 28). This is thought to be because the complexation of Trp and WPI stabilized Trp and blocked its access to DPPH. The effect of reducing DPPH scavenging ability has been shown in previous studies, where the binding of whey protein concentrate and quercetin led to the separation of quercetin from DPPH through the hydrophobic cavity of the whey protein concentrate, resulting in reduced DPPH scavenging ability.

[0153] ABTS is a water-soluble radical and can be scavenged by antioxidants when its absorbance decreases. Due to the difference in solubility, the relative radical scavenging ability of ABTS is higher than that of DPPH, and therefore, the radical scavenging ability of ABTS was demonstrated at low sample concentrations (0.05 mg / mL). Free Trp showed strong relative ABTS radical scavenging ability and was not affected by the treatment (Figure 33). The ABTS radical scavenging ability of WPI-Trp was significantly reduced by pH shift combined with heating (p<0.05), which suggested that the most significant content of free Trp was encapsulated (Figure 31). Therefore, it was found that pH shift combined with heat treatment induces the strongest complexation of WPI-Trp nanoparticles. Nevertheless, the reduced antioxidant capacity due to Trp encapsulation occurred because Trp complexed within the protein and steric hindrance inhibited access to the radical.

[0154] 3.8 Molecular docking

[0155] Molecular docking was used to investigate the interaction mechanism between Trp and WPI. Docking was investigated for simulated nanocomplexes of WPI-Trp and the three major WPI protein species (α-LA, β-LG, and BSA), particularly regarding hydrogen bonding and hydrophobic interactions (Figure 34). The amine group in Trp could form two hydrogen bonds with the Glu-49 and Gln-43 residues in α-LA, while other amino acids (Ile-41, Gln-54, His-32, Val-42, Thr-33, Trp-104, Tyr-103, and Phe-53) formed hydrophobic pockets for complexing with Trp (Figure 34, Panel A). In the case of β-LG, Trp-19 and Glu-44 formed two hydrogen bonds with the amine group in the indole ring, and Glu-158 formed another hydrogen bond with the amino group of Trp (Figure 34, Panel B). The amino acid residues of β-LG (Thr-18, Tyr-20, Glu-157, Gln-159, and Leu-156) contributed to the hydrophobic interaction with Trp. For BSA, two hydrogen bonds were formed between amino acid residues (Leu-189 and Ser-192), and Arg-458, Ile-455, Leu-454, Ala-193, Arg-435, Tyr-451, and Ser-428 were responsible for the hydrophobic interaction during binding (Figure 34, Panel C). Regarding binding affinity (Table 3), α-LA was predicted to have the highest binding score (-7.9 kcal / mol), followed by BSA (-6.6 kcal / mol), and then β-LG (-5.8 kcal / mol), indicating that α-LA exhibited the best binding performance with Trp. Therefore, although not bound by any particular theory, the molecular docking results obtained by the inventors suggest that the formation of the WPI-Trp complex was mainly promoted by hydrophobic interactions and hydrogen bonding.

[0156] [Table 3]

[0157] 3.9 Proposed mechanism for WPI-Trp NP formation

[0158] While not bound by any specific theory, the inventors propose the following mechanism for WPI-Trp NP formation based on collected data. This process is thought to begin with the unfolding and refolding of whey protein during a pH shift, causing the protein to enter a molten globule state, where internal hydrophobic residues are exposed (Figure 35). Of these exposed residues, methionine and proline can bind to Trp. Next, the free Trp can be encapsulated within the hydrophobic cavities of the protein. Then, heat treatment of WPI-Trp after the pH shift can induce aggregation. During the heating process, the whey protein may denature, and interactions between exposed hydrophobic residues, exposure of reactive SH groups, and exchange reactions between SH and SS may occur, all of which can contribute to the complexation of WPI-Trp NPs.

[0159] While not bound by any specific theory, pH shift treatment promotes the exposure of hydrophobic residues, such as those containing internal phosphors (tyrosine, tryptophan, and phenylalanine), thereby increasing the binding sites for Trp. While not bound by any specific theory, heat treatment promotes the aggregation of WPI, thereby further encapsulating Trp. Encapsulation of added Trp was confirmed based on aggregation-induced luminescence. Evaluation of the prepared particles demonstrated that combining pH shift and heat treatment produces WPI-Trp nanoparticles with lower heat sensitivity than particles produced by heating alone. Non-covalent interactions, including hydrogen bonds and hydrophobic interactions, were the main driving forces for the complexation of WPI-Trp nanoparticles.

[0160] The foregoing is illustrative of the present invention and should not be construed as limiting the present invention. The present invention is defined by the appended claims, and equivalents of the claims should also be included within the present invention.

Claims

1. A method for preparing particles, To prepare a protein in a composition (e.g., an aqueous composition); The pH of the above composition is adjusted to a basic pH, and then, Adjusting the pH of the aforementioned composition to an acidic pH; and then, Optionally, the composition may be heated. This is how to form particles containing the protein. The method, including the method described above.

2. The method according to claim 1, further comprising adding an activator to the composition before adjusting the pH of the composition, wherein the particles comprise the protein and the activator.

3. The method according to claim 1 or 2, wherein the protein is selected from dairy proteins (e.g., milk proteins), plant proteins, and / or animal (e.g., meat) proteins, and optionally the protein is a milk protein.

4. The method according to any one of claims 1 to 3, wherein the protein is selected from the group consisting of whey protein isolate, alpha-lactalbumin, lysozyme, cytochrome c, apomyoglobin, and staphylococcal nuclease, and any combination thereof, and optionally the protein is whey protein isolate.

5. The method according to any one of claims 2 to 4, wherein the activator is selected from the group consisting of tryptophan, leucine, phenylalanine, cysteine, tyrosine, vitamin E, and any combination thereof, and optionally the activator is tryptophan.

6. The method according to any one of claims 1 to 5, wherein the composition is an aqueous composition comprising the protein and optionally the activator, wherein optionally the protein and / or the optional activator are dissolved in the aqueous composition.

7. The method according to any one of claims 2 to 6, wherein the composition is an aqueous composition comprising the protein and the activator, and optionally the protein and the activator are dissolved in the aqueous composition.

8. The method according to any one of claims 2 to 7, wherein the composition contains the protein and the activator in a weight ratio of about 2:1 to about 30:1 (protein:activator), and optionally the weight ratio is about 5:1 (protein:activator).

9. The method according to any one of claims 1 to 8, wherein the composition has a total solids content in the range of about 1 w / v% to about 10 w / v%, and optionally the composition has a total solids content of about 2.5 w / v%.

10. The method according to any one of claims 1 to 9, wherein adjusting the pH of the composition to the basic pH includes adjusting the pH of the composition from about 8, about 9, or about 10 to about 11, about 12, or about 13, and optionally adjusting the pH of the composition to the basic pH includes adjusting the pH of the composition to about 11.

11. The method according to any one of claims 1 to 10, wherein the pH of the composition is adjusted to the acidic pH, which is done from about 15 minutes, about 20 minutes, or about 25 minutes to about 30 minutes, about 35 minutes, or about 40 minutes after the pH of the composition has been adjusted to the basic pH, and optionally the method further comprises mixing the composition at the basic pH for a first time from about 15 minutes, about 20 minutes, or about 25 minutes to about 30 minutes, about 35 minutes, or about 40 minutes in response to the adjustment of the pH of the composition to the basic pH.

12. The method according to any one of claims 1 to 11, wherein adjusting the pH of the composition to the acidic pH includes adjusting the pH of the composition to a pH of less than 7, and optionally adjusting the pH of the composition to the acidic pH includes adjusting the pH of the composition from about 2, about 3, about 4, or about 5 to about 6, or about 6.

5.

13. The method according to any one of claims 1 to 12, wherein the method comprises heating the composition for about 10, about 15, about 20, about 25, or about 30 minutes to about 35, about 40, about 45, about 50, about 55, or about 60 minutes, and optionally the method comprises heating the composition for about 20 minutes.

14. The method according to any one of claims 1 to 13, wherein the method comprises heating the composition to a temperature in the range of about 50, about 55 or about 60°C to about 65, about 70, about 75 or about 80°C, and optionally the method comprises heating the composition to a temperature in the range of about 50, about 55 or about 60°C to about 65, about 70, about 75 or about 80°C for about 10, about 15, about 20, about 25 or about 30 minutes to about 35, about 40, about 45, about 50, about 55 or about 60 minutes.

15. The method according to any one of claims 1 to 14, wherein adjusting the pH of the composition to the basic pH comprises adding a base to the composition, wherein the base is optionally NaOH.

16. The method according to any one of claims 1 to 15, wherein adjusting the pH of the composition to the acidic pH comprises adding an acid to the composition, wherein the acid is optionally HCl.

17. The method according to any one of claims 1 to 16, wherein the particles have a particle size distribution in the range from about 50, about 100, about 200, or about 300 nm to about 400, about 500, about 600, or about 700 nm, along with a polydispersity index of less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than 0.25, or less than 0.2, and / or the particles have an average particle size (e.g., average particle diameter) in the range from about 50, about 75, or about 100 nm to about 125, about 150, or about 200 nm.

18. The method according to any one of claims 1 to 17, further comprising dehydrating the particles, and optionally further comprising freeze-drying or spray-drying the composition after adjusting the pH of the composition to the acidic pH and / or optionally heating the dehydrating particles.

19. The method according to any one of claims 2 to 18, wherein the activator is present in the particles in an amount from about 0% by weight, about 5% by weight, or about 10% by weight to about 15% by weight, about 20% by weight, or about 25% by weight of the particles, and / or the protein is present in the particles in an amount from about 75% by weight, about 80% by weight, or about 85% by weight to about 90% by weight, about 95% by weight, or about 100% by weight of the particles.

20. A particle, said particle is Proteins; and, Optionally, activator Includes, Here, if the activator is present in the particles, the activator is also present within the protein (for example, within the tertiary structure of the protein), and optionally the activator is nonspecifically bound to the protein (for example, via hydrophobic interactions, electrostatic interactions, hydrogen bonds, etc.). The aforementioned particles.

21. Proteins; and, Activating agent Includes, Here, the activator is present within the protein (for example, within the tertiary structure of the protein), and optionally, the activator is nonspecifically bound to the protein (for example, via hydrophobic interactions, electrostatic interactions, hydrogen bonds, etc.). The particle according to claim 20.

22. The particle according to claim 20 or 21, wherein, if the activator is present, the activator is located within a region of the tertiary structure (e.g., within a tertiary fold) that includes at least one nonspecific hydrophobic interaction between two or more amino acid residues, and optionally, the activator is located within a hydrophobic pocket of the protein.

23. The particle according to any one of claims 20 to 22, wherein, when the activator is present, the activator is present within the protein core of the protein.

24. The particle according to any one of claims 20 to 23, wherein, when the activator is present, the activator is a plurality of activators, and at least one of the plurality of activators is present within the protein, and optionally, an additional activator from the plurality of activators is present on the surface of the protein.

25. The particle according to any one of claims 20 to 24, wherein the protein is selected from dairy protein (e.g., milk protein), plant protein and / or animal (e.g., meat) protein, and optionally the protein is milk protein.

26. The particle according to any one of claims 20 to 25, wherein the protein has a molten globule state and / or a bifoliate structure.

27. The particle according to any one of claims 20 to 26, wherein the protein is selected from whey protein isolate, α-lactalbumin, lysozyme, cytochrome c, apomyoglobin, staphylococcal nuclease, and any combination thereof, and optionally the protein is whey protein isolate.

28. The particle according to any one of claims 20 to 27, wherein the protein has about 100 amino acids to about 200, about 300, about 400 or about 500 amino acids and / or has a molecular weight from about 10 kDa to about 20, about 30, about 40 or about 50 kDa.

29. The particle according to any one of claims 20 to 28, wherein the protein has an isoelectric point (pI) of about 4 to about 5 or about 5.5, and optionally, the protein has a pI of about 4.2 to about 4.5 or about 5.

2.

30. The particle according to any one of claims 20 to 29, wherein the protein comprises two domains and / or the protein comprises one or more (e.g., 1, 2, 3, 4, or more) intramolecular disulfide bonds at a pH between about 5 and about 9, and optionally the protein comprises at least one disulfide crosslink connecting the two domains of the protein.

31. The particle according to any one of claims 20 to 30, wherein the structure of the protein comprises approximately 15% to 30% α-helices, approximately 5% to 30% β-sheets, and approximately 5% to 25% β-turns, and optionally approximately 30% to 75% of the structure is disordered.

32. The particle according to any one of claims 20 to 31, wherein the protein has an amino acid sequence having sequence identity with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than that with one or more of the sequence ID numbers 1 to 3.

33. The particle according to any one of claims 20 to 32, wherein the protein is a plurality of proteins, and optionally about 5, about 10 or about 20 to about 25, about 30, about 40 or about 50 proteins are present in the particle.

34. The particles according to any one of claims 20 to 33, wherein, if the activator is present, the activator is an organic compound having a molecular weight ranging from about 70, about 100, about 150, or about 200 g / mol to about 250, about 300, about 400, or about 500 g / mol.

35. The particles according to any one of claims 20 to 34, wherein, in the presence of the activator, the activator has a solubility in water of about 15 mg / mL at 25°C or below and / or a pKa of about 1.5 to about 3 and / or a pI of about 5 to about 6.5, and optionally, the activator has a solubility in water of about 10 mg / mL at 25°C or below and / or a pKa of about 2.7 or about 2.8 to about 2.9 or about 3 and / or a pI of about 5.7 or about 5.8 to about 5.9, about 6 or about 6.

1.

36. The particle according to any one of claims 20 to 35, wherein, if the activator is present, the activator is selected from tryptophan, leucine, phenylalanine, cysteine, tyrosine, vitamin E, and any combination thereof, and optionally the activator is tryptophan.

37. The particle according to any one of claims 20 to 36, wherein the particle, when optionally measured using a microscopy method (e.g., scanning electron microscopy (SEM) and / or transmission electron microscopy (TEM) and / or dynamic light scattering (DLS)), has a diameter ranging from about 50, about 75, or about 100 nm to about 125, about 150, or about 200 nm, and optionally, the particle has a diameter of about 110 nm or about 125 nm.

38. The particle according to any one of claims 20 to 37, wherein the protein is present in the particle in an amount of about 75% to about 100% by weight of the particle, and the activator is present in the particle in an amount of about 0% to about 25% by weight of the particle.

39. The particles according to any one of claims 20 to 38, wherein the activator is present in the particles in an amount ranging from about 10% by weight, 11% by weight, about 12% by weight, about 13% by weight or about 14% by weight to about 15% by weight, about 16% by weight, about 17% by weight, about 18% by weight, about 19% by weight or about 20% by weight.

40. The particles according to any one of claims 20 to 39, wherein the protein and the activator are each soluble in water at a temperature of about 25°C and a pH of about 11, and / or the protein and / or activator have a negative charge in water at a pH of about 11.

41. The particles according to any one of claims 20 to 40, wherein the particles have increased activity and / or function (e.g., increased antioxidant activity) compared to the activity and / or function of the protein alone.

42. The particles according to any one of claims 20 to 41, wherein when stored in a closed container at approximately 4°C for approximately 1 or 2 months, the size of the particles (e.g., diameter) remains within approximately ±20% of their original size.

43. The particles according to any one of claims 20 to 42, wherein the particles optionally have a free sulfhydryl (SH) group content ranging from about 5, about 6, about 7, about 8, about 9 or about 10 μmol SH / g to about 11, about 12, about 13, about 14 or about 15 μmol SH / g when measured using Elman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) i.e., DTNB) and UV-Vis spectroscopy.

44. When the aforementioned particles are optionally measured using 8-anilino-1-naphthalene sulfonate (ANS) as a fluorescent probe, the result is approximately 4 × 10⁻⁶. 7 ~Approx. 7×10 7 Particles according to any one of claims 20 to 43, having surface hydrophobicity.

45. The particle according to any one of claims 20 to 44, wherein the particle has an increased intrinsic fluorescence intensity compared to the intrinsic fluorescence intensity of the protein and / or the activator alone (for example, when the protein and / or activator are not present in the particle).

46. The particles according to any one of claims 20 to 44, wherein, after exposure to a temperature of approximately 70°C for approximately 20 minutes, the amounts of α-helices, β-sheets, β-turns, and disordered tertiary and / or secondary structures present in the particles remain within approximately ±5%, approximately ±10%, approximately ±15%, approximately ±20%, approximately ±25%, approximately ±30%, or approximately ±35% of the amounts of α-helices, β-sheets, β-turns, and disordered tertiary and / or secondary structures present in the particles before exposure (for example, the amounts of α-helices, β-sheets, β-turns, and disordered tertiary and / or secondary structures present in the particles during their initial formation and / or immediately before exposure).

47. A plurality of particles comprising the particles described in any one of claims 20 to 46.

48. The plurality of particles according to claim 47, wherein the plurality of particles have a Dv(50) ranging from about 50, about 75 or about 100 nm to about 125, about 150 or about 200 nm.

49. The plurality of particles according to claim 47 or 48, wherein the plurality of particles have a polydispersity index (PDI) of less than about 0.5, optionally less than about 0.

3.

50. A composition comprising a carrier (e.g., water and / or oil) and particles prepared according to any one of claims 1 to 19 and / or particles according to any one of claims 20 to 46 and / or a plurality of particles according to any one of claims 47 to 49, wherein optionally, when the activator is present in the particles and the particles or the plurality of particles are present in the composition in an amount of about 100 mg per 1 mL of water, less than 30% of the activator is present free in the composition.

51. The composition according to claim 50, wherein the composition is a dispersion and optionally, there are no visible aggregates in the composition (for example, the composition is transparent and not cloudy or opaque).

52. An article comprising particles prepared in accordance with any one of claims 1 to 19 and / or particles according to any one of claims 20 to 46 and / or a plurality of particles according to any one of claims 47 to 49 and / or a composition according to any one of claims 50 to 51.

53. The article according to claim 52, wherein the article is a food (e.g., infant formula, dairy products, etc.), a nutritional supplement, a therapeutic drink, and / or a cosmetic.

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