Lactoferrin complex, composition containing same, and method of producing and using same

Abstract

A complex comprising lactoferrin and whey protein hydrolysate, compositions comprising the complex, and methods of making and using the complex are described. The composition of the present invention may comprise lactoferrin and whey protein hydrolysate, and may be an aqueous composition.

Description

[Technical Field]

[0001] Statement Regarding Electronic Submission of Sequence Listings The sequence listing in XML text format entitled 1213-7WO_ST26.xml (size 4,100 bytes, generated on October 17, 2023 and submitted herewith) is hereby incorporated by reference in its entirety.

[0002] The present invention relates to complexes comprising lactoferrin and whey protein hydrolysate, compositions comprising such complexes, and methods of making and using such complexes. [Background technology]

[0003] Lactoferrin (LF) is an iron-binding multifunctional protein that occurs in many biological secretions, including milk. LF has iron-binding / transfer properties, antibacterial properties, anti-inflammatory properties, and anticarcinogenic properties. LF promotes cell growth, detoxifies harmful free radicals, and has antibacterial, antiviral, anti-inflammatory, and anticarcinogenic properties. Due to its multiple biological functions, LF is incorporated into many commercialized products, including infant formula, nutritional supplements, therapeutic beverages, and cosmetics. Summary of the Invention [Problem to be solved by the invention]

[0004] However, LF is sensitive to denaturation induced by heat treatment, especially under neutral pH conditions, which leads to structural changes and loss of biological functionality. [Means for solving the problem]

[0005] A first aspect of the present invention is directed to a complex comprising lactoferrin and whey protein hydrolysate, wherein the lactoferrin and the whey protein hydrolysate are associated via electrostatic interactions.

[0006] An additional aspect of the invention is directed to compositions comprising the conjugates of the invention. In some embodiments, the compositions are aqueous compositions.

[0007] A further aspect of the present invention is directed to a method for preparing the complex of the present invention, comprising providing a composition comprising lactoferrin and whey protein hydrolysate at a pH ranging from about 5, about 5.5, or about 5.8 to about 6, about 6.5, or about 7; and mixing the composition, thereby providing the complex.

[0008] Further aspects of the present invention are directed to articles comprising the conjugates of the invention, the compositions of the invention, and / or the conjugates prepared according to the methods of the invention. In some embodiments, the articles are food products (e.g., infant formula, dairy products, etc.), dietary supplements, therapeutic beverages, and / or cosmetics.

[0009] It should be noted that aspects described with respect to one embodiment may be incorporated into a different embodiment even if not specifically described therein. That is, all aspects and / or features of any embodiment may be combined in any manner and / or combination. 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 apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments, however, such description is merely illustrative of the present invention. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows a schematic diagram illustrating an exemplary method for preparing a binary complex of the present invention using lactoferrin (LF) and whey protein hydrolysate (WPH), according to some embodiments of the present invention. [Figure 2] FIG. 2 is a graph illustrating the effect of pH and mixing ratio on the turbidity of mixtures of lactoferrin and whey protein hydrolysate. [Figure 3] FIG. 3 is a graph illustrating the effect of pH and mixing ratio on the turbidity of mixtures of lactoferrin and whey protein hydrolysate. [Figure 4] FIG. 4 is a graph illustrating the effect of pH and mixing ratio on the turbidity of mixtures of lactoferrin and whey protein hydrolysate. [Figure 5] FIG. 5 is a graph illustrating the effect of pH and mixing ratio on the turbidity of mixtures of lactoferrin and whey protein hydrolysate. [Figure 6]FIG. 6 is a graph illustrating the effect of pH and mixing ratio on the average particle size of complexes obtained from mixtures of lactoferrin and whey protein hydrolysate. [Figure 7] FIG. 7 is a graph illustrating the effect of pH and mixing ratio on the average particle size of complexes obtained from mixtures of lactoferrin and whey protein hydrolysate. [Figure 8] FIG. 8 is a graph illustrating the effect of pH and mixing ratio on the average particle size of complexes obtained from mixtures of lactoferrin and whey protein hydrolysate. [Figure 9] FIG. 9 is a graph illustrating the effect of pH and mixing ratio on the average particle size of complexes obtained from mixtures of lactoferrin and whey protein hydrolysate. [Figure 10] FIG. 10 is a graph illustrating the effect of blend ratio on particle size distribution of lactoferrin, whey protein hydrolysate, or a mixture thereof at pH 5.8. [Figure 11] FIG. 11 is a graph illustrating the effect of blend ratio on particle size distribution of lactoferrin, whey protein hydrolysate, or a mixture thereof at pH 5.8. [Figure 12] FIG. 12 is a graph illustrating the effect of blend ratio on particle size distribution of lactoferrin, whey protein hydrolysate, or a mixture thereof at pH 5.8. [Figure 13] FIG. 13 is a graph illustrating the effect of blend ratio on particle size distribution of lactoferrin, whey protein hydrolysate, or a mixture thereof at pH 5.8. [Figure 14] FIG. 14 is a graph illustrating the effect of blend ratio on particle size distribution of lactoferrin, whey protein hydrolysate, or a mixture thereof at pH 5.8. [Figure 15] FIG. 15 is a graph illustrating the effect of blend ratio on particle size distribution of lactoferrin, whey protein hydrolysate, or a mixture thereof at pH 5.8. [Figure 16]FIG. 16 is a graph illustrating the effect of pH and mixing ratio on the zeta potential of mixtures of lactoferrin and whey protein hydrolysate. [Figure 17] FIG. 17 is a graph illustrating the effect of pH and mixing ratio on the zeta potential of mixtures of lactoferrin and whey protein hydrolysate. [Figure 18] FIG. 18 is a graph illustrating the effect of pH and mixing ratio on the zeta potential of mixtures of lactoferrin and whey protein hydrolysate. [Figure 19] FIG. 19 is a graph illustrating the effect of pH and mixing ratio on the zeta potential of mixtures of lactoferrin and whey protein hydrolysate. [Figure 20] Figure 20 shows the effect of concentration (1% w / v to 10% w / v), mixing temperature (45°C), and time (0, 10, 20, or 30 minutes) on the turbidity of LF-WPH mixtures at ratios of 1:1 and 1:2 (LF:WPH). [Figure 21] Figure 21 shows the effect of concentration (1% w / v to 10% w / v), mixing temperature (45°C), and time (0, 10, 20, or 30 min) on the turbidity of LF-WPH mixtures at ratios of 1:1 and 1:2 (LF:WPH). [Figure 22] Figure 22 shows the effect of concentration (1 w / v% to 10 w / v%), mixing temperature (45°C), and time (0 min, 10 min, 20 min, or 30 min) on the average particle size of LF-WPH mixtures at ratios of 1:1 and 1:2 (LF:WPH). [Figure 23] Figure 23 shows the effect of concentration (1 w / v% to 10 w / v%), mixing temperature (45°C), and time (0 min, 10 min, 20 min, or 30 min) on the average particle size of LF-WPH mixtures at ratios of 1:1 and 1:2 (LF:WPH). [Figure 24] Figure 24 shows the effect of concentration (1% w / v to 10% w / v), mixing temperature (45°C), and time (0, 10, 20, or 30 min) on the zeta potential of LF-WPH mixtures at ratios of 1:1 and 1:2 (LF:WPH). [Figure 25]Figure 25 shows the effect of concentration (1% w / v to 10% w / v), mixing temperature (45°C), and time (0, 10, 20, or 30 min) on the zeta potential of LF-WPH mixtures at ratios of 1:1 and 1:2 (LF:WPH). [Figure 26] FIG. 26 shows optical microscope and scanning electron microscope images of mixed samples of lactoferrin and whey protein hydrolysate at pH 5.8 and different mixing ratios. [Figure 27] Figure 27 shows small angle X-ray scattering (SAXS) curves (Figure 27) and corresponding pair distance distribution functions (PDDFs) (Figures 28 to 32) evaluated based on indirect Fourier transformation for lactoferrin, whey protein hydrolysate, and lactoferrin-whey protein hydrolysate complexes at pH 5.8 and mixing ratios of 2:1 and 1:1, along with radius of gyration (Rg) and largest intermolecular distance (Dmax) values. [Figure 28] Figure 28 shows the small angle X-ray scattering (SAXS) curves (Figure 27) and the corresponding pair distance distribution functions (PDDFs) (Figures 28 to 32) evaluated based on indirect Fourier transformation for lactoferrin, whey protein hydrolysate, and lactoferrin-whey protein hydrolysate complexes at pH 5.8 and mixing ratios of 2:1 and 1:1, along with the radius of gyration (Rg) and largest intermolecular distance (Dmax) values. [Figure 29]Figure 29 shows the small angle X-ray scattering (SAXS) curves (Figure 27) and the corresponding pair distance distribution functions (PDDFs) (Figures 28 to 32) evaluated based on indirect Fourier transformation of lactoferrin, whey protein hydrolysate, and lactoferrin-whey protein hydrolysate complexes at pH 5.8 and mixing ratios of 2:1 and 1:1, along with the radius of gyration (Rg) and largest intermolecular distance (Dmax) values. [Figure 30] Figure 30 shows the small angle X-ray scattering (SAXS) curves (Figure 27) and the corresponding pair distance distribution functions (PDDFs) (Figures 28 to 32) evaluated based on indirect Fourier transformation for lactoferrin, whey protein hydrolysate, and lactoferrin-whey protein hydrolysate complexes at pH 5.8 and mixing ratios of 2:1 and 1:1, along with the radius of gyration (Rg) and largest intermolecular distance (Dmax) values. [Figure 31]Figure 31 shows the small angle X-ray scattering (SAXS) curves (Figure 27) and the corresponding pair distance distribution functions (PDDFs) (Figures 28 to 32) evaluated based on indirect Fourier transformation for lactoferrin, whey protein hydrolysate, and lactoferrin-whey protein hydrolysate complexes at pH 5.8 and mixing ratios of 2:1 and 1:1, along with the radius of gyration (Rg) and largest intermolecular distance (Dmax) values. [Figure 32] Figure 32 shows the small angle X-ray scattering (SAXS) curves (Figure 27) and the corresponding pair distance distribution functions (PDDFs) (Figures 28 to 32) evaluated based on indirect Fourier transformation for lactoferrin, whey protein hydrolysate, and lactoferrin-whey protein hydrolysate complexes at pH 5.8 and mixing ratios of 2:1 and 1:1, along with the radius of gyration (Rg) and largest intermolecular distance (Dmax) values. [Figure 33] Figure 33 shows optical microscope images of the supernatant or sediment of mixtures of lactoferrin and whey protein hydrolysate at pH 5.8 and at lactoferrin:whey protein hydrolysate ratios of 2:1 (Figure 33) and 1:1 (Figure 34) after incubation overnight at 4°C. [Figure 34] Figure 34 shows optical microscope images of the supernatant or sediment of mixtures of lactoferrin and whey protein hydrolysate at pH 5.8 and at lactoferrin:whey protein hydrolysate ratios of 2:1 (Figure 33) and 1:1 (Figure 34) after incubation overnight at 4°C. [Figure 35]FIG. 35 shows optical images of samples of pure lactoferrin, lactoferrin / whey protein hydrolysate complex that had been centrifuged and freeze-dried, and lactoferrin / whey protein hydrolysate complex that had been freeze-dried directly without centrifugation in PBS (10 mM, pH 7) before and after heat treatment. [Figure 36] FIG. 36 is a graph illustrating the turbidity in PBS (10 mM, pH 7) of a pure lactoferrin sample and a centrifuged and lyophilized lactoferrin / whey protein hydrolysate complex before and after heat treatment. [Figure 37] FIG. 37 is a graph illustrating the turbidity in PBS (10 mM, pH 7) of a pure lactoferrin sample and a sample of lactoferrin / whey protein hydrolysate complex that was directly lyophilized without centrifugation, before and after heat treatment. [Figure 38] FIG. 38 is a graph illustrating the average particle size in PBS (10 mM, pH 7) of a pure lactoferrin sample and a centrifuged and freeze-dried lactoferrin / whey protein hydrolysate complex sample before and after heat treatment. [Figure 39] FIG. 39 is a graph illustrating the average particle size in PBS (10 mM, pH 7) of a pure lactoferrin sample and a lactoferrin / whey protein hydrolysate complex sample that was directly freeze-dried without centrifugation, before and after heat treatment. [Figure 40] FIG. 40 is a graph illustrating the lactoferrin loading ratio in PBS (10 mM, pH 7) before and after heat treatment in a pure lactoferrin sample and in a centrifuged and lyophilized lactoferrin / whey protein hydrolysate complex sample. [Figure 41] FIG. 41 is a graph illustrating the lactoferrin loading ratio in PBS (10 mM, pH 7) before and after heat treatment in a pure lactoferrin sample and in a sample of lactoferrin / whey protein hydrolysate complex that was directly lyophilized without centrifugation. [Figure 42] FIG. 42 is a graph illustrating the lactoferrin retention percentage in PBS (10 mM, pH 7) before and after heat treatment in pure lactoferrin samples and in centrifuged and lyophilized lactoferrin / whey protein hydrolysate complex samples. [Figure 43] FIG. 43 is a graph illustrating the lactoferrin retention percentage in PBS (10 mM, pH 7) before and after heat treatment in pure lactoferrin samples and in samples of lactoferrin / whey protein hydrolysate complex that were directly lyophilized without centrifugation. [Figure 44] Figure 44 is an image of an SDS-PAGE gel in PBS (10 mM, pH 7) of a pure lactoferrin (LF) sample and a centrifuged and freeze-dried lactoferrin / whey protein hydrolysate complex (LF-WPH) sample before and after heat treatment. [Figure 45] Figure 45 shows images of an SDS-PAGE gel in PBS (10 mM, pH 7) of a pure lactoferrin (LF) sample and a lactoferrin / whey protein hydrolysate complex sample that was directly freeze-dried without centrifugation (LF-WPH direct mix) before and after heat treatment. [Figure 46] Figure 46 shows optical images in PBS (10 mM, pH 7) of a pure lactoferrin sample, a lactoferrin-whey protein hydrolysate complex sample that was centrifuged and freeze-dried, and a lactoferrin-whey protein hydrolysate complex sample that was freeze-dried directly without centrifugation, before and after oil bath heat treatment. [Figure 47] FIG. 47 is a graph illustrating the turbidity in PBS (10 mM, pH 7) of a pure lactoferrin sample and a centrifuged and lyophilized lactoferrin / whey protein hydrolysate complex sample before and after oil bath heat treatment. [Figure 48]Figure 48 is a graph illustrating the turbidity in PBS (10 mM, pH 7) of a pure lactoferrin sample and a sample of lactoferrin / whey protein hydrolysate complex that was directly lyophilized without centrifugation, before and after oil bath heat treatment. [Figure 49] FIG. 49 is a graph illustrating the turbidity in PBS (10 mM, pH 7) of a pure lactoferrin sample and a centrifuged and lyophilized lactoferrin / whey protein hydrolysate complex sample before and after oil bath heat treatment. [Figure 50] Figure 50 is a graph illustrating the average particle size in PBS (10 mM, pH 7) of a pure lactoferrin sample and a lactoferrin / whey protein hydrolysate complex sample that was directly freeze-dried without centrifugation, before and after oil bath heat treatment. [Figure 51] FIG. 51 is a graph illustrating the lactoferrin loading ratio in PBS (10 mM, pH 7) in a pure lactoferrin sample and in a centrifuged and lyophilized lactoferrin / whey protein hydrolysate complex sample before and after oil bath heat treatment. [Figure 52] FIG. 52 is a graph illustrating the lactoferrin loading ratio in PBS (10 mM, pH 7) before and after oil bath heating in a pure lactoferrin sample and in a sample of lactoferrin / whey protein hydrolysate complex that was directly lyophilized without centrifugation. [Figure 53] FIG. 53 is a graph illustrating the lactoferrin retention percentage in PBS (10 mM, pH 7) before and after oil bath heat treatment in pure lactoferrin samples and in centrifuged and freeze-dried lactoferrin / whey protein hydrolysate complex samples. [Figure 54]Figure 54 is a graph illustrating the lactoferrin retention percentage in PBS (10 mM, pH 7) before and after oil bath heat treatment in pure lactoferrin samples and in samples of lactoferrin / whey protein hydrolysate complex that were directly lyophilized without centrifugation. [Figure 55] Figure 55 is a graph illustrating the growth of Staphylococcus aureus as indicated by OD625nm at 37°C in PBS (10 mM, pH 7) in the presence of pure lactoferrin, pure whey protein hydrolysate, or lactoferrin / whey protein hydrolysate complex, before and after heat treatment. [Figure 56] Figure 56 is a graph illustrating the growth of Escherichia coli as indicated by OD625nm at 37°C in PBS (10 mM, pH 7) in the presence of pure lactoferrin, pure whey protein hydrolysate, or lactoferrin / whey protein hydrolysate complex, before and after heat treatment. [Figure 57] Figure 57 is a graph illustrating the growth of Staphylococcus aureus as indicated by OD625nm at 37°C in PBS (10 mM, pH 7) in the presence of pure lactoferrin, pure whey protein hydrolysate, or lactoferrin / whey protein hydrolysate complexes after heat treatment at 75°C for 2 minutes. [Figure 58] Figure 58 is a graph illustrating the growth of Escherichia coli as indicated by OD625nm at 37°C in PBS (10 mM, pH 7) in the presence of pure lactoferrin, pure whey protein hydrolysate, or lactoferrin / whey protein hydrolysate complexes after heat treatment at 75°C for 2 minutes. [Figure 59]Figure 59 is a graph illustrating the growth of Staphylococcus aureus as indicated by OD625nm at 37°C in PBS (10 mM, pH 7) in the presence of pure lactoferrin, pure whey protein hydrolysate, or lactoferrin / whey protein hydrolysate complexes after heat treatment at 90°C for 2 minutes. [Figure 60] Figure 60 is a graph illustrating the growth of Escherichia coli as indicated by OD625nm at 37°C in PBS (10 mM, pH 7) in the presence of pure lactoferrin, pure whey protein hydrolysate, or lactoferrin / whey protein hydrolysate complexes after heat treatment at 90°C for 2 minutes. [Figure 61] FIG. 61 shows graphs demonstrating the effect of pH and blend ratio on turbidity, zeta potential, and mean particle size of LF-WPH blends according to some embodiments of the present invention. [Figure 62] FIG. 62 shows graphs demonstrating the effect of pH and blend ratio on turbidity, zeta potential, and mean particle size of LF-WPH (BIOZATE® 9) blends according to some embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present 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 practiced or all the features that may be added to the present invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be omitted from that embodiment. Thus, the present invention contemplates that, in some embodiments of the invention, any one feature or combination of features described herein may be excluded or omitted. Additionally, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, without departing from the invention. Thus, the following description is intended to illustrate some specific embodiments of the invention, but is not intended to exhaustively identify 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 dictates 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 excluded or omitted. For example, if the present specification states that a composition includes component A, component B, and component C, it is specifically intended that any of A, B, or C, or any combination thereof, can be omitted and removed.

[0015] As used in the detailed description of the present 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 dictates 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 word "about" when referring to a measurable value, such as an amount or concentration, is meant to encompass 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 encompass X and a variation of ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of X. Ranges of measurable values ​​provided herein can encompass 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 is intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein as if it 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 herein, 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 compositions of the present invention means that the scope of the claim should be construed to include the specific materials or steps recited in the claim and that 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 interpreted as equivalent to "comprise."

[0022] As used herein, the words "increase," "increase," "enhance," "enhance," "improve," and "enhance" (and 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 quantity (e.g., a control value).

[0023] As used herein, the terms "reduction," "reduced," "reduce," "reduce," and "decrease" (as well as grammatical variations thereof) refer to a decrease of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, for example, compared to another measurable characteristic or amount (e.g., a control value). In some embodiments, the decrease may result in no or essentially no detectable activity or amount (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) is 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 reduced) relative to a reference nucleotide sequence or reference polypeptide, respectively, that is and the sequences identified above should be understood to mean a nucleotide sequence or polypeptide comprising, consisting essentially of, and / or consisting of a nucleotide sequence or polypeptide 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" (sequence identity) refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout the window of alignment of the 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 documents: Computational Molecular Biology (Lesk, AM, ed.), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.), Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.), Stockton Press, New York (1991).

[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. "Sequence 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 determined using one of the sequence comparison algorithms described below or by visual inspection. In some embodiments of the present invention, the substantial identity exists over a region of contiguous nucleotides of the nucleotide sequences of the present invention that is about 10 to about 20 nucleotides, about 10 to about 25 nucleotides, about 10 to about 30 nucleotides, about 15 to about 25 nucleotides, about 30 to about 40 nucleotides, about 50 to about 60 nucleotides, about 70 to about 80 nucleotides, about 90 to about 100 nucleotides, or more, 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, or 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 to 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 needed, 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, for example, GCG 登録商標 Wisconsin Package 登録商標(Accelrys Inc., San Diego, CA). The "identity fraction" of an aligned segment between 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 percent sequence identity is expressed as the percent 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] A complex comprising lactoferrin and whey protein hydrolysate is provided according to an embodiment of the present invention.The complex of the present invention can be a two-component complex, comprising two different components, for example, lactoferrin and whey protein hydrolysate, each of which is different from each other (for example, different in chemical structure).In some embodiments, the complex of the present invention comprises two or more (for example, 2, 3, 4, or more) different components, each of which is different from each other (for example, different in chemical structure), lactoferrin and whey protein hydrolysate.In the complex of the present invention, the lactoferrin and the whey protein hydrolysate can associate with each other through electrostatic interaction.

[0031] The complexes of the present invention may comprise one or more lactoferrin molecules (e.g., proteins) and one or more whey protein hydrolysate molecules (e.g., proteins and / or peptides), which may associate with each other via electrostatic interactions. The complexes of the present invention may have a zeta potential of from about -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, or 0 mV to about +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, or +15 mV, optionally when present in a composition (e.g., water and / or buffer) having a pH of from about 5.8 or about 6 to about 6.2 or about 6.5. In some embodiments, the complexes of the invention can have a zeta potential of about -10, about -9, about -8, about -7, about -6, about -5, about -4, about -3, about -2, about -1, about 0, about +1, about +2, about +3, about +4, about +5, about +6, about +7, about +8, about +9, about +10, about +11, about +12, about +13, about +14, or about +15 mV, when present in a composition (e.g., water and / or buffer) optionally having a pH of about 5.8 or about 6 to about 6.2 or about 6.5. In some embodiments, the complexes of the invention can have a zeta potential of about 0 mV, when present in a composition (e.g., water and / or buffer) optionally having a pH of about 5.8 or about 6 to about 6.2 or about 6.5.

[0032] The complexes of the present invention may contain lactoferrin and whey protein hydrolysate in an amount of about 1 w / w%, 5 w / w%, 10 w / w%, 15 w / w%, 20 w / w%, 25 w / w%, 30 w / w%, 35 w / w%, 40 w / w%, 45 w / w%, 50 w / w%, 55 w / w%, 60 w / w%, 65 w / w%, 70 w / w%, 75 w / w%, 80 w / w%, 85 w / w%, 90 w / w%, 95 w / w%, 96 w / w%, 97 w / w%, 98 w / w%, 99% or 100 w / w% of the complex. In some embodiments, the complexes of the present invention may comprise whey protein hydrolysate in an amount of about 1 w / w%, about 5 w / w%, about 10 w / w%, about 15 w / w%, about 20 w / w%, about 25 w / w%, about 30 w / w%, about 35 w / w%, about 40 w / w%, about 45 w / w%, about 50 w / w%, about 55 w / w%, about 60 w / w%, about 65 w / w%, about 70 w / w%, or about 75 w / w% of the complex. In some embodiments, the complexes of the present invention may comprise whey protein hydrolysate in an amount from about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% w / w to about 50%, about 55%, about 60%, about 65%, about 70%, or 75% w / w of the complex. In some embodiments, the complexes of the present invention may contain lactoferrin in an amount from about 1 w / w%, about 5 w / w%, about 10 w / w%, about 15 w / w%, about 20 w / w%, about 25 w / w%, about 30 w / w%, about 35 w / w%, about 40 w / w%, or about 45 w / w% to about 50 w / w%, about 55 w / w%, about 60 w / w%, about 65 w / w%, about 70 w / w%, about 75 w / w%, about 80 w / w%, about 85%, or 90 w / w% of the complex. In some embodiments, the complexes of the present invention may contain lactoferrin in an amount from about 40 w / w%, about 45 w / w%, about 50 w / w%, about 55 w / w%, about 60 w / w%, or about 65 w / w% to about 70 w / w%, about 75 w / w%, about 80 w / w%, about 85 w / w%, or about 90 w / w% of the complex.In some embodiments, the complexes of the present invention may contain lactoferrin in an amount of about 1 w / w%, about 5 w / w%, about 10 w / w%, about 15 w / w%, about 20 w / w%, about 25 w / w%, about 30 w / w%, about 35 w / w%, about 40 w / w%, about 45 w / w%, about 50 w / w%, about 55 w / w%, about 60 w / w%, about 65 w / w%, about 70 w / w%, about 75 w / w%, about 80 w / w%, about 85 w / w%, or about 90 w / w% of the complex.

[0033] In some embodiments, the complexes of the present invention may comprise lactoferrin in an amount of about 25 w / w%, about 30 w / w%, about 35 w / w%, about 40 w / w%, about 45 w / w%, or about 50 w / w%, about 55 w / w%, about 60 w / w%, about 65 w / w%, about 70 w / w%, about 75 w / w%, about 80 w / w%, about 85 w / w%, or 90% w / w of the complex, and whey protein hydrolysate in an amount of about 25 w / w%, about 30 w / w%, about 35 w / w%, or about 40 w / w% to about 45 w / w%, about 50 w / w%, about 55 w / w%, about 60 w / w%, about 65 w / w%, about 70 w / w%, or about 75 w / w% of the complex. In some embodiments, the complexes of the present invention may contain about 50 w / w% or about 65 w / w% to about 75 w / w% lactoferrin and about 25 w / w% to about 50 w / w% whey protein hydrolysate. In some embodiments, the complexes of the present invention may contain about 50 w / w% lactoferrin and about 50 w / w% whey protein hydrolysate.

[0034] One or more (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 or more) lactoferrin molecules may be present in the complexes of the invention. Lactoferrin present in and / or used to prepare the complexes of the invention may have a net positive charge, optionally at a pH of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8, or at a pH less than about 7 or about 8. In some embodiments, lactoferrin present in and / or used to prepare the complexes of the invention may have a zeta potential of greater than about +1 mV, greater than about +2 mV, greater than about +3 mV, greater than about +4 mV, greater than about +5 mV, greater than about +6 mV, greater than about +7 mV, greater than about +8 mV, greater than about +9 or greater than about +10 mV to about +11 mV, about +12 mV, about +13 mV, about +14 mV, about +15 mV, about +16 mV, about +17 mV, about +18 mV, about +19 mV or about +20 mV, optionally when present in a composition (e.g., water and / or buffer) having a pH of about 4, about 4.5 or about 5 to about 6, about 6.5 or about 7.

[0035] Lactoferrin present in and / or used to prepare the complexes of the present invention may have a globular structure. In some embodiments, lactoferrin present in and / or used to prepare the complexes of the present invention may have an elongated structure consisting of two globular lobes. In some embodiments, lactoferrin present in and / or used to prepare the complexes of the present invention has a radius of gyration (R) of about 4 nm, about 5 nm, about 6 nm, about 7 nm, or about 8 nm, as optionally measured by small angle X-ray scattering (SAXS). g In some embodiments, the lactoferrin present in and / or used to prepare the complexes of the invention has a radius of gyration (R gIn some embodiments, the lactoferrin present in and / or used to prepare the complexes of the invention has an intermolecular distance (D) of about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm, as can optionally be measured by small angle X-ray scattering (SAXS). max ). In some embodiments, the lactoferrin present in the complexes of the present invention and / or used to prepare the complexes of the present invention has an intermolecular distance of about 21.2 nm. In some embodiments, the lactoferrin present in the complexes of the present invention and / or used to prepare the complexes of the present invention is soluble in water at a pH of 8 or less and / or a pH of greater than 9, for example, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 9, about 9.5, or about 10. In some embodiments, the lactoferrin present in the complexes of the present invention and / or used to prepare the complexes of the present invention has a pI in the pH range of about 7 to about 9, optionally about 8. In some embodiments, the lactoferrin present in the complexes of the present invention and / or used to prepare the complexes of the present invention is a dairy protein. As used herein, "dairy protein" refers to a protein naturally found in dairy products and / or milk, and / or a protein derived from such a naturally occurring protein, having an amino acid sequence with at least 70% sequence identity to the amino acid sequence of the naturally occurring protein. For example, in some embodiments, the dairy protein is naturally found in milk (e.g., animal milk), and / or the protein is isolated from milk, or the protein is synthetically prepared, having an amino acid sequence with at least 70% sequence identity to the amino acid sequence of the naturally occurring protein. The lactoferrin present in and / or used to prepare the complexes of the present invention may be obtained from and / or derived from an animal, such as a mammal (e.g., cow, goat, sheep, or human).

[0036] Exemplary lactoferrin that may be present in and / or used to prepare the complexes of the invention include, but are not limited to, bovine lactoferrin or human lactoferrin. In some embodiments, the lactoferrin present in the complexes of the invention has an amino acid sequence that has 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 SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the lactoferrin present in the complexes 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 SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the lactoferrin present in the complexes of the invention has an amino acid sequence having about 100% sequence identity to SEQ ID NO:1 and / or SEQ ID NO:2.

[0037] One or more (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50, or more) whey protein hydrolysates may be present in a complex of the invention. In some embodiments, a complex of the invention comprises two or more whey protein hydrolysates, which may be identical to one another or different from one another. In some embodiments, a complex of the invention comprises one or more whey protein hydrolysate molecules (e.g., individual whey protein hydrolysate compounds) that are the same.

[0038] The whey protein hydrolysate present in and / or used to prepare the complexes of the present invention may have a net negative charge, optionally at a pH of about 2, about 3, about 4, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5 or about 8 and / or at a pH below about 7 or below about 8. In some embodiments, whey protein hydrolysates present in and / or used to prepare the complexes of the invention may have a zeta potential of about −20 mV to about −1 mV, for example, a zeta potential of about −20, about −19, about −18, about −17, about −16, about −15, about −14, about −13, about −12, about −11, about −10, about −9, about −8, about −7, about −6, about −5, about −4, about −3, about −2, or about −1, when present in a composition (e.g., water and / or buffer) optionally having a pH of from about 5, about 5.5, or about 5.8 to about 6.2 or about 6.5. Whey protein hydrolysates present in and / or used to prepare the complexes of the invention may have a pI at a pH of from about 3, about 3.5, or about 4 to about 4.5 or about 5. In some embodiments, whey protein hydrolysates present in and / or used to prepare the complexes of the invention may have a pI at a pH of about 4.5. In some embodiments, whey protein hydrolysates present in and / or used to prepare the complexes of the invention may be soluble in water at a pH of about 8 or less, such as about 7.5, about 7, about 6.5, about 6, about 5.5, about 5, about 4.5, about 4, about 3.5, or about 3.

[0039] In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the invention has a radius of gyration of about 1 nm, about 2 nm, about 3 nm, about 4 nm, or about 5 nm, as may optionally be measured by small angle X-ray scattering (SAXS). In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the invention has a radius of gyration (R g In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the invention has an intermolecular distance (D) of about 3 nm, about 4 nm, about 5 nm, about 6 nm, or about 7 nm, as can optionally be measured by small angle X-ray scattering (SAXS). max In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the invention has an intermolecular distance (D max )

[0040] As used herein, "whey protein hydrolysate" refers to whey protein (e.g., whey protein concentrate and / or whey protein isolate) that has been enzymatically hydrolyzed, where the hydrolysis rate (i.e., degree of hydrolysis) refers to the amount of hydrolysis. The degree of hydrolysis (DF) of a whey protein hydrolysate can be measured by methods known in the art, such as, but not limited to, by measuring the percentage ratio of the number of broken peptide bonds to the total number of bonds per unit weight of whey protein, and / or by measuring the ratio of released alpha-NH groups to total alpha-NH groups in the initial protein substrate (e.g., whey protein). As one skilled in the art will recognize, whey protein hydrolysate differs from whey protein concentrate and / or isolate. In some embodiments, whey protein isolate and / or whey protein concentrate are used to prepare the whey protein hydrolysate. Proteases and / or peptidases capable of hydrolyzing whey protein and / or its peptides can be used to prepare the whey protein hydrolysate. In some embodiments, the enzymes used to prepare the whey protein hydrolysate are trypsin, pepsin, chymotrypsin, fungal protease, papain, and / or commercially available protease mixtures, such as Protex 6L protease. The whey protein hydrolysate present in and / or used to prepare the complexes of the present invention may have a degree of hydrolysis of about 8%, about 8.5%, about 9%, about 9.5%, about 10%, or about 10.5% to about 11%, about 11.5%, about 12%, about 12.5%, or about 13%. In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the present invention may have a degree of hydrolysis of about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, or about 13%.In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the invention may have a degree of hydrolysis of about 9% to about 11%. In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the invention may have a degree of hydrolysis of about 10% or about 11%. In some embodiments, the whey protein hydrolysate is a commercially available whey protein hydrolysate, such as BIOZATE, available from Agropur. 登録商標 9, but is not limited to these.

[0041] The whey protein hydrolysate present in and / or used to prepare the complexes of the present invention may comprise a mixture of peptides and / or amino acids. In some embodiments, the whey protein hydrolysate (WPH) present in and / or used to prepare the complexes of the present invention comprises alanine in an amount of about 4 g to about 6 g per 100 g of WPH, arginine in an amount of about 1 g to about 3.5 g per 100 g of WPH, and aspartic acid and / or arginine in an amount of about 10 g to about 12.5 g per 100 g of WPH. cysteine ​​is present in an amount of about 1 g to about 4 g per 100 g of WPH, glutamic acid and / or glutamine is present in an amount of about 14 g to about 18 g per 100 g of WPH, glycine is present in an amount of about 0.5 g to about 3 g per 100 g of WPH, histidine is present in an amount of about 1 g to about 3 g per 100 g of WPH, and isoleucine is present in an amount of about 4 g to about 7 g per 100 g of WPH; Leucine is present in an amount of about 10 g to about 14 g per 100 g of WPH, lysine is present in an amount of about 8 g to about 12 g per 100 g of WPH, methionine is present in an amount of about 1 g to about 4 g per 100 g of WPH, phenylalanine is present in an amount of about 1 g to about 5 g per 100 g of WPH, proline is present in an amount of about 3 g to about 6 g per 100 g of WPH, and serine is present in an amount of about 1 g to about 10 g per 100 g of WPH. The WPH may have an amino acid profile in which threonine is present in an amount of about 3 g to about 6 g per 100 g of WPH, tryptophan is present in an amount of about 1, about 2, or about 3 g to about 4, about 5, or about 6 g per 100 g of WPH, tyrosine is present in an amount of about 1 g to about 5 g per 100 g of WPH, and / or valine is present in an amount of about 4 g to about 7 g per 100 g of WPH.In some embodiments, the whey protein hydrolysate present in and / or used to prepare the complexes of the invention comprises tryptophan in an amount of about 1 gram, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9 grams, about 10 grams, about 11 grams, about 12 grams, about 13 grams, about 14 grams, about 15 grams, about 16 grams, about 17 grams, about 18 grams, about 19 grams or about 20 grams per 100 grams of whey protein hydrolysate.

[0042] In some embodiments, whey protein hydrolysates of the present invention may comprise a mixture of peptides and / or amino acids, with about 75%, about 80%, about 85% or more by weight of the mixture having a molecular weight (e.g., average molecular weight) of about 10 kilodaltons, about 11 kilodaltons, about 12 kilodaltