Protein particles containing active agents and methods of making and using same - Patents.com

Abstract

Described herein are particles that comprise a protein and an active agent, such as particles that comprise alpha-lactalbumin and tryptophan. Methods of making and using the particles that comprise a protein and an active agent are also described herein.

Description

[Technical field]

[0001] STATEMENT REGARDING ELECTRONIC SUBMISSION OF SEQUENCE LISTINGS The sequence listing in XML text format entitled 1213-4WO_ST26.xml (size 4,507 bytes, generated on April 25, 2023 and submitted herewith) is incorporated herein by reference in its entirety.

[0002] The present invention relates to particles comprising a protein and an active agent, and to methods of making and using such particles. [Background technology]

[0003] Tryptophan (Trp) is a non-polar aromatic essential amino acid that can be obtained exclusively from dietary intake. It contributes to normal growth, protein synthesis, and the synthesis of important neurotransmitters and biomolecules. Tryptophan plays an important role in regulating neurobehavioral actions, such as appetite, mood, sleep, and pain perception. Tryptophan and tryptophan-containing peptides have shown various bioactive properties related to disease management, including psychological / cognitive function, antihypertensive, antioxidant, antidiabetic, and satiety. However, the use of tryptophan is limited due to its stability in food matrices and its noticeable bitter taste due to its non-polar and aromatic residues. Among free amino acids, tryptophan has the lowest bitter taste threshold (BTT: 4 mmol / L) (Di Pizio & Nicoli, 2020 Molecules (Basel, Switzerland), 25(20), doi.org / 10.3390 / olecules25204623). It is noteworthy that tryptophan is easily oxidized and degraded by changes in pH and temperature, affecting its bioactivity. Heat treatment induces the Maillard reaction between the primary amino group of tryptophan and reducing carbohydrates. In addition, tryptophan can be further degraded by oxidative species generated during the Maillard reaction. Similarly, lipid-derived oxidation products have been reported to reduce the bioavailability of tryptophan in storage studies (Nielsen et al., 1985). Summary of the Invention [Means for solving the problem]

[0004] A first aspect of the invention is directed to a particle comprising a protein; and an active agent, wherein the active agent is present within the protein (e.g., within the tertiary structure of the protein). In some embodiments, the active agent is non-specifically bound to the protein (e.g., via hydrophobic interactions, electrostatic interactions, hydrogen bonds, etc.).

[0005] A second aspect of the invention is directed to a plurality of particles, each particle of the plurality of particles comprising a protein and an active agent, wherein the active agent is present within the protein (e.g., within the tertiary structure of the protein).

[0006] Another aspect of the invention is directed to a composition comprising a carrier (eg, water and / or oil) and the particles of the invention.

[0007] A further aspect of the present invention is directed to a method of preparing a particle, the method comprising homogenizing or sonicating a composition comprising a protein and an active agent for about 1 minute to about 2 hours, wherein the composition has a pH of about 10 to about 12, thereby providing the particle, and the above method.

[0008] A further aspect of the invention is directed to a food product comprising the particles of the invention.

[0009] It should be noted that aspects described with respect to one embodiment may be incorporated in a different embodiment even if not specifically described with respect thereto. That is, all aspects and / or features of any embodiment may be combined in any manner and / or combination. The applicant reserves the right to modify the claims as originally filed and / or to file new claims accordingly, including the right to amend the claims as originally filed to rely on and / or incorporate features of any other claim or claims, even if not originally so claimed. These and other objects and / or aspects of the present invention are described in detail in the specification set forth below. Further features, advantages and details of the present invention will be appreciated by those skilled in the art from the accompanying drawings and the detailed description of the preferred embodiment which follows, however such description is merely illustrative of the present invention. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of an exemplary method for forming an α-lactalbumin-tryptophan (α-La-Trp) complex according to some embodiments of the present invention. [Diagram 2] FIG. 2 shows graphs of particle size distribution by intensity, volume and number measured at 25° C. and pH 11 and pH 7, respectively, for an α-La-Trp complex with a weight ratio of α-LA to Trp of 20:1. [Diagram 3] FIG. 3 shows graphs of particle size distribution by intensity, volume and number for an α-La-Trp complex with a weight ratio of α-LA to Trp of 15:1, measured at 25° C. and pH 11 and pH 7, respectively. [Figure 4] FIG. 4 shows graphs of particle size distribution by intensity, volume and number measured at 25° C. and pH 11 and pH 7, respectively, for an α-La-Trp complex with a weight ratio of α-LA to Trp of 10:1. [Diagram 5]FIG. 5 shows graphs of particle size distribution by intensity, volume and number for α-La-Trp complex with a weight ratio of α-LA to Trp of 5:1, measured at 25° C. and pH 11 and pH 7, respectively. [Figure 6] Figure 6 shows the average size (particle diameter, nm (d.nm)) (open squares) and polydisperse index (PDI, solid squares) of α-La-Trp particles prepared with a weight ratio of α-La to Trp of 20:1 at pH 11 under various pressure conditions for 30 min. [Figure 7] FIG. 7 is a graph of the average size (d.nm) (open squares) and PDI (polydispersity index, solid squares) of α-La-Trp particles prepared with a weight ratio of α-La to Trp of 20:1, at pH 11, under a pressure of 30,000 psi, and with various recirculation times. [Figure 8] FIG. 8 shows a graph of particle size distribution for α-La-Trp complex nanoparticles formed at 30,000 psi for 40 minutes (run 10), 20,000 psi for 30 minutes (run 12), and 40,000 psi for 30 minutes (run 14) in a weight ratio of 5:1, with strength and volume measured at 25° C., pH 11, and pH 7, respectively. [Figure 9] FIG. 9 shows a graph of particle size distribution for α-La-Trp complex nanoparticles formed at 30,000 psi for 40 minutes in a 5:1 weight ratio, with strength, volume and count measured at 25° C., pH 11 and pH 7, respectively. [Figure 10-1] Figure 10-1 shows graphs from a particle stability analysis of α-La-Trp complex nanoparticles formed at 30,000 psi for 40 minutes in a weight ratio of 5:1. Panels A and B of Figure 10-1 show the effect of storage time on particle size at pH 11 and pH 7, respectively. [Figure 10-2] Figure 10-2 shows graphs from particle stability analysis of α-La-Trp complex nanoparticles formed at 30,000 psi for 40 minutes in a weight ratio of 5:1. Panels C and D of Figure 10-2 show the effect of temperature on particle size at pH 11 and pH 7, respectively. [Figure 11] FIG. 11 is another schematic diagram of an exemplary method for forming an α-lactalbumin-tryptophan (α-La-Trp) complex, according to some embodiments of the present invention. [Figure 12] FIG. 12 shows graphs of particle size distribution for α-lactalbumin (α-La) by intensity, volume and number measured at 25° C., pH 11 and pH 7, respectively, before (panels A1-A3 of FIG. 12) and after (panels B1-B3 of FIG. 12) sonication. [Figure 13] FIG. 13 shows graphs of particle size distribution for tryptophan (Trp) by intensity, volume, and number measured at 25° C., pH 11, and pH 7, respectively, before (FIG. 13, panels A1-A3) and after (FIG. 13, panels B1-B3) sonication. [Figure 14] FIG. 14 shows graphs of particle size distribution of α-La-Trp by intensity, volume, and number measured at 25° C., pH 11, and pH 7, respectively, before (FIG. 14, panels A1-A3) and after (FIG. 14, panels B1-B3) sonication. [Figure 15] FIG. 15 is an illustration of an exemplary homogenizer setup, according to some embodiments of the present invention. [Figure 16] FIG. 16 shows graphs of the ABTS scavenging activity of α-lactalbumin (FIG. 16, panel A), tryptophan (FIG. 16, panel B), and α-lactalbumin-tryptophan (FIG. 16, panel C) before and after HPH treatment. [Figure 17] FIG. 17 is a graph of the intrinsic fluorescence of lactalbumin-tryptophan complexes before and after high-pressure homogenization (HPH). [Figure 18]Figure 18 shows the graphs of the average size (open squares) and PDI (polydispersity index, solid squares) of α-LA-Trp-NPs prepared under different conditions. Panel A of Figure 18 is a graph of the effect of pressure (α-LA / Trp ratio 20:1 at pH 11 with 30 min of recirculation). Panel B of Figure 18 is a graph of the effect of α-LA / Trp ratio (30 min under HPH pressure 206.8 Mpa). Panel C of Figure 18 is a graph of the effect of recirculation time (α-LA / Trp ratio 5:1 at pH 11 under HPH pressure 206.8 Mpa). [Figure 19] Figure 19 shows a graph of particle size distribution of mixtures of α-LA, Trp and α-LA-Trp without HPH at pH 11, and the same mixtures when the pH is shifted from 11 to 7 and from 11 to 3 at 25°C. α-La (α-lactalbumin), Trp (tryptophan). [Figure 20] FIG. 20 shows a graph of the fluorescence intensity as a result of HPH-induced self-assembly of α-LA and Trp (Panel A of FIG. 20), as well as the changes in fluorescence and turbidity during long-term treatment with HPH (Panel B of FIG. 20). [Figure 21] FIG. 21 shows the JMP output for an effect summary of all factors and factor combinations selected by the model, ranked based on their individual p-values ​​(Panel A of FIG. 21), as well as predictive profiler and desirability plots showing the effect of independent variables on the size (Y1) and Trp fluorescence intensity (Y2) of α-LA-Trp-NP at pH 11, and those at pH 7 (Y3 and Y4) (Panel B of FIG. 21). [Figure 22] FIG. 22 shows a graph of the fluorescence spectra (excitation wavelength 295 nm) of α-LA, Trp and α-LA-Trp mixtures with and without HPH treatment at pH 11 and pH 7 (Panel A of FIG. 22), a schematic diagram of the proposed mechanism of α-LA-Trp-NPs formation based on the fluorescence data (Panel B of FIG. 22), and scanning electron micrographs of α-LA (Panel C, images C1 and C4 of FIG. 22), Trp (Panel C, images C2 and C5 of FIG. 22) and α-LA-Trp (Panel C, images C3 and C6 of FIG. 22) mixtures after HPH treatment. [Diagram 23] FIG. 23 shows a graph of particle size distribution at 25° C. for α-LA-NP, Trp-NP, and α-LA-Trp-NP formed by HPH treatment at pH 11, 7, and 3. [Figure 24] Figure 24 shows particle thermal stability analysis of α-LA-NP, Trp-NP and α-LA-Trp-NP after HPH treatment. NPs were formed at α-LA / Trp ratio of 5:1, 206.8 MPa, 40 min, pH 11, and in the sample with pH shift, the pH shifted from 11 to 7. [Diagram 25] 25 shows a graph of particle freeze-thaw stability analysis and freeze-thaw-thermal stability analysis of α-LA-NP, Trp-NP, and α-LA-Trp-NP made through HPH processing. The NPs were made at α-LA / Trp ratio of 5:1, 206.8 MPa, 40 min, pH 11, and in the samples with pH shift, the pH shifted from 11 to 7 and 3. [Figure 26] Figure 26 shows SEM images of freeze-dried α-LA (panel A, image A1 in Figure 26), Trp (panel A, image A2 in Figure 26), and a mixture of α-LA and Trp (panel A, image A3 in Figure 26) without high-pressure homogenization treatment (NOHPH) and with high-pressure homogenization treatment (HPH) (panel A, images A4 to A6 in Figure 26), as well as a graph showing the particle size distribution and PDI value (in parentheses) of freeze-dried α-LA, Trp, and a mixture of α-LA and Trp after redispersion in water and PBS buffer (panel B in Figure 26). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The invention will now be described herein below with reference to the accompanying drawings and examples in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented or all the features that may be added to the invention. For example, features illustrated with respect to one embodiment may be incorporated in other embodiments, and features illustrated with respect to a particular embodiment may be omitted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any one or combination of features described herein may be excluded or omitted. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, which do not depart from the invention. Thus, the following description is intended to illustrate some specific embodiments of the invention, and is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the present invention.

[0013] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is set forth.

[0014] Unless the context indicates otherwise, it is specifically intended that the various features of the present invention described herein can be used in any combination.Moreover, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein can be eliminated or omitted.For example, when the present specification states that a composition comprises component A, component B and component C, it is specifically intended that any of A, B or C, or combinations thereof, can be omitted and removed.

[0015] As used in the detailed description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0016] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0017] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to include not only the stated value, but also a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. For example, "about X", where X is a measurable value, is meant to include X and a variation of ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of X. Ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​within the range.

[0018] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y", and phrases such as "about X to Y" mean "about X to about Y".

[0019] The recitation of ranges of values ​​herein, unless otherwise stated herein, is intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if each separate value were individually set forth herein. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0020] As used in this specification, the words "comprises" and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0021] As used herein, the transitional phrase "consisting essentially of" as applied to the compositions of the present invention means that the claims should be construed to include the particular materials or steps recited in the claims, and which do not materially affect one or more of the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" as used in the claims of the present invention is not intended to be construed as the equivalent of "comprise."

[0022] As used herein, the words "increase", "increase", "enhance", "enhance", "enhance", and "enhance" (as well as grammatical variations thereof) refer to 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 as compared to another measurable characteristic or amount (e.g., a control value).

[0023] As used herein, the words "reduction", "reduced", "reduce", "reduce" and "decrease" (as well as grammatical variations thereof) refer to a decrease of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%, for example, compared to another measurable characteristic or amount (e.g., a control value). In some embodiments, the decrease may result in no detectable activity or amount, or essentially no decrease (i.e., an insignificant amount, e.g., less than about 10%, or even less than 5%).

[0024] A "portion" or "fragment" of a nucleotide sequence or polypeptide (including a domain) refers to a nucleotide sequence or polypeptide (e.g., one or more nucleotides or one or more peptides) of reduced length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues) relative to a reference nucleotide sequence or reference polypeptide, respectively, that is with respect to the above nucleotide sequences or polypeptides comprising, consisting essentially of, and / or consisting of nucleotide sequences or polypeptides of consecutive residues that are identical or nearly identical (e.g., 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), respectively.

[0025] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout the window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods, including, but not limited to, those described in the following references: Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., 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).

[0026] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent identity" can refer to the percentage of identical amino acids in an amino acid sequence compared to a reference polypeptide.

[0027] As used herein, in the context of two nucleic acid molecules, nucleotide sequences, or protein sequences, the phrase "substantially identical" or "substantial identity" is used interchangeably with the phrase "substantially identical" or "substantial identity." "identity" refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have at least about 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% nucleotide or amino acid residue identity as measured using one of the sequence comparison algorithms described below or by visual inspection. In some embodiments of the invention, the substantial identity exists over a region of contiguous nucleotides of the nucleotide sequences of the invention that is about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides, or more nucleotides in length, and any range therebetween up to the full length of the sequence. In some embodiments, the nucleotide sequences can be substantially identical over at least about 20 nucleotides (e.g., 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, a substantially identical nucleotide or protein sequence performs substantially the same function as the nucleotide (or encoded protein sequence) to which it is substantially identical.

[0028] In sequence comparison, typically, one sequence serves as a reference sequence, with which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for one or more test sequences relative to the reference sequence based on the designated program parameters.

[0029] Optimal alignment of sequences for aligning a 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 by using these algorithms, such as the GCG 登録商標 Wisconsin Package 登録商標(Accelrys Inc., San Diego, CA). The "identity fraction" of an aligned segment of a test sequence and a 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 of the reference sequence). The percentage of sequence identity is expressed as the percentage of identity multiplied by 100. Comparison of one or more polynucleotide sequences can be performed on full-length polynucleotide sequences or portions thereof, or on longer polynucleotide sequences. For purposes of the present invention, "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0030] Particles comprising a protein and an active agent associated with the protein are provided according to embodiments of the present invention. In some embodiments, the active agent is present within the protein present in the particle of the present invention, and / or the active agent is present on the surface of the protein present in the particle of the present invention. In some embodiments, the active agent is present within the tertiary structure of the protein. In some embodiments, the active agent is non-specifically bound to the protein, for example, via hydrophobic interactions, electrostatic interactions, hydrogen bonds, and / or the like. 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) can be present in the particle 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) active agents are present between two or more protein molecules that are associated with each other (e.g., via non-specific interactions). In some embodiments, the particles of the present invention are homogeneous polygonal particles.

[0031] The particles of the invention may comprise one or more (e.g., 1, 5, 10, 20, 30, 40, 50 or more) active agents, which may be the same 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 the same or different from each other. In some embodiments, the particles of the invention comprise an active agent that is in a region of the tertiary structure of the protein (e.g., in a tertiary fold). In some embodiments, the active agent is in a region of the tertiary structure of the protein (e.g., in a tertiary fold) that comprises at least one non-specific hydrophobic interaction between two or more amino acid residues. In some embodiments, the active agent is in a hydrophobic pocket of the protein. In some embodiments, the active agent is in the protein core of the protein. In some embodiments, the active agent is in a folded region of the protein, optionally with zero solvent accessibility. In some embodiments, when a particle of the present invention comprises multiple active agents (wherein the active agents in the multiple active agents may be the same as or different from one another), at least one active agent of the multiple active agents may be present within the protein, and one or more active agents of the multiple active agents may be present on the surface of the protein.

[0032] Exemplary proteins of the 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 protein," "milk protein," "plant protein," "animal protein," and "meat protein" refer to proteins that are naturally found in dairy, milk, plants, animals, and meat, respectively, and / or proteins that are derived from such naturally occurring proteins such that they have an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of the naturally occurring protein. For example, in some embodiments, the dairy, milk, plant, animal, or meat protein is naturally found in dairy, milk, plants, animals, or meat, respectively, and / or the protein is isolated from dairy, milk, plants, animals, or meat, respectively, or the protein is synthetically prepared such that it has an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of the naturally occurring protein. In some embodiments, the protein is a milk protein, such as, but not limited to, α-lactalbumin, β-lactoglobulin and / or lactoferrin. In some embodiments, whey protein isolate (WPI) containing α-lactalbumin and β-lactoglobulin is used to prepare the particles of the present invention, and the particles of the present invention contain one or more α-lactalbumins and one or more β-lactoglobulins.

[0033] The proteins used to prepare the particles of the invention may have a molten globule state and / or may have a bilobal structure. The proteins may contain two or more (e.g., 2, 3, 4, 5, or more) domains and / or at a pH of about 5 to about 9, the proteins may contain one or more (e.g., 1, 2, 3, 4, or more) intramolecular disulfide bonds. In some embodiments, the proteins contain at least two domains and the proteins contain at least one disulfide bridge connecting the two domains of the protein. In some embodiments, the proteins are monomeric. In some embodiments, the protein has about 100 amino acids to about 200 amino acids, about 300 amino acids, about 400 amino acids or about 500 amino acids, and / or a molecular weight of about 10,000 kDa to about 20,000 kDa, about 30,000 kDa, about 40,000 kDa or about 50,000 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 10,000, about 15,000 kDa, about 20,000 kDa, about 25,000 kDa, about 30,000 kDa, about 35,000 kDa, about 40,000 kDa, about 45,000 kDa, or about 50,000 kDa. The protein may have an isoelectric point (pI) of 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, or about 5. In some embodiments, the protein has a pI of about 4.2 to about 4.5.The protein may have a tertiary structure comprising α-helices in an amount of about 10% or about 15% to about 20%, about 25% or about 30% of the total tertiary structure (optionally calculated by the percentage of the number of amino acids present in α-helices relative to the total number of amino acids in the protein), and β-sheets in an amount of about 1% or about 5% to about 10%, about 15%, about 20% or about 25% (optionally calculated by the percentage of the number of amino acids present in β-sheets relative to the total number of amino acids in the protein), and / or a disordered tertiary structure in an amount of about 50%, about 55%, or about 60% to about 65%, about 70% or about 75% of the total tertiary structure (optionally calculated by the percentage of the number of amino acids present in unordered tertiary structure relative to the total number of amino acids in the protein).

[0034] 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 a protein (e.g., one or more protein molecules) in a total amount of about 75%, about 80%, about 85%, to about 90%, about 95%, about 99%, or about 100% by weight of the particle, and an active agent (e.g., one or more active agents) in a total amount of about 0.1%, about 0.5%, about 1%, or about 5% to about 10%, about 15%, about 20%, or about 25% by weight of the particle.

[0035] Further exemplary proteins that may be present in the particles of the present invention include, but are not limited to, α-lactalbumin, lysozyme, cytochrome c, apomyoglobin, staphylococcal nuclease, β-lactoglobulin, lactoferrin, and any combination thereof. In some embodiments, the particles of the present invention comprise α-lactalbumin (e.g., bovine α-lactalbumin and / or human α-lactalbumin). The protein of the present invention can be derived from any source (e.g., plant, animal, etc.). In some embodiments, the protein is obtained and / or derived from an animal source, such as a mammal (e.g., bovine or human). In some embodiments, the particles of the present invention comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 α-lactalbumin molecules. In some embodiments, a protein present in a particle of the invention has an amino acid sequence having 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 about 100% sequence identity to one or more of SEQ ID NOs:1-3. In some embodiments, a protein present in a particle of the invention has an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to one or more of SEQ ID NOs:1-3.In some embodiments, a protein present in a particle of the invention has an amino acid sequence having about 100% sequence identity to one or more of SEQ ID NOs:1-3.

[0036] The active agent used to prepare the particles of the present invention can be an organic compound, such as, but not limited to, an amino acid.In some embodiments, the active agent has a molecular weight of 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 active agent can have a solubility in water of about 15 mg / mL or less at 25°C, for example, about 15 mg / mL or less, about 14 mg / mL or less, about 13 mg / mL or less, about 12 mg / mL or less, about 11 mg / mL or less, about 10 mg / mL or less, about 9 mg / mL or less, about 8 mg / mL or less, about 7 mg / mL or less, about 6 mg / mL or less or about 5 mg / L or less. In some embodiments, the active agent may have a solubility in water at 25° C. of about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 or about 10 mg / mL to about 11 mg / L, about 12 mg / L, about 13 mg / L, about 14 or about 15 mg / L. In some embodiments, the active agent has a pKa of about 1.5, about 2, about 2.5, about 2.6, about 2.7 or about 2.8 to about 2.9, about 3, about 3.2 or about 3.5, and / or a pI of about 5, about 5.5, about 5.6, about 5.7 or about 5.8 to about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4 or about 6.5. Exemplary active agents 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 active agent present in the particles of the invention is tryptophan.

[0037] The particles of the present invention may have a size in at least one dimension (e.g., diameter) of about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 125 or about 150 nm to 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, about 800 nm, about 850 or about 900 nm, optionally as 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 particle has a size (e.g., diameter) in at least one dimension of about 25 nm, 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, about 800 nm, about 850 nm or about 900 nm.In some embodiments, the particle has a size (e.g., diameter) in at least one dimension of about 230 nm.In some embodiments, the particle is a nanoparticle. In some embodiments, the particles of the present invention, the particles prepared according to the methods of the present invention, and / or the particles present in the compositions of the present invention have a Dv(50) of about 175 nm or about 200 nm to about 225 nm, about 250 nm, about 275 nm, about 300 nm, or about 325 nm, optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS. In some embodiments, the particles of the present invention, the particles prepared according to the methods of the present invention, and / or the particles present in the compositions of the present invention have a Dv(50) of about 175 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, or about 325 nm, optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS.In some embodiments, the particles of the present invention have a polydispersity index (PDI) of less than about 0.5 (e.g., less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1). In some embodiments, the particles of the present invention have a PDI of less than 0.3 or less than 0.2.

[0038] In some embodiments, the protein and / or active agent used to prepare the particles of the present invention is dissolved in water at a temperature of about 25° C. and a pH of about 11. In some embodiments, the active agent and / or protein is dissolved in water in an amount of 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 / L or about 400 mg / L at a temperature of about 25° C. and a pH of about 11. The protein and / or active agent may have a negative charge in water at a pH of about 11.

[0039] Particles of the invention comprising a protein and an active agent may have improved (e.g., increased) storage, stability, activity, and / or function compared to the storage, stability, activity, and / or function of the protein alone (e.g., the protein not present in a particle of the invention and not associated with the active agent). In some embodiments, the size in at least one dimension of the particle (e.g., diameter) remains within ±20% of its original size (e.g., the size of the particle at initial formation and / or the size at the first day of storage) in response to storage in a sealed container at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months at about 4° C. to about 10° C. For example, at an initial time point (e.g., beginning on day 1 of the storage period), the particles may have a diameter of about 225 nm, and after storage 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 beginning on day 1 of the storage period, the particles may have a size that increases or decreases by about 20% or less. Thus, a particle having a starting size of about 225 nm may have a size at the end of the storage period in the range of about 180 nm to about 270 nm. In some embodiments, in response to storage 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 particles of the present invention have at least one dimension of size (e.g., diameter) that increases by an amount less than about 20% compared to their original size. In some embodiments, dried particles (e.g., freeze-dried particles and / or spray-dried particles and / or particles containing water in an amount of about 0% to about 5% by weight of the dried particles) 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 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, the particles present in a composition (e.g., water and / or buffer) 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, the size (e.g., diameter) of the particles in the composition is measured at the end of the storage period.

[0040] In some embodiments, the particle of the present invention and / or the particles of the present invention are stable in that they have one peak particle size distribution in the composition at a pH of about 7 and / or in the composition at a pH of about 11, optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS. In some embodiments, the particle of the present invention and / or the particles of the present invention are stable in that they have one peak particle size distribution in the composition at a pH of about 7 and one peak particle size distribution in the composition at a pH of about 11, optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS. In some embodiments, the composition comprising the particles (e.g., a composition having a pH of about 3, about 7, and / or about 11) has two or more peak particle size distributions, optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS, indicating that one of the particles is not stable. In some embodiments, the particles of the present invention are stable in that they do not fall out of (e.g., do not precipitate) and / or do not aggregate in the compositions of the present invention. In some embodiments, the particles of the present invention are stable in that they do not fall out of (e.g., do not precipitate) and / or do not aggregate in the compositions of the present invention when the pH in the composition is adjusted (e.g., from a pH of about 11 to a pH of about 7).

[0041] In some embodiments, the particles of the invention have increased freeze-thaw stability compared to the freeze-thaw stability of the protein alone (i.e., the protein not present in the particles of the invention) and / or the active agent alone (i.e., the active agent not present in the particles of the invention). As used herein, "freeze-thaw stability" can refer to the stability and / or properties of a material (e.g., a particle of the invention, an active agent alone, or a protein alone) before and after exposure to freezing (i.e., 0°C) or lower, followed by exposure to temperatures above freezing (i.e., above 0°C). Freeze-thaw stability can be determined by comparing the stability and / or properties of a material before freezing (i.e., before exposure to temperatures below 0°C) with the stability and / or properties of the same material after freezing and thawing (i.e., after exposure to temperatures below 0°C and after exposure to temperatures above 0°C). In some embodiments, one or more properties of the particles of the present invention before freezing and after exposure to temperatures above freezing can be compared with the same one or more properties of the protein alone before and after the same conditions, optionally where the protein is the same protein present in the particles.In some embodiments, one or more properties of the particles of the present invention before freezing and after exposure to temperatures above freezing can be compared with the same one or more properties of the active agent alone before and after the same conditions, optionally where the active agent is the same active agent present in the particles.In some embodiments, one or more properties of the first particle of the present invention before freezing and after exposure to temperatures above freezing can be compared with the same one or more properties of the second particle of the present invention before and after the same conditions, optionally where the first particle and the second particle contain the same protein and / or active agent, but are different in some aspects (e.g., are prepared differently and / or have different sizes and / or different concentrations of the protein and / or active agent). In some embodiments, freeze-thaw stability may be determined by comparing the stability and / or properties of the material before freezing (i.e., before exposure to temperatures below 0° C.) with the stability and / or properties of the material after freezing and thawing (i.e., after exposure to temperatures below 0° C. and above 0° C.).In some embodiments, freeze-thaw stability is determined by measuring the change, if any, in particle size (e.g., diameter), polydispersity index, and / or particle size distribution, and / or by one or more properties of the material as a dry product and / or in an aqueous composition (e.g., the presence and / or amount of precipitates and / or aggregates (e.g., agglomerates) of the material).

[0042] In some embodiments, the particles of the present invention have improved freeze-thaw stability. In some embodiments, after a change in temperature (e.g., a change in storage temperature, an increase in temperature, and / or a change in temperature from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C), at least one dimension of the particles of the present invention remains within about ±20% of its original size (e.g., the size of the particle at the time of initial formation and / or the size before the temperature change), optionally within about ±15% of its original size, optionally at a pH of about 7 to about 11 (e.g., a pH of about 7 or about 11). For example, prior to a change in temperature from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C, the particles (optionally at a pH of about 7 or about 11) may have a diameter of about 230 nm, and after a change in temperature from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C, the particles (optionally at a pH of about 7 or about 11) may have an increase or decrease in size of about 20% or less, optionally an increase or decrease in size of about 15% or less. Thus, optionally at a pH of about 7 or about 11, a particle having a size of 230 nm prior to the change in temperature may have a size after the change in temperature in the range of about 185 nm to about 275 nm. In some embodiments, after a change in temperature from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C, optionally at a pH of about 7 or about 11, the particles of the present invention have a size in at least one dimension (e.g., diameter) that increases by less than about 20% compared to its original size, and optionally increases by less than about 15% compared to its original size. In some embodiments, the particles of the present invention are stable and / or remain stable (e.g., the particles do not fall out of (e.g., precipitate) from the composition and / or do not aggregate with other particles) before, during, and / or after the change in temperature (e.g., from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C).

[0043] In some embodiments, the particles of the invention have increased freeze-thaw thermal stability compared to the freeze-thaw thermal stability of the protein alone (i.e., the protein not present in the particles of the invention) and / or the active agent alone (i.e., the active agent not present in the particles of the invention). As used herein, "freeze-thaw thermal stability" can refer to the stability and / or properties of a material (e.g., a particle of the invention, an active agent alone, or a protein alone) before and after exposure to a temperature at or below freezing temperature (i.e., 0°C) followed by exposure to a temperature above freezing temperature (i.e., above 0°C), including heat treatment (i.e., exposure to a temperature of at least 55°C for at least 25 minutes). Freeze-thaw thermal stability can be determined by comparing the stability and / or properties of a material before freezing (i.e., before exposure to 0°C or below) with the stability and / or properties of the same material after freezing and thawing (i.e., after exposure to 0°C or below and above 0°C, including heat treatment). In some embodiments, one or more properties of a particle of the present invention before freezing and after heat treatment can be compared to the same one or more properties of a protein alone before and after the same conditions, optionally where the protein is the same protein present in the particle. In some embodiments, one or more properties of a particle of the present invention before freezing and after heat treatment can be compared to the same one or more properties of an active agent alone before and after the same conditions, optionally where the active agent is the same active agent present in the particle. In some embodiments, one or more properties of a first particle of the invention before freezing and after heat treatment may be compared to the same one or more properties of a second particle of the invention before and after the same conditions, optionally where the first particle and the second particle contain the same protein and / or active agent but differ in some aspects (e.g., are formulated differently and / or have different sizes and / or different concentrations of the protein and / or active agent). In some embodiments, freeze-thaw thermal stability may be determined by comparing the stability and / or properties of the material before freezing (i.e., before exposure to below 0° C.) with the stability and / or properties of the material after freezing and thawing (including heat treatment) (i.e., after exposure to below 0° C. and above 0° C., including heat treatment). In some embodiments, freeze-thaw thermal stability is determined by measuring the change, if any, in particle size (e.g., diameter), polydispersity index, and / or particle size distribution, and / or by one or more properties of the material as a dry product and / or in an aqueous composition (e.g., the presence and / or amount of precipitates and / or aggregates (e.g., agglomerates) of the material).

[0044] In some embodiments, the particles of the present invention have improved freeze-thaw thermal stability. In some embodiments, after a change in temperature (e.g., a change in storage temperature, an increase in temperature, and / or a change in temperature from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C), followed by heat treatment (e.g., heat treatment at about 55°C, about 60°C, or about 65°C for about 25 minutes, about 30 minutes, or about 35 minutes), at least one dimension of the particle remains within about ±20% of its original size (e.g., the size of the particle at the time of initial formation and / or the size before the temperature change), optionally at a pH of about 7 to about 11 (e.g., a pH of about 7 or about 11), and optionally within about ±15% of its original size. For example, the particles may have a diameter of about 230 nm, and after the change in temperature and heat treatment, the particles may have an increase or decrease in size of about 20% or less, optionally an increase or decrease in size of about 15% or less, optionally at a pH of about 7 or about 11. Thus, optionally at a pH of about 7 or about 11, particles having a size of 230 nm before the change in temperature and heat treatment may have a size in the range of about 170 nm to about 290 nm after the change in temperature and heat treatment. In some embodiments, after a change in temperature from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C, followed by heat treatment at about 55°C, about 60°C, or about 65°C for about 25 minutes, about 30 minutes, or about 35 minutes, the particles of the invention (optionally at a pH of about 7 or 11) have a size in at least one dimension (e.g., diameter) that is increased by an amount of less than about 20% compared to its original size, optionally increased by an amount of less than about 15% compared to its original size. In some embodiments, the particles of the present invention are stable (e.g., the particles do not fall out of the composition (e.g., precipitate) and / or aggregate with other particles) when the particles are exposed to a change in temperature (e.g., a change from about -20°C, about -15°C, or about -10°C to about 15°C, about 20°C, or about 25°C) followed by heat treatment (e.g., heat treatment for about 25 minutes, about 30 minutes, or about 35 minutes at about 55°C, about 60°C, or about 65°C). In some embodiments, the particles of the present invention have a synergistic effect.As used herein, "synergistic," "synergy," or grammatical variations thereof, refers to a combination that exhibits an effect that is greater than the effect expected from the sum of the effects of the individual parts of the combination. For example, the term "synergistic" or "synergistic" with respect to the particles of the present invention may refer to a combination of a protein and an active agent, and / or two different method steps for preparing the particles of the present invention that result in a property and / or effect that is greater than the effect expected from the sum of the individual parts alone. In some embodiments, the particles of the present invention prepared using a high-pressure homogenization step and a pH-shifting step may have a stability (e.g., colloidal stability, pH stability, thermal stability, freeze-thaw stability, and / or freeze-thaw thermal stability) that is greater than the sum of the stability of a particle containing the same amount of the same protein and active agent prepared by the same high-pressure homogenization step alone and the stability of a particle containing the same amount of the same protein and active agent prepared by the same pH-shifting step alone.

[0045] In some embodiments, the particles of the present invention have increased activity and / or function (e.g., increased antioxidant activity) compared to the activity and / or function of the protein alone. In some embodiments, the particles of the present invention have improved function. In some embodiments, the particles of the present invention improve the properties of the protein and / or active agent present in the particle. For example, in some embodiments, the particles of the present invention can reduce the bitterness of the active agent (e.g., tryptophan) by, for example, increasing the bitter taste threshold (BTT), optionally measured by Di Pizio & Nicoli, 2020 Molecules (Basel, Switzerland), 25(20), doi.org / 10.3390 / molecules25204623 (which is incorporated herein for bitterness and / or methods for measuring bitter taste threshold (BTT)).

[0046] According to some embodiments, a composition is provided comprising the particles of the present invention. In some embodiments, the composition comprises a plurality of particles of the present invention. In some embodiments, the composition comprises the particles of the present invention and a carrier. The carrier can 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, for example, in an amount of about 5% to about 20%). One or more additives can be present in the compositions of the present invention. Exemplary additions include, but are not limited to, pectin and / or gums. In some embodiments, the compositions of the present invention do not include masking agents, flavoring agents, cyclodextrins (e.g., β-cyclodextrin), and / or physical barriers optionally configured to mask or reduce the taste of the active agent present in the particles of the present invention. In some embodiments, the composition is not a gel (e.g., a hydrogel, such as a 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., a hydrogel, such as a protein hydrogel) or an emulsion. In some embodiments, the compositions of the present invention do not include agents (e.g., masking agents) that are not constructed and / or designed to provide a desired flavor or taste of the composition, but rather are constructed and / or designed to reduce the bitterness and / or off-taste of an active agent present in the composition.

[0047] In some embodiments, the composition of the present invention is a food product, a dietary supplement, a therapeutic drink, and / or a cosmetic product. In some embodiments, the particles of the present invention can be present in a food product. In some embodiments, the food product is a dairy product (e.g., milk, yogurt, etc.).

[0048] The active agent present in the particles of the present invention, when present in the carrier and / or composition of the present invention, can remain associated with the particles and / or proteins present in the particles (e.g., remain complexed with the particles and / or proteins present in the particles, within the particles and / or proteins present in the particles, etc.). In some embodiments, about 30% or less of the total amount of active agent 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 active agent present in the particles of the present invention provided in the carrier and / or composition and / or used to prepare the particles of the present invention can be present in the carrier and / or composition as free active agent (i.e., active agent not associated with the particles and / or proteins). Thus, for a particle containing a given amount of active agent, when the particle is added to a carrier and / or composition, the amount of free active agent present in the carrier and / or composition may be about 30% or less than the given amount of active agent present in the particle. In some embodiments, a composition comprising water and the particles of the present invention (where the particles are present in an amount of about 100 mg per mL of water) contains free active agent in an amount of about 0%, about 1%, about 2%, about 5%, about 10%, or about 15% by weight to about 20%, about 25%, or about 30% by weight of the total amount of active agent present in the particle. 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 (e.g., does not have visible lumps, aggregates, or particles) in the composition. In some embodiments, the composition is clear and not cloudy or opaque. In some embodiments, the composition may appear cloudy, but there is no precipitate and / or aggregates.

[0049] A method for preparing the particles of the present invention is provided according to some embodiments of the present invention.In some embodiments, the method comprises homogenizing and / or sonicating a composition comprising a protein and an active agent for about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes or about 30 minutes to about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes or about 120 minutes, wherein the composition has a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12, thereby providing the particles.In some embodiments, the homogenizing and / or sonicating is carried out for about 10 minutes to about 50 minutes. The composition comprising the protein and the active agent may be homogenized and / or sonicated for about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes. In some embodiments, the composition has a pH of about 10, about 10.5, about 11, about 11.5, or about 12. In some embodiments, the composition has a pH of about 11. In some embodiments, the protein and / or active agent in the composition are negatively charged, optionally before, during, and / or after the composition is homogenized and / or sonicated. In some embodiments, the homogenizing and / or sonicating is carried out at a temperature of about 15°C or about 20°C to about 25°C or about 30°C, optionally at a temperature of about 15°C, about 20°C, about 25°C or about 30°C.

[0050] In some embodiments, compositions used to prepare particles of the invention having a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12 (e.g., optionally about 11) may contain protein and active agent 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, or 10:1 to 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:active agent). In some embodiments, the composition comprises protein and active agent in a weight ratio of about 5:1 to about 20:1 (protein:active agent). In some embodiments, compositions used to prepare the particles of the invention, having a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12 (e.g., optionally about 11), contain protein and active agent 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:active agent). In some embodiments, the composition used to prepare the particles, having a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12 (e.g., optionally about 11), has a solids content of about 1 w / v%, about 2 w / v%, about 3 w / v%, about 4 w / v%, about 5 w / v%, about 6 w / v%, about 7 w / v%, about 8 w / v%, about 9 w / v%, or about 10 w / v% to about 11 w / v%, about 12 w / v%, about 13 w / v%, about 14 w / v%, about 15 w / v%, about 16 w / v%, about 17 w / v%, about 18 w / v%, about 19% or about 20 w / v%.In some embodiments, the composition used to prepare the particles, having a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12 (e.g., optionally about 11), has a solids content of about 1 w / v%, about 2 w / v%, about 3 w / v%, about 4 w / v%, about 5 w / v%, about 6 w / v%, about 7 w / v%, about 8 w / v%, about 9 w / v%, about 10 w / v%, about 11 w / v%, about 12 w / v%, about 13 w / v%, about 14 w / v%, about 15 w / v%, about 16 w / v%, about 17 w / v%, about 18 w / v%, about 19 w / v% or about 20 w / v%.

[0051] In compositions used to prepare the particles of the invention having a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12 (e.g., optionally about 11), the active agent is present in an amount of 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 about 50 mg / mL to about 10 mg / mL. and / or the protein may be present in an amount of 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, about 50 mg / mL, 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, about 100 mg / mL, about 150 mg / mL or about 200 mg / mL. About 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, 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 to about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, about 260 mg / mL, about 270 mg / mL, about 280 mg / mL, about 290 mg / mL, about 300 mg / mL, about 310 mg / mL, about 320 mg / mL, about 330 mg / mL, about 340 mg / mL, about 350 mg / mL, about 360 mg / mL, about 370 mg / mL, about 380 mg / mL, about 390 mg / mL, about 400 mg / mL, about 410 mg / mL, about 420 mg / mL, about 430 mg / mL, about 440 mg / mL, about 450 mg / mL, about 460 mg / mL, about 470 mg / mL, about 480 mg / mL, about 490 mg / mL, about 500 mg / mL, about 510 mg / mL, about 520 mg / mL, about 53 It may be present in an amount of about 40 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, about 260 mg / mL, about 270 mg / mL, about 280 mg / mL, about 290 mg / mL or about 300 mg / mL. For example, in some embodiments, the active agent (e.g., tryptophan) may be dissolved in an amount of about 30 mg / mL to about 50 mg / mL in a composition having a pH of about 11, and the protein (e.g., alpha-lactalbumin and / or WPI) may be dissolved in the same composition in an amount of about 50 mg / mL or about 100 mg / mL to about 200 mg / L or about 300 mg / L. In some embodiments, alpha-lactalbumin is dissolved in a composition having a pH of about 11 in an amount of about 200 mg / mL or less, such as from about 100 mg / mL to about 200 mg / mL.In some embodiments, the WPI is dissolved in an amount of about 50 mg / mL or less, e.g., about 10 mg / mL to about 50 mg / mL, in a composition having a pH of about 11. In some embodiments, the lactoferrin is dissolved in an amount of about 10 mg / mL or less, e.g., about 1 mg / mL to about 10 mg / mL, in a composition having a pH of about 11.

[0052] Homogenization of compositions having a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12 and comprising a protein and an active agent can be performed using methods and / or homogenizers known in the art. In some embodiments, homogenizing the compositions of the present invention comprises homogenizing the composition at a pressure of about 5,000 psi, about 10,000 psi, about 15,000 psi, or about 20,000 psi to about 25,000 psi, about 30,000 psi, about 35,000 psi, about 40,000 psi, or about 45,000 psi. In some embodiments, homogenizing the composition comprises homogenizing the composition at a pressure in the range of about 20,000 psi to about 40,000 psi. In some embodiments, homogenizing the composition comprises homogenizing the composition at a pressure of about 5,000 psi, about 10,000 psi, about 15,000 psi, about 20,000 psi, about 25,000 psi, about 30,000 psi, about 35,000 psi, about 40,000 psi, or about 45,000 psi. In some embodiments, the homogenizer used in the method of the present invention may comprise the device shown in FIG. 15. In some embodiments, the homogenizer may be a device sold by Bee International (USA), such as, but not limited to, a BEE Emulsifying Cell sold by Bee International (USA). In some embodiments, the method of the present invention comprises homogenizing using a homogenizer and / or device that provides in-line cavitation. In some embodiments, the methods of the invention include homogenizing a composition comprising a protein and an active agent using a homogenizer and / or device in which the composition is delivered through a nozzle at pressure (e.g., a pressure of about 5,000 psi to about 45,000 psi), thereby providing a high velocity jet stream (e.g., a jet stream having a velocity of up to about 70 mL / min).In some embodiments, the conveying and / or flowing of the composition through the inlet of the homogenizer and / or the nozzle of the device may be laminar, e.g., gentle process, or turbulent, e.g., pre-mixing. The nozzle of the homogenizer and / or the device may have an orifice with a diameter of about 0.1 mm, which may induce high shear. The conveying of the composition of the present invention through the nozzle may provide a rapid acceleration with a pressure drop upon exiting the orifice of the nozzle, which may induce cavitation. After the composition leaves the nozzle, the composition may flow and / or be sent into an absorption cell that includes one or more orifices with a diameter of about 0.5 mm or more. Without wishing to be bound by any particular theory, the kinetic energy of the fluid jet in such homogenization processes may be absorbed within the composition and / or within particles present in the composition, optionally by manipulating the flow and / or converting its velocity into shear, cavitation and / or impact forces. In some embodiments, homogenizing the compositions of the present invention includes exposing the composition to high pressure homogenization (HPH) and / or high shear forces, cavitation and turbulence.

[0053] Sonicating the composition having a pH of about 10 or about 10.5 to about 11, about 11.5 or about 12 and comprising a protein and an active agent can be performed using methods and / or sonicators known in the art. In some embodiments, sonicating the composition comprises sonicating the composition at a frequency of about 15 kHz or about 20 kHz to about 25 kHz or about 30 kHz with an amplitude of about 40%, 45% or about 50% to about 55%, about 60%, about 65% or about 70%. In some embodiments, sonicating the composition comprises sonicating the composition at a frequency of about 15 kHz, about 20 kHz, about 25 kHz or about 30 kHz with an amplitude of about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or about 70%.

[0054] The methods of the invention may include adjusting the pH of the composition to a pH of about 6.5 to about 7.5 after homogenizing and / or sonicating the composition. In some embodiments, the pH of the composition is adjusted to a pH of about 7. An acid and / or a base (e.g., an organic acid and / or an organic base) may be used to adjust the pH of the composition.

[0055] The method of the present invention may provide particles of the present invention having a particle size distribution of 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 an average particle size (e.g., average particle diameter) of about 100 nm, about 125 nm, about 150 nm, or about 175 nm to about 200 nm, about 225 nm, about 250 nm, about 275 nm, or about 300 nm. In some embodiments, the particles can have an average particle size (e.g., average particle diameter) of about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, or about 300 nm. In some embodiments, the particles can have an average particle size (e.g., average particle diameter) of about 230 nm.

[0056] In some embodiments, the method of the present invention comprises dehydrating the particles of the present invention and / or the composition in which the particles are present. Dehydrating the particles of the present invention and / or the composition comprising the particles of the present invention can be carried out using methods and / or devices known in the art. In some embodiments, dehydrating the particles of the present invention and / or the composition comprising the particles of the present invention comprises freeze-drying and / or spray-drying the particles and / or the composition.

[0057] The method 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 response to adjusting the pH of the composition in which the particles are present. In some embodiments, the method of the present invention includes reducing the size (e.g., diameter, optionally average diameter) of the particles of the present invention by 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 by 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 is adjusted from a pH of about 11 to a pH of about 7.

[0058] In some embodiments, the method of the present invention comprises administering a therapeutically effective amount of the particles of the present invention and / or the compositions of the present invention to a subject.As used herein, the term "therapeutically effective amount" refers to an amount of the particles and / or compositions of the present invention that induces a therapeutically useful response in a 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.

[0059] As used herein, "treat", "treating" or "treatment" (and grammatical variations thereof) refers to any type of treatment that provides benefit to a subject, and may mean that the severity of the subject's condition is alleviated, at least partially improved, or improved, and / or some alleviation, reduction, or decrease in at least one clinical symptom associated with the subject's condition is achieved, and / or a delay in the progression of the condition occurs. In some embodiments, the quality of sleep and / or the severity of symptoms associated with mental health in the subject may be reduced in the subject compared to the severity of the symptoms in the absence of the method of the present invention. In some embodiments, the particles of the present invention and / or the compositions of the present invention are administered to a subject to improve sleep quality (e.g., increase the length of sleep and / or the time of rapid eye movement (REM) sleep, reduce sleep interruptions, etc.), improve mental health, and / or treat these diseases and / or their symptoms.

[0060] In some embodiments, the particles of the present invention and / or the compositions of the present invention can 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 some benefit is provided to the subject. In some embodiments, a treatment effective amount can be achieved by administering the compositions of the present invention.

[0061] As used herein, the terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to avoidance, reduction and / or delay of onset of symptoms associated with a disease, disorder or condition, and / or reduction of the severity of onset of symptoms associated with a disease, disorder or condition, compared to that which would occur in the absence of the method of the present invention. Prevention may be complete, e.g., the complete absence of symptoms. The prevention may also be partial, such that the onset and / or severity of onset of symptoms in a subject is less than that which would occur in the absence of the method of the present invention. In some embodiments, the particles of the present invention and / or the compositions of the present invention are administered to a subject to prevent a disease, disorder or condition.

[0062] In some embodiments, the particles of the present invention and / or the compositions of the present invention can be administered in a prevention effective amount. As used herein, a "prophylactically effective" amount is an amount sufficient to prevent symptoms associated with a disease, disorder or condition (as defined herein) in a subject. Those skilled in the art will understand that the level of prevention need not be complete, as long as some benefit is provided to the subject. In some embodiments, a prevention effective amount can be achieved by administering the compositions of the present invention.

[0063] The present invention finds use in both veterinary and medical applications. Subjects suitable for treatment with the methods 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., simians and humans), non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas), and the like, and mammals in utero. Any mammal in need of being treated according to the present invention is suitable. Human subjects of both sexes and at any stage of development (i.e., neonates, infants, juveniles, adolescents, 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.Human subjects include both males and females of all ages, including fetal subjects, neonatal subjects, infant subjects, young subjects, adolescent subjects, adult subjects, and geriatric subjects, as well as pregnant subjects.In certain embodiments of the present invention, the subject is a human adolescent and / or adult.

[0064] The methods of the invention may be carried out on animal subjects, in particular mammalian subjects, such as mice, rats, dogs, cats, livestock and horses, for veterinary purposes and / or for drug screening and drug development purposes.

[0065] In some embodiments, the subject is "in need of" or "in need of" the methods of the invention, e.g., the subject has a finding typically associated with a disease, disorder or condition, is suspected of having a disease, disorder or condition, and / or the subject has a disease, disorder or condition.

[0066] The present invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the invention claimed, but rather to illustrate certain embodiments. Any variations of the exemplified methods that occur to those skilled in the art are intended to fall within the scope of the present invention.

[0067] Working Example

[0068] Example 1:

[0069] Bovine milk α-lactalbumin powder was kindly provided by Agropur, USA (batch number, JE 0001-21-414, purity 92.5%). Pure tryptophan was purchased from Sigma (St. Louis, MO, USA). All water used was Milli-Q water. All other chemicals were of analytical grade.

[0070] The procedure used in this example for the formation of α-lactalbumin-tryptophan (α-La-Trp) complex is shown in FIG. 1. Trp was mixed with α-La at various α-La to Trp weight ratios (20:1, 15:1, 10:1, and 5:1). The mixture of α-La and Trp was completely dissolved in Milli-Q water by adjusting the pH to 11 using 1N NaOH solution (final total solids concentration of α-La and Trp was 10 w / v%). The mixture was then passed through a high pressure homogenizer (HPH) (Nano DeBEE, Bee International, USA) at pressures ranging from 1000 psi to 40000 psi, and homogenized (e.g., recirculated through the homogenizer) for 5-60 minutes. The recovered α-La-Trp suspension was returned to neutral pH (pH 7) using 1N hydrochloric acid solution. The effects of α-La to Trp ratio, HPH pressure and circulation time on complex formation were investigated based on particle size, particle size distribution and polydispersity index (PDI).

[0071] A Box-Behnken design (BBD) consisting of 15 runs was applied to evaluate the formation conditions. Three extraction factors (α-La to Trp ratio, HPH pressure and circulation time) were examined. As shown in Table 1 below, three equally spaced levels were selected for α-La to Trp weight ratio (i.e., 15:1, 10:1, and 5:1), pressure (i.e., 20000 psi, 30000 psi, and 40000 psi), and circulation time (i.e., 20 min, 30 min, and 40 min). The average particle size and PDI values ​​at pH 11 and pH 7 were selected as the responses for this study. The evaluation was performed using JMP pro 16 (SAS Institute Inc. NC, USA).

[0072] [Table 1]

[0073] Selected samples were used for stability analysis. Storage stability tests were conducted at 4°C for 1 day, 7 days, 14 days, 21 days and 28 days. Particle size and particle size distribution were measured. Color and physical state were visually described by taking pictures. Thermal stability of the complexes was measured under different processing conditions: 63°C for 30 minutes and 90°C for 2 minutes based on particle size, particle size distribution and polydispersity index (PDI).

[0074] 1. Effect of the weight ratio of α-LA to Trp on the particle size of α-La-Trp complexes

[0075] The effect of weight ratios of α-LA to Trp (20:1, 15:1, 10:1 and 5:1) on the particle size of α-La-Trp complex was studied under HPH pressure of 30000 psi for 30 minutes. A clear difference was observed in the appearance of the mixtures of α-La and Trp with and without HPH treatment. Without HPH treatment, the mixtures appeared transparent, whereas after HPH treatment the mixtures had a cloudy / colloidal appearance indicating particle formation.

[0076] The α-La-Trp complex nanoparticles showed colloidal suspension without any visible aggregates. When the particle size of the colloidal suspension was measured by DLS (FIGS. 2-5), those samples produced by HPH at pH 11 show a narrow particle size distribution with an average particle size range of 270-293 nm for all ratios tested. When the pH was adjusted to 7, the particle size distributions of the 20:1, 15:1 and 10:1 ratios shifted slightly to a smaller range with an average particle size of about 200 nm. Without wishing to be bound by any particular theory, this is believed to be due to enhanced molecular associations (e.g., hydrophobic interactions, electrostatic interactions, hydrogen bonds) during the pH shift. However, the particle size distribution of the 5:1 ratio split into two peaks, which is believed to be due to the higher amount of tryptophan present during the formation of the particles compared to the other weight ratios, without wishing to be bound by any particular theory. This indicated that particles formed at a 5:1 ratio were stable at pH 11, but not at pH 7 using these particle formation conditions. Therefore, the minimum α-LA to Trp weight ratio for the 30 min, 30,000 psi HPH conditions should be 5:1 (α-La:Trp).

[0077] 2. Effect of HPH pressure and recirculation time on the particle size

[0078] The effect of pressure on the particle size of α-La-Trp complex was investigated at pH 11 for 30 min with α-LA to Trp ratio of 20:1. As shown in FIG. 6, there was an obvious difference in the suspensions formed after HPH under different pressure conditions. Differences were also observed in the appearance of the suspensions formed after HPH under different pressure conditions. The particle size and PDI value increased as the pressure increased from 1000 psi to 5000 psi, and then decreased when the pressure exceeded 5000 psi. The particle size of α-La-Trp complex formed under high pressure (above 10000 psi) tends to be smaller and more uniform (PDI<0.3) than that formed under low pressure. Without wishing to be bound by any particular theory, this is believed to be due to the high shear force, cavitation and turbulence under high pressure, which may promote uniform dynamics during particle formation. Therefore, HPH pressure of 20000-40000 psi was suitable for the subsequent BBD experiments.

[0079] The effect of recirculation time on the particle size of α-La-Trp complex was investigated at pH 11 under a pressure of 30000 psi with an α-LA to Trp ratio of 20:1. As shown in Figure 7, the particle size and PDI value increased as the pressure decreased from 5 to 30 psi, and then reached a plateau of particle size of 280 nm at 30 min. As a result, a recirculation time range of 20 to 40 min was considered for further BBD experiments.

[0080] 3. Preparation of α-La-Trp Complex Particles Using the Box-Behnken Design

[0081] Based on the above findings, a three-level, three-factor Box-Behnken design was implemented to determine which combination of HPH variables would result in the smallest and most stable α-La-Trp complex particles. The Box-Behnken design matrix, as well as the mean particle size and PDI values ​​under different experimental conditions, are shown in Table 2 below. All results were placed in a Box-Behnken design in JMP to obtain the predicted values ​​and final conclusions. All responses in these experiments were significantly fitted to quadratic models (p<0.0001) and there was no significant lack of fit (p>0.05). The fit of the model to the experimental data was verified using error analysis. As can be seen from Table 2, the predicted values ​​are close to the experimental values. The errors are 0.5-21.4 nm and 0.001-0.048 for mean particle size and PDI, respectively, i.e., the developed model fits the experimental data well.

[0082] [Table 2]

[0083] Interestingly, the α-La-Trp complex particles become stable when using different combinations of HPH pressure and recirculation time (e.g., 30000psi for 40 minutes, 20000psi for 30 minutes, and 40000psi for 30 minutes), as demonstrated by one peak particle size distribution at pH 7 at α-LA to Trp ratio of 5:1 (see Table 2, Runs 10, 12, and 14, and Figure 8). With the main aim of encapsulating more tryptophan into the particles and considering the operability in the actual processing procedure, a confirmation experiment was carried out under the following conditions: α-La to Trp ratio of 5:1, 30000psi pressure for 40 minutes. As shown in Figure 9, under these conditions, the predicted values ​​are close to the experimental values. This confirms that the model is suitable for predicting process behavior.

[0084] 4. Stability of α-La-Trp Complex Nanoparticles

[0085] The α-La-Trp complex nanoparticles were stored at 4° C. in a sealed container for up to 3 months. The effect of storage time on the size of the α-La-Trp complex nanoparticles and their stability in water at pH 11 and pH 7 is shown in FIG. 10, Panel A and FIG. 10, Panel B. These α-La-Trp complex nanoparticles were prepared by HPH at α-La-Trp weight ratio of 5:1, 30000 psi, 40 min. As shown in FIG. 10, Panel A, at pH 11, the particle size distribution of the α-La-Trp complex nanoparticles shifted slightly to a larger size range during 14 days of storage based on intensity and volume, while a peak of small size particles was observed by number. This may be due to particle swelling and / or aggregation and release of small particles during storage at pH 11. However, the particle size distribution of the α-La-Trp complex nanoparticles remained stable at pH 7 (FIG. 10, Panel B), and the particle size did not change significantly upon storage. Without wishing to be bound by any particular theory, this indicates that the pH shift from pH 11 to pH 7 stabilized the α-La-Trp complex nanoparticles produced by HPH.

[0086] The effect of temperature on the stability of α-La-Trp complex nanoparticles was determined under different heat treatment conditions: 63°C for 30 min and 90°C for 2 min. As shown in Figure 10, panel C, after incubation at 63°C for 30 min, the particle size of α-La-Trp complex nanoparticles at pH 11 shifted to a larger size range due to swelling and / or aggregation of the particles. Similar results were observed for samples incubated at 90°C for 2 min. However, for α-La-Trp complex nanoparticles at pH 7 (Figure 10, panel D), the particle size distribution remained unchanged upon heating. This further confirms the high stability of α-La-Trp complex nanoparticles after pH shift (from pH 11 to pH 7).

[0087] Example 2:

[0088] Bovine milk α-lactalbumin powder was kindly provided by Agropur, USA (batch number, JE 0001-21-414, purity 92.5%). Pure tryptophan was purchased from Sigma (St. Louis, MO, USA). All water used was Milli-Q water. All other chemicals were of analytical grade.

[0089] The procedure used for the formation of α-La-Trp complex is shown in FIG. 11. Trp was mixed at a 20:1 weight ratio of α-La to Trp. The mixture of α-La and Trp was completely dissolved in Milli-Q water (final total solids concentration of α-La and Trp was 10 w / v%) by adjusting the pH to 11 using 1N NaOH solution. The mixture was then sonicated for 5 min using a 20 kHz sonicator (VibraCell, Sonics and Materials Inc.) equipped with a probe transducer and a 1 / 2" (13 mm) flat tip. After sonication, the pH of the α-La-Trp suspension was returned to neutral (pH 7) using 1N HCl solution.

[0090] Clear solutions were observed upon dissolving α-La, Trp, or a mixture of α-La and Trp at pH 11. The solution of α-La alone did not change after sonication and pH shift treatment. However, clear differences were observed in the appearance of Trp and the mixture of α-La and Trp with and without sonication. Trp alone and the mixture of α-La and Trp showed colloidal dispersions without any visible aggregates. These colloidal dispersions remained stable in that the particles did not fall out of the composition even when the pH of the composition was adjusted to 7.

[0091] The particle size distribution of α-La before and after sonication was measured by dynamic light scattering (DLS). As shown in FIG. 12, α-La had an average size of 6.5±0.1 nm at H 11 and was mainly small particles. When the pH was adjusted from 11 to 7, it shifted to a larger size range with an average size of 15.5±3.6 nm. After sonication, both large and small particles were observed for α-La, indicating a very polydisperse particle size distribution. The average size of α-La increased to 169.8±55.8 nm. Without wishing to be bound by any particular theory, this indicates that ultrasonic irradiation may promote the association (e.g., hydrophobic interactions, electrostatic interactions, hydrogen bonds) between α-La molecules. When the pH was returned to 7, the average particle size decreased to 41.9±42.4 nm due to protein folding during the pH shift.

[0092] The particle size distribution of Trp before and after sonication was measured by DLS. As shown in Figure 13, Trp at pH 11 was very polydisperse with a broad particle size distribution. When the pH was adjusted from pH 11 to pH 7, the particle size shifted to a larger range (Figure 13, panel A1). During sonication, Trp molecules associate to form Trp-Trp complex particles. The particle size distribution became more uniform with an average size of 215.1 ± 4.4 nm (Figure 13, panels B1-B3). When the pH was adjusted from pH 11 to pH 7, the average size of Trp particles increased to 270 ± 7.0 nm due to aggregation of the particles.

[0093] The particle size distribution of α-La-Trp before and after sonication was measured by DLS. As shown in FIG. 14, the mixture of α-La and Trp at pH 11 before sonication showed a similar particle size distribution as α-La with an average size of 6.8±0.1 nm. When adjusted from pH 11 to pH 7, the particle size shifted to a slightly larger range (average size: 7.9±0.7 nm) (FIG. 14, panel A1). During sonication, α-La and Trp molecules associated to form α-La and Trp complex particles. The particle size distribution became more uniform with an average size of 363.9±11.4 nm (FIG. 14, panels B1-B3). However, when the pH was adjusted from pH 11 to pH 7, the average size of α-La-Trp particles decreased to 211.7±5.7 nm, which is believed to be due to the folding of protein molecules within the particles, without wishing to be bound by any particular theory. Without wishing to be bound by any particular theory, the presence of Trp may promote particle formation of the α-La molecules, while the presence of the α-La molecules may promote molecular association during the pH shift due to folding of the protein molecules.

[0094] Example 3:

[0095] Samples of lyophilized α-La-Trp particles (5:1 by weight (La:Trp), 100 mg / mL total solids concentration, formed at 30,000 psi for 40 min) were redispersed in buffer (phosphate-buffered saline (PBS), pH 7) and diluted to various concentrations (0.01, 0.05, 0.1, 0.2, 0.5, 1.0 mg / mL) for ABTS+- antioxidant activity assay. ABTS+- was prepared by mixing potassium persulfate (2.6 mmol / L) with ABTS solution (7.4 mmol / L) and allowed to stand at room temperature for 16 h in the dark. The radical solution was adjusted to an absorbance of 0.70 ± 0.02 at 734 nm in phosphate-buffered saline (PBS, pH 7.4). Samples (20 μL) of various concentrations were mixed with 200 μL of ABTS+- solution at room temperature for 20 min. The absorbance was measured at 734 nm. The ABTS scavenging activity was calculated using the following formula:

[0096]

number

[0097] The antioxidant activity of α-lactalbumin was increased after HPH treatment (panel A of FIG. 16). Tryptophan showed very strong antioxidant activity, with about 100% ABTS radical scavenging activity at concentrations above 0.1 mg / mL (panel B of FIG. 16). At a very low concentration of 0.01 mg / mL, the antioxidant activity of tryptophan was increased by HPH treatment. The ABTS radical scavenging activities of the composite nanoparticles did not change significantly compared to the α-lactalbumin-tryptophan mixture without HPH treatment (panel C of FIG. 16). However, they were substantially greater than α-La alone (panels A and C of FIG. 16). At a concentration of 0.5 mg / mL, the ABTS radical scavenging activity of the composite was approximately 100%, while that of α-La was 20%. Overall, tryptophan enrichment significantly increased the antioxidant activity of α-lactalbumin. HPH treatment did not significantly affect the antioxidant activity of tryptophan present in α-La-Trp particles.

[0098] Example 4:

[0099] Fluorescence experiments were designed and performed to measure the intrinsic fluorescence intensity of a mixture of α-La and Trp before and after HPH treatment. The particle formation conditions used were a weight ratio (La:Trp) of 5:1, and a total solid concentration of 100 mg / mL, at 30,000 psi and pH 11 for 40 min. The control sample was a mixture of α-La and Trp before HPH, with a weight ratio (La:Trp) of 5:1, and a total solid concentration of 100 mg / mL, at pH 11. Fluorescence spectra were performed by a spectrofluorophotometer (SHIMADZU, RF-6000, Japan) using a quartz cell with an optical path length of 1.0 cm in a thermostat bath. The excitation wavelength was set at 295 nm, and the emission spectra were recorded from 310 to 400 nm. The data were collected with a wavelength resolution of 0.5 nm. As shown in FIG. 17, after HPH, the protein and tryptophan molecules were tightly associated, and the intensity of the intrinsic fluorescence increased.

[0100] Example 5:

[0101] The storage stability of α-La-Trp particles (formed with a weight ratio (La:Trp) of 5:1 and a total solids concentration of 100 mg / mL, at 30,000 psi, for 40 minutes) was investigated for 3 months at 4° C. As shown in FIG. 10, panels A and B, the size of the particles remained stable after storage at 4° C. for 3 months. The size change of the particles was less than 15% of their original size (increased from 223.8 nm to 249.9 nm).

[0102] Example 6:

[0103] 1. Introduction

[0104] A combined high pressure homogenization (HPH) and pH shift treatment was applied to a mixture of α-lactalbumin (α-LA) and tryptophan (Trp) to produce nanoparticles (α-LA-Trp-NPs). Processing parameters, such as α-LA / Trp ratio, HPH pressure and recirculation time, were investigated to determine their effects on the physicochemical properties of α-LA-Trp-NPs. The desired α-LA / Trp ratio (5:1), HPH pressure (206.8 MPa), and recirculation time (40 min) were found to produce small α-LA-Trp-NPs (243.0 ± 7.2 nm) with a narrow particle size distribution. The formation of α-LA-Trp-NPs was found to follow a controlled HPH-induced aggregation mechanism in which soluble, unfolded proteins and amino acids gradually associate and grow into larger particle sizes, aggregates, and become insoluble. Comparison of the size and morphology of α-LA-Np with that of α-LA-Trp-NP indicates that the presence of Trp significantly affects the size and morphology of the NPs in the dry form. The freeze-dried α-LA-Trp-NPs could be easily redispersed in Milli-Q water and phosphate-buffered saline (PBS) buffer to form uniform nanoparticle (NP) dispersions. By using a combination of HPH and pH shift, the thermal stability, freeze-thaw stability, and freeze-thaw thermal stability of α-LA-Trp-NPs were improved. Compared to Trp alone NPs, the complex NPs showed better freeze-thaw stability and retained particle properties even after heat treatment at 63°C for 30 min after freeze-thaw cycles. α-LA-Trp-NPs were also observed to have remarkable stability against pH changes and heat treatment at 63°C for 30 min and 90°C for 2 min.

[0105] 2. Materials and Methods

[0106] 2.1.Materials

[0107] Bovine milk α-lactalbumin (α-LA) powder was provided by Agropur, USA (batch number, JE 0001-21-414, purity 92.5%). Pure tryptophan was purchased from Sigma (reagent grade ≥ 98%, St. Louis, MO, USA). Hydrochloric acid (HCl, ACS grade) and sodium hydroxide (NaOH, 98%) were purchased from Fisher Scientific (Hampton, NH, USA). All water used was Milli-Q water. Milli-Q water (18.2 MΩ / cm) was produced using a Millipore water purification system (Millipore Sigma, Burlington, MA, USA).

[0108] 2.2. Preparation of α-LA-Trp-NPs

[0109] α-LA-Trp-NP was prepared using high pressure homogenization-pH shift technique. Briefly, a mixture of α-LA and Trp was completely dissolved in Milli-Q water by adjusting the pH to 11 using 1N NaOH solution. The mixture was passed through a high pressure homogenizer (Nano DeBee, Bee International, inc. USA) and recirculated. After recirculation, the pH of the collected α-LA-Trp-NPs dispersion was adjusted to neutral (pH 7) using 1N hydrochloric acid solution. For comparison, control samples, i.e., α-LA-NP without Trp and Trp-NP without α-LA, were prepared using the same procedure.

[0110] 2.3. Formulation and process parameters of α-LA-Trp-NPs

[0111] The formulation (α-LA to Trp ratio) and process parameters (HPH pressure and recirculation time) were investigated based on the physicochemical properties of α-LA-Trp-NPs, including particle size, particle size distribution, polydispersity index (PDI) and tryptophan fluorescence intensity (TFI). A solids concentration of 10 w / v% was selected after a series of preliminary tests (data not shown), where the maximum level of solids concentration with a uniform particle size distribution was observed by DLS and the PDI value was less than 0.3. Different α-LA to Trp w / w% ratios (20:1, 15:1, 10:1, 5:1, 4:1 and 3:1) were tested while keeping the HPH pressure (206.8 MPa) and recirculation time (30 min) constant. While keeping the mass ratio and recirculation time (30 min) constant, α-LA-Trp-NPs were prepared at various HPH pressures (6.9 MPa, 34.5 MPa, 68.9 MPa, 137.9 MPa, 206.8 MPa, and 275.8 MPa) that were systematically varied. Finally, by keeping the HPH pressure (206.8 MPa) and mass ratio constant, the effect of recirculation time (5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min) was investigated.

[0112] 2.4. Measurement of particle size, particle size distribution and polydispersity index

[0113] The particle size, particle size distribution and PDI values ​​of NPs were determined by dynamic light scattering (DLS) using a Zetasizer (Nano S, Malvern Instruments, Worcestershire, UK). The measurements were performed at a scattering angle of 173° and a temperature of 25°C, with 15 runs performed for each measurement. The particle size distribution can also be reflected by the PDI value, which ranges from 0 to 1. A smaller PDI value indicates a narrower size distribution. In most cases, monodispersed particle systems had a PDI less than 0.3. All measurements were performed in triplicate.

[0114] 2.5. Turbidity measurements

[0115] The turbidity of the α-LA-Trp mixture was measured using a UV-Vis light spectrophotometer (UV-2600, SHIMADZU Co., Japan). The sample solution was analyzed at room temperature, and the transmittance was measured at 600 nm. Milli-Q water was used as a blank reference (transmittance 100%). The turbidity (T) was calculated as follows (Equation 1 below):

[0116]

number

[0117] 2.6. Tryptophan fluorescence spectrum analysis

[0118] Tryptophan fluorescence spectra of the mixture of α-La and α-LA-Trp were performed using a spectrofluorometer (SHIMADZU, RF-6000, Japan) according to a previous method with some modifications (Zhu et al., (2021) Physicochemical and functional properties of a novel xanthan gum-lysozyme nanoparticle material prepared by high pressure homogenization. LWT, 143, 111136.). Briefly, the fluorescence excitation wavelength was set at 295 nm, and the emission was recorded in the range of 310-500 nm. Data were collected with a step resolution of 0.5 nm. During the formation of nanoparticles, the changes in the fluorescence intensity of the protein and its combination with tryptophan were monitored, including the fluorescence intensity of the α-LA-Trp mixture under various HPH conditions, the fluorescence intensity of the α-LA, Trp and α-LATrp mixture at pH 11 before and after HPH, and the fluorescence intensity after returning the pH to 7.

[0119] 2.7. Scanning Electron Microscopy (SEM)

[0120] The morphology of α-LA, Trp, and α-LA-Trp and the mixture with and without HPH was observed using SEM (Zeiss Gemini 500, Jena, Germany). The freeze-dried samples were directly mounted on the SEM plate with a carbon conductive tab before coating, while the liquid samples (without freeze-drying) were diluted with PBS (10 mM, pH 7) to a solid concentration of 1 mg / mL and dropped onto the SEM plate with a carbon conductive tab and dried in vacuum at room temperature. All samples were coated with carbon using a sputter coater (Denton Desk V, New Jersey, USA) and then scanned by SEM at 1 keV and imaged by a high-efficiency secondary electron detector with a 20.0 μm aperture.

[0121] 2.8. Stability analysis

[0122] 2.8.1 pH stability

[0123] The effects of HPH treatment and pH shift on the pH stability of α-LA, Trp, and mixtures of α-LA and Trp were investigated and compared. The pH of all dispersions at pH 11 with and without HPH was adjusted to pH 7 and pH 3 by adding 1.0 M HCl. Appearance, turbidity, particle size, particle size distribution, and PDI were measured and recorded.

[0124] 2.8.2 Temperature stability

[0125] The effects of HPH treatment and pH shift on the thermal stability of α-LA, Trp, and α-LA-Trp mixtures were investigated and compared. Samples at pH 11 with and without HPH, and after pH shift at pH 7 and pH 3, were incubated at 63°C for 30 minutes (pasteurization) and at 90°C for 2 minutes (flash pasteurization), and then cooled to 25°C. Appearance, turbidity, particle size, particle size distribution, and PDI were measured and recorded.

[0126] 2.8.3 Freeze-thaw stability and freeze-thaw thermal stability

[0127] The effects of HPH treatment and pH shift on the freeze-thaw stability and freeze-thaw thermal stability of α-LA, Trp, and a mixture of α-La and Trp were investigated and compared. Samples at pH 11 with and without HPH, and samples after pH shift at pH 7 were stored at -20°C overnight, and then transferred into a water bath set at 25°C for 2 hours until completely thawed. After the freeze-thaw cycle, the appearance, turbidity, particle size, and particle size distribution were recorded. The freeze-thawed samples were further heated at 63°C for 30 minutes to investigate the freeze-thaw thermal stability.

[0128] 2.9. Redispersion of Lyophilized NPs

[0129] Lyophilized α-LA-NP, Trp-NP, and α-LA-Trp-NP (10 mg) were redispersed in 10 mL of Milli-Q water or PBS buffer (10 mM, pH 7) and stirred for 2 h. The distribution and PDI of NPs in the dispersions were measured by DLS.

[0130] 2.10.Statistical analysis

[0131] All experiments were performed in triplicate. Results are presented as the mean ± SD (standard deviation). Significance between means was established using one-way ANOVA (OriginPro 9.0.0, OriginLab Northampton, MA, USA).

[0132] JMP Pro 16.0.0 software (SAS Institute Inc., Cary, NC, USA) was used to develop the experimental design and to analyze the data. To perform sensitivity analysis of the process parameters, we adopted a Design of Experiment (DOE) approach. For this study, a Full Factorial Design (3K) strategy was selected, and two variables (K=2) were analyzed: HPH pressure (X1, MPa) and recirculation time (X2, min). The responses investigated were particle size (Y1) and TFI (Y2) at pH 11, and particle size (Y3) and TFI (Y4) at pH 7.

[0133] 3. Results and Discussion

[0134] HPH and pH shift were used to form nanoparticles containing α-LA and Trp. Without wishing to be bound by any particular theory, the results indicate that at high pH, ​​the protein is unfolded, providing openings and areas for incorporating Trp. HPH mixed the protein with Trp molecules to form nanoparticles. The pH of the nanoparticle solution was then subsequently lowered, and it is believed that the protein was forced to fold around Trp, creating smaller NPs with high Trp fluorescence.

[0135] 3.1. Effect of HPH on the formation of α-LA-Trp-NPs

[0136] We used a screening test to explore the effect of HPH on the hydrodynamic particle size of α-LA-Trp-NP, HPH pressure and recirculation time, and varying α-LA / Trp ratio. The average particle size (open square) and PDI value (polydispersity index, solid square) of α-LA-Trp-NP prepared under various conditions were measured using DLS (Figure 18). The effect of pressure on the particle size of α-LA-Trp-NP was investigated at pH 11 for 30 min, with a ratio of α-LA to Trp of 20:1 (w / w) (Panel A of Figure 18). The particle size and PDI value of α-LA-Trp-NP increased as the pressure increased from 6.9 MPa to 34.5 MPa, and then decreased when the pressure was above 34.5 MPa (Panel A of Figure 18). In general, the particle size of α-LA-Trp-NPs formed under high pressure (above 68.9 MPa) was smaller and more uniform (PDI<0.3) than that formed under low pressure. This is probably due to the increased shear force, cavitation and turbulence under high pressure, which promotes protein aggregation and forms colloidal particles derived from protein aggregation. The average particle size of the formed α-LA-Trp-NPs slightly decreased from 436 nm to 271 nm as the HPH pressure was increased from 68.9 MPa to 275.8 MPa. The PDI decreased from 0.29 at 68.9 MPa to 0.15 at 206.8 MPa, but then increased slightly to 0.19 at 275.8 MPa. Without wishing to be bound by any particular theory, this is believed to be due to the particles becoming more polydisperse with increasing HPH pressure, which allows greater intermolecular interactions to be available at low pressure and break down at high pressure.

[0137] The effect of α-LA / Trp ratio (20:1 w / w, 15:1 w / w, 10:1 w / w, 5:1 w / w, 4:1 w / w and 3:1 w / w) on the particle size of α-LA-Trp-NPs was carried out under HPH pressure of 206.8 Mpa for a recirculation time of 30 min. In the range of α-LA to Trp ratios of 20:1 and 5:1, we did not observe significant changes in particle size and PDI values, but these NPs showed a narrow particle size distribution with an average particle size of 290 nm (Panel B of FIG. 18). When the Trp content was further increased to α-LA / Trp ratios of 4:1 and 3:1, the particle size significantly decreased and was not uniform (PDI>0.3). Therefore, an α-LA / Trp ratio of 5:1 was selected for further testing. The particle size and PDI value of La-Trp-NP decreased with increasing recirculation time from 5 to 30 min, and then reached a plateau of average particle size of about 290 nm after 30 min (panel C of FIG. 18). Comparing the particle size of the α-LA-Trp mixture (α-LA / Trp at 5:1 w / w) not subjected to HPH at pH 11 (FIG. 19), the particle size of La-Trp-NP increased from 7 nm and PDI value of 0.37 (FIG. 19) to 332 nm and PDI value of 0.26 after 5 min of HPH. This result indicates that HPH at a pressure of 206.8 MPa induces aggregation of α-LA and Trp molecules within 5 min to form large particles. This is consistent with the visual difference observed in turbidity from the appearance of colloidal dispersions formed after HPH under various recirculation times.

[0138] 3.2. Detection of aggregates via tryptophan fluorescence

[0139] The aggregation mechanism of the α-LA-Trp mixture was examined by monitoring both the changes in turbidity and the changes in Trp fluorescence during HPH. Trp fluorescence is associated with aggregation. When the aggregation of Trp molecules increases, the Trp chromophore increases or turns on, and when aggregation is low, Trp turns off or shows low fluorescence (Liu, Wolstenholme, et al., 2018). Initially, the α-LA-Trp mixture (5:1 α-LA:Trp, w / w) at pH 11 without HPH showed weak fluorescence emission due to the pH-dependent unfolding of the protein (Panel A of FIG. 20). However, after 50 min of HPH treatment at pH 11, the fluorescence intensity increased about 35-fold. The fluorescence intensity was positively correlated with the turbidity of the α-LA-Trp aggregates (Panel B of FIG. 20), indicating that HPH can induce the aggregation of Trp and α-LA. Therefore, we were able to use fluorescence to monitor the aggregation of α-LA-Trp and to select the desired NP formation conditions (see Section 3). The mechanism of NP formation by HPH and the pH shift monitored by Trp fluorescence was further investigated and is discussed in Section 3.4 below.

[0140] 3.3. Investigation of HPH status

[0141] The HPH conditions aimed at producing uniform α-LA-Trp-NPs with small particle size and α-LA / Trp ratio of 5:1 were investigated at pH 11 and pH 7. Particle size, PDI and Trp fluorescence intensity (TFI) at pH 11 and pH 7 were measured for nine treatment combinations of HPH pressure (137.9, 206.8 and 275.8 MPa) and recirculation time (20, 30 and 40 min) (Table 3). These nine treatments represent a full factorial design (FFD) determined using the Design of Experiments function in JMP software. Based on the results of our ANOVA analysis, the correlation coefficients (R2) of all models were in the range of 0.87-0.97, and the p-values ​​were 0.0003-0.0108 (p<0.05), which correlated well with the actual experimental data. The results showed that particle size and TFI were significantly affected by HPH pressure (p<0.05) at both pH 11 and pH shift from pH 11 to pH 7 (pH 11→7), but HPH and recirculation time did not significantly affect each other (p>0.05) (Table 3 and Panel A of FIG. 21). Our goal was to minimize nanoparticle size with uniform particle size distribution (minimize PDI) and increase TFI at both pH 11 and pH 7. Based on these criteria, the predicted desirable conditions were as follows: HPH pressure 200.9 MPa (29,138 PSI) and HPH time 40 min (Panel B of FIG. 21). These conditions had the highest desirability value of 0.81 (Panel B of FIG. 21). Due to the constraints of the equipment setup, we used a HPH pressure of 206.8 MPa (30,000 PSI) and a recirculation time of 40 minutes.

[0142] [Table 3]

[0143] The average particle size of α-LA-Trp-NP obtained under the desired conditions was 291 nm at pH 11 and 239 nm at pH 7. This result indicates that the particles formed at pH 11 remain uniform when the pH is shifted to neutral conditions. The slight decrease in particle size with pH shift is attributed to molecular associations, such as hydrophobic interactions, electrostatic interactions, and hydrogen bonds, which are enhanced during the pH shift, resulting in a denser structure in which the protein molecules are more highly folded within the nanoparticles at pH 7. In addition, the change in the pH of the solution changed the environment of α-LA-Trp-NP, which affected the particle size of α-LA-Trp-NP. This is consistent with the TFI data, which decreased after the TFI was shifted to pH 7. Due to the increase in protein molecular interactions, the self-aggregation of Trp decreased at pH 7, and subsequently, the TFI decreased. Overall, the particles formed by HPH were stable with a uniform particle size distribution at both pH 11 and pH 7. It is noted that the pressure and time of HPH have a significant effect on the particle size and PDI value of α-LA-Trp-NPs (p<0.05).

[0144] Finally, in all the following experiments in which α-LA-NP and Trp-NP were prepared for comparison, HPH conditions of 206.8 MPa and 40 min were used. The average size (Z-average) and PDI values ​​of α-LA-NP, Trp-NP and α-LA-Trp-NP in the dispersions obtained under the desired conditions are shown in Table 4 below. Under these conditions, the particle size of α-LA-Trp-NP was 283.0±6.9 nm and 243.0±7.2 nm, with a narrow particle size distribution (PDI<0.2) at pH 11 and pH 7, respectively, which was consistent with the predicted results (Panel B of FIG. 21). It is also interesting that under the same conditions, NPs were formed from α-LA and Trp, respectively. The particle size of α-LA-NP was almost the same as that of α-LA-Trp-NP, while Trp-NP showed a small particle size of approximately 210 nm at both pH 11 and pH 7. This is mainly due to the self-assembly behavior of both α-LA and Trp.

[0145] [Table 4]

[0146] α-LA, Trp and a mixture of α-LA and Trp were suspended in water and dissolved by adjusting the pH to 11. At high pH, ​​the intermolecular attractions of α-LA, such as disulfide bonds, hydrophobic interactions and electrostatic interactions, are reduced and its solubility is increased. The reduced intermolecular attractions in α-LA increase the possibility for the formation of new intramolecular interactions, bonds and structures between proteins and Trp. Once the solution was dissolved, we evaluated the effect of pH shift with and without HPH.

[0147] Shifting the pH from pH 11 to pH 7 without HPH did not change the appearance of α-LA, Trp, and the mixed solution of α-LA and Trp, and they all remained clear solutions. The particle size of α-LA and the α-LA-Trp mixture without HPH (NOHPH) increased slightly during the pH shift, which is associated with the formation of soluble aggregates with a size range of 2-10 nm (Figure 19). The presence of Trp in α-LA slightly increased the particle size of α-LA at both pH 11 and 7, thereby indicating the complex formation between α-LA and Trp.

[0148] When HPH was applied to a pH 11 solution of α-LA, Trp and α-LA-Trp, the samples were colloidal insoluble dispersions. The average particle size increased from about 2 nm for NOHDH (FIG. 19) to about 280 nm for HDH (Table 4). This suggests that α-LA, Trp and α-LA-Trp complexes aggregated under high pressure, high shear and cavitation to form larger sized particles. The formed NPs were maintained after shifting the pH from 11 to 7, but the particle size was smaller, which may be attributed to protein shrinkage at lower pH. Without wishing to be bound by any particular theory, the inventors concluded that HPH plays an important role in the formation of α-LA-Trp-NPs, and the NPs are robust under the pH shift but slightly reduced in size.

[0149] 3.4. Potential mechanisms of NP formation by HPH and pH shift

[0150] Panel A of FIG. 22 shows the fluorescence spectroscopy of α-LA, Trp and α-LA-Trp mixtures with and without HPH treatment at pH 11 and pH 7. The excitation wavelength was 295 nm. The emission peak of the α-LA-Trp (5:1 w / w) mixture was observed in the wavelength range of 320-500 nm (Panel A of FIG. 22). At pH 11 without HPH, the α-LA, Trp and α-LA-Trp mixtures showed weak fluorescence emission at pH 11. However, when HPH was applied, the emission intensity of the same samples prepared at pH 11 increased by about 50 times for α-LA, about 45 times for Trp, and about 35 times for α-LA-Trp, respectively. Therefore, we speculated that HPH increased the aggregation, which may be the trigger of the turn on mechanism of the Trp chromophore for all samples.

[0151] The results remained consistent when the pH was adjusted from pH 11 to pH 7: the NOHPH sample was weakly fluorescent, and the HPH sample showed significantly higher fluorescence intensity at pH 7 (Figure 22, Panel A). This is consistent with the visual appearance of the samples, where the NOHPH sample was a clear molecular solution, while after HPH, a cloudy colloidal dispersion showed aggregation. This is also consistent with the particle size results, where submicron-sized particles were observed in the HPH-treated samples. According to previous reports (Stanciuc et al., 2012; Toptygin et al., (2002). Effect of the Solvent Refractive Index on the Excited-State Lifetime of a Single Tryptophan Residue in a Protein. The Journal of Physical Chemistry B, 106(14), 3724-3734; Vivian & Callis, (2001). Mechanisms of Tryptophan Fluorescence Shifts in Proteins. Biophysical Journal, 80(5), 2093-2109), exposing internal tryptophan residues to the protein surface results in a decrease in fluorescence intensity due to solvent quenching. However, buried tryptophan residues are less affected by the presence of solvent and therefore show strong fluorescence.Second, previous studies on the fluorescence of the Trp chromophore have shown that free Trp molecules are largely non-fluorescent due to rapid nonradiative decay via twisted-intramolecular charge transfer (TICT), which is blocked when the molecule is locked in place by proteins, protein aggregates, and self-assemblies, resulting in high fluorescence (Carayon et al., (2016). Conjugates of Benzoxazole and GFP Chromophore with Aggregation-Induced Enhanced Emission: Influence of the Chain Length on the Formation of Particles and on the Dye Uptake by Living Cells. Small, 12(47), 6602-6612; Liu, et al. (2018). Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence. Journal of the American Chemical Society, 140(24), 7381-7384). Therefore, without wishing to be bound by any particular theory, we propose that α-LA-Trp-NPs formed by HPH may have Trp molecules deeply buried within the protein structure as aggregates, which reduces the effect of solvent quenching and inhibits TICT, thus resulting in higher fluorescence (Panel B of FIG. 22).

[0152] The slight decrease observed in the fluorescence maximum emission intensity and wavelength of the α-LA-Trp mixture by shifting the pH from 11 to 7 may be due to the difference in molecular / aggregate structure. The particle size decreased after shifting from about 283 nm at pH 11 to about 243 nm at pH 7 (Table 4). This means that more of the Trp in the mixture may be exposed to the solvent, and thus the fluorescence was affected by solvent-induced quenching. Studies of pH-dependent disassembly and reassembly of proteins have proposed that proteins unfold and disassemble at high pH, ​​and then they refold and reassemble at neutral pH when the pH is shifted to low pH (Tang, 2020; Tang et al.). Therefore, without wishing to be bound by any particular theory, we believe that upon pH shift from 11 to 7, α-LA-Trp-NPs contract and a decrease in fluorescence intensity is observed due to solvent quenching of Trp close to the surface of the protein (Figure 22 Panel B).

[0153] SEM micrographs of NOHPH and HPH samples of α-LA, Trp and α-LA-Trp dispersions (1 mg / mL) show the observed differences in the morphology of the samples (Panel C of FIG. 22). The NOHPH samples showed small size aggregates, while large size aggregates were observed in the case of the HPH samples. As shown in FIG. 3C1, the NOHPH sample of α-LA showed spherical particles with a size of about 40 nm (Panel C of FIG. 22), while the particles of the HPH sample were larger with a size of about 80 nm (Panel C of FIG. 22). The particle size in the dry state was much smaller than the hydrodynamic particle size measured by DLS shown in Table 2 due to the dehydration of the particles and the difference in the measurement technique used. Similar observations were seen in the case of NOHPH and HPH samples of Trp and NOHPH and HPH samples of α-LA-Trp mixture; the relative size of the HPH samples to the NOHPH samples was consistently larger. However, the mixture of α-LA-Trp showed a different morphology from either α-LA or Trp. The structure showed higher crystallinity and had a less spherical shape. The HPH sample of α-LA-Trp showed homogeneous polygonal particles with a uniform size of about 180 nm. These particles were significantly different in both morphology and size compared to α-LA and Trp. This is further evidence of the formation of a complex between α-LA and Trp, and is consistent with the results of Trp fluorescence.

[0154] 3.5. Colloidal stability of α-LA-Trp-NPs at various pH levels

[0155] The dispersion stability of NOHHPH and HPH samples after pH shift from 11 to neutral (pH 7) and to highly acidic conditions (pH 3) was investigated by visual observation and particle size measurement (Figure 23 and Table 4). In the case of NOHPH samples, the pH shift did not change the appearance of the mixtures of α-LA, Trp and α-LA-Trp, which were all clear solutions at pH 11, 7 and 3.

[0156] Visually, the colloidal dispersion remained unchanged during the shift in pH from 11 to 7 or from 11 to 3 in the case of HPH samples. The appearance and turbidity of the colloidal dispersion did not change significantly (Table 4). This is consistent with the particle size results (Figure 23), where all NPs showed larger size particles compared to their NOHPH samples. The pH stability of all NPs at pH 11, 7, and 3 indicated that the intermolecular interactions within the NPs induced by HPH were stable at those pH values. The PDI value of α-LA-Trp-NP at pH 3 increased significantly, and a peak of small size particles between 20 and 100 nm was observed. The small size of the particles means that the particles were partially decomposed during the shift in pH from 11 to 3. Comparing the NPs formed from α-LA and Trp alone, the NPs formed from their mixture (α-LA-Trp-NP) had low stability at pH 3. These results indicate that the interactions induced by the aggregation of α-LA and the aggregation of Trp alone exhibited better stability at pH 3 than the interactions of their mixtures, thereby resulting in little release of small particles from larger ones. Overall, the NPs formed by HPH were stable over a wide pH range.

[0157] 3.6. Effect of HPH and pH shift on the thermal stability of α-LA-Trp-NPs

[0158] To evaluate the thermal stability of HPH self-assembled α-LA-NP, Trp-NP and α-LA-Trp-NP mixtures, all solutions were incubated at 63°C for 30 min and 90°C for 2 min to approximate food processing conditions. Their appearance, turbidity, particle size and particle size distribution were determined. Heat treatment did not affect the appearance of colloidal dispersions of α-LA-Trp-NPs and their counterparts α-LA-NP and Trp-NP at both pH 11 and pH 7. The physical appearance of the solutions is consistent with the absence of change in the measured turbidity (Table 5 below). We speculated that the particles formed by HPH might remain intact during heat treatment, which was supported by the results of particle size measurements (Figure 24 and Table 5). The particle size distribution of all samples after treatment was 70 nm to 3000 nm, which was larger than the particle size of the molecular forms of α-LA and Trp (<10 nm, Figure 19). This suggested that the intermolecular or intramolecular associations induced by HPH could be maintained upon heating. The average particle size (Z-average) of α-LA-Trp-NP at pH 11 increased from about 290 nm to about 540 nm (63°C, 30 min) and about 520 nm (90°C, 2 min). However, the average particle size of α-LA-Trp-NP (at pH 7) due to pH shift did not change after incubation at 63°C for 30 min (240 nm to 260 nm) and at 90°C for 2 min (240 nm to 250 nm) (Table 5). The PDI value of α-LA-Trp-NP did not change significantly (PDI<0.2) upon heating at pH 11, indicating that the particles in the dispersion were uniform and had a narrow particle size distribution (Figure 24). The increase in particle size after heating is believed to be due to the swelling of the particles. The increase in particle size of α-LA-Trp-NP after heat treatment at pH 11 was greater than that of α-LA-NP. For example, the particle size of α-LA-Trp-NP increased from about 283 nm to about 540 nm in response to heating at 63°C for 30 min, whereas the particle size of α-LANP increased from about 289 nm to about 450 nm.This may be because Trp, which is complexed with proteins, exists in NPs, resulting in fewer protein-protein interactions during HPH at pH 11, promoting the swelling of α-LA-Trp-NPs upon heating. However, the particle size of Trp-NPs did not change significantly upon heating at both pH 11 and 7. This is because the intermolecular forces between Trp molecules, such as π-π stacking, are stronger, which may require more energy to swell. An interesting fact is that the particle size and particle size distribution of α-LA-NPs and α-LA-Trp-NPs did not change significantly upon heating when the pH shifted from 11 to 7 (Table 5 and Figure 24). This property of α-LA-NPs and α-LA-Trp-NPs at pH 7 may be due to certain inter- or intramolecular associations, such as hydrophobic interactions, electrostatic interactions, or hydrogen bonds, formed during the pH shift that provide protection from swelling and thermal degradation.

[0159] Overall, all NPs showed excellent thermal stability at both pH 11 and pH 7 based on slight changes in turbidity, particle size, and PDI. This is because the protein unfolded significantly at pH 11 and NPs formed from the aggregation of α-LA and Trp molecules through surface-active groups, but no additional aggregation occurred during heating due to the unavailability of surface-active groups. The thermal stability of these Trp-containing NPs is a useful property for many commercial applications that include Trp-enriched ingredients in formulations exposed to high temperature processing, especially in beverages and other liquid-type food matrices and nutritional product applications.

[0160] [Table 5]

[0161] 3.7. Effect of HPH and pH shift on freeze-thaw stability and freeze-thaw thermal stability of α-LA-Trp-NPs at various pH

[0162] The physical stability of our NPs-based delivery systems in freeze-thaw cycles and freeze-thaw thermal cycles is important for products intended for use as food ingredients. We subjected our α-LA-Trp-NP, α-LA-NP and Trp-NP to freeze-thaw stability and freeze-thaw thermal stability conditions designed to mimic or exceed food industry conditions. In the case of freeze-thaw cycles, the samples were exposed to extreme changes in storage temperature from -20°C to 25°C, and then, in the case of freeze-thaw thermal cycles, the samples underwent additional heat treatment at 63°C for 30 minutes.

[0163] The particle size of α-LA-NPs increased slightly after freeze-thaw treatment at pH 11 and then grew again after heat treatment (Figure 25 and Table 5). Increases in protein particle size after freeze-thaw have been reported and are generally attributed to protein aggregation caused by ice crystals growing during freezing (Chen et al., (2022). High internal phase Pickering emulsions stabilized by tannic acid-ovalbumin complexes: Interfacial property and stability. Food Hydrocolloids, 125, 107332). The increase in particle size at pH 11 after heating was consistent with the results for samples without freeze-thaw, and no significant differences in particle size and PDI values ​​were observed (Figure 24 and Table 5). This is believed to be because the protein aggregates that occurred during freezing were subsequently broken down during heating, and the NPs grew in size due to swelling. However, no significant change in particle size of α-LA-NP was observed after freeze-thaw treatment cycles or freeze-thaw thermal cycles after shifting from pH 11 to pH 7. This result indicated that freeze-thaw cycles did not significantly affect the colloidal dispersion stability of α-LA-NP, and therefore, the particle stability of α-LA-NP was improved by the pH shift.

[0164] Conversely, Trp-NP exhibited significantly different behavior, exhibiting freeze-thaw stability at pH 11 but poor freeze-thaw stability at pH 7 (Figure 25). After freeze-thawing in Trp-NP dispersions at pH 7, particle settling was visually observed, which coincided with an increase in turbidity (Table 5).

[0165] The particle size of α-LA-Trp-NP at pH 11 was not significantly affected by freeze-thaw cycles (Figure 25 and Table 5). However, after heat treatment at pH 11, the particle size increased slightly, but the size distribution remained narrow. Without wishing to be bound by any particular theory, the presence of Trp in NP may improve the freeze-thaw stability of α-LA-Trp-NP by preventing protein aggregation during freezing. Compared to Trp-NP, no visible aggregates were observed in the α-LA-Trp-NP dispersion after freeze-thaw treatment at pH 7 and after freeze-thaw heat treatment. At pH 7, the particle size of α-LA-Trp-NP was uniform and did not change significantly depending on freeze-thaw treatment and freeze-thaw heat treatment. This suggested that the presence of α-LA enhances the freeze-thaw stability and freeze-thaw heat stability of Trp through complex formation due to the synergistic effect of HPH and pH shift.

[0166] 3.8. Redispersibility of freeze-dried α-LA-Trp-NPs

[0167] The effect of freeze-drying on our NPs and their redispersibility in aqueous solutions was investigated. All NPs showed signs of aggregation after freeze-drying, with only minor differences between NOHPH and HPH samples (Panel A of FIG. 26). However, small particles were observed on the surface of the aggregates with HPH of α-LA (Panel A of FIG. 26) and α-LA-Trp mixture (Panel A of FIG. 26). In a similar manner, the self-organization behavior of tryptophan molecules was observed upon freeze-drying, which resulted in irregular shapes without HPH (Panel A of FIG. 26). However, tryptophan molecules with HPH were observed to self-organize into well-ordered networks with nanosheet-like morphology (Panel A of FIG. 26).

[0168] We also investigated the redispersion of NPs in aqueous solutions. The dried powders were redispersed in both Milli-Q water and PBS buffer (10 mM, pH 7.0), and their particle size distributions and PDIs were determined (Panel B of FIG. 26). All NPs freeze-dried in Milli-Q water and in PBS buffer showed nanoscale particle sizes and narrow particle size distributions (PDI<0.3). The average particle size was slightly smaller for all samples compared to the particle size measured before freeze-drying at pH 7 (Table 4 and FIG. 23). This is thought to be related to the tendency of molecules in NPs to form strong intermolecular associations during freeze-drying, thereby forming a strong network within the NPs (Dong et al., 2021). Since the particle sizes were measured by DLS in Milli-Q water and in PBS buffer, the strong network formed during freeze-drying prevented the hydration of freeze-dried NPs, thereby resulting in smaller and narrower particle size distributions, compared to those without freeze-drying.

[0169] The foregoing is illustrative of the present invention and is not to be construed as limiting the present invention. The present invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

1. A particle, said particle is Proteins; and, Activating agent Includes, Here, the activator is present within the protein. The aforementioned particles.

2. The particle according to claim 1, wherein the activator is located within a region of the tertiary structure in which at least one nonspecific hydrophobic interaction is provided between two or more amino acid residues.

3. The particle according to claim 1, wherein the activator is located within the protein core of the protein.

4. The particle according to claim 1, wherein the activator is a plurality of activators, and at least one of the plurality of activators is present in the protein.

5. The particle according to claim 1, wherein the protein is selected from dairy protein, plant protein, or animal protein.

6. The particle according to claim 1, wherein the protein has a molten globule state and / or a bilobal structure.

7. The particle according to claim 1, wherein the protein has about 100 to about 500 amino acids and / or a molecular weight of about 10,000 kDa to about 50,000 kDa.

8. The particle according to claim 1, wherein the protein has an isoelectric point (pI) of about 4 to about 5.

9. The protein comprises two domains and / or, The particles according to claim 1, wherein the protein has a pH of approximately 5 to approximately 9 and contains one or more intramolecular disulfide bonds.

10. The particle according to claim 1, wherein the tertiary structure of the protein comprises an α-helix in an amount of about 15% to about 30% and a β-sheet in an amount of about 5% to about 25%.

11. The particles according to claim 1, selected from α-lactalbumin, lysozyme, cytochrome c, apomyoglobin, and Staphylococcus nuclease.

12. The particle according to claim 1, wherein the protein has an amino acid sequence having at least 70% sequence identity with one or more of SEQ ID NO: 1 to 3.

13. The particle according to claim 1, wherein the protein is a plurality of proteins.

14. The particles according to claim 1, wherein the activator is an organic compound having a molecular weight of about 70 g / mol to about 500 g / mol.

15. The particles according to claim 1, wherein the activator has a solubility in water of about 15 mg / mL or less at 25°C, and / or has a pKa of about 1.5 to about 3 and / or a pI of about 5 to about 6.

5.

16. The particles according to claim 1, wherein the activator is selected from tryptophan, leucine, phenylalanine, cysteine, tyrosine, vitamin E, and any combination thereof.

17. The particle according to claim 1, wherein the particle has a diameter of about 25 nm to about 900 nm.

18. The particle according to claim 1, wherein the protein is present in the particle in an amount of about 75% to about 100% by weight, and the activator is present in the particle in an amount of about 1% to about 25% by weight.

19. The particle according to claim 1, 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.

20. The particles according to claim 1, wherein the size of the particles remains within ±20% of their original size when stored in a sealed container at approximately 4°C to approximately 10°C for less than approximately 6 months.

21. The particle according to claim 1, wherein the particle has increased freeze-thaw stability compared to the freeze-thaw stability of the protein alone.

22. The particle according to claim 1, wherein, after a change in temperature, the size of the particle remains within ±20% of its original size.

23. The particle according to claim 1, wherein the particle has increased freeze-thaw thermal stability compared to the freeze-thaw thermal stability of the protein alone.

24. The particle according to claim 1, wherein, after a change in temperature, and subsequently after heat treatment, the size of the particle remains within ±20% of its original size.

25. The particle according to claim 1, wherein the particle has increased activity and / or function compared to the activity and / or function of the protein alone.

26. A plurality of particles comprising the particles described in any one of claims 1 to 25.

27. The plurality of particles according to claim 26, wherein the plurality of particles have a Dv(50) of about 175 nm to about 325 nm.

28. The plurality of particles according to claim 26, wherein the plurality of particles have a polydispersity index (PDI) of less than about 0.

5.

29. A composition comprising a carrier and the particles described in claim 1 or a plurality of particles including the particles described in claim 1, wherein less than 30% of the activator is free in the composition when the particles or plurality of particles are present in the composition in an amount of about 100 mg per 1 mL of water.

30. The composition according to claim 29, wherein the composition is a dispersion.

31. A method for preparing particles, the method is The process involves homogenizing or sonicating a composition containing a protein and an activator for about 1 minute to about 2 hours, wherein the composition has a pH of about 10 to about 12, thereby providing the particles. The aforementioned method.

32. The method according to claim 31, wherein the method comprises homogenizing the composition at a pressure of about 5,000 psi to about 45,000 psi.

33. The method according to claim 31, wherein the method comprises ultrasonically treating the composition at a frequency of about 15 kHz to about 30 kHz and an amplitude of about 40% to about 70%.

34. The method according to claim 31, wherein the composition comprises protein and activator in a weight ratio of about 2:1 to about 30:1 (protein:activator).

35. The method according to claim 31, wherein the composition has a solid content of about 1 w / v% to about 20 w / v%.

36. The method according to claim 31, wherein the homogenization or ultrasonic treatment is performed for about 10 minutes to about 50 minutes at a temperature in the range of about 15°C to about 30°C.

37. The method according to claim 31, further comprising homogenizing or sonicating the composition and then adjusting the pH of the composition to about 6.5 to about 7.

5.

38. The method according to claim 31, wherein the particles have a particle size distribution in the range of about 50 nm to about 700 nm, having a polydispersity index of less than about 0.5, and / or the particles have an average particle size of about 100 nm to about 300 nm.

39. The method according to claim 31, further comprising dehydrating the particles.

40. The method according to claim 31, further comprising reducing the size of the particles.

41. The method according to claim 31, wherein the particles are the particles described in claim 1, and / or the method provides a composition comprising a plurality of particles including the particles described in claim 1, and / or a carrier, and the particles described in claim 1 or a plurality of particles including the particles described in claim 1, wherein less than 30% of the activator is free in the composition when the particles or plurality of particles are present in the composition in an amount of about 100 mg per 1 mL of water.

42. The particles according to claim 1; a plurality of particles including the particles according to claim 1; a carrier and a composition comprising the particles according to claim 1 or a plurality of particles including the particles according to claim 1, wherein less than 30% of the activator is free in the composition when the particles or plurality of particles are present in the composition in an amount of about 100 mg per 1 mL of water; and / or an article comprising particles prepared according to claim 31.

43. The article according to claim 42, wherein the article is a food, a nutritional supplement, a therapeutic beverage, and / or a cosmetic.

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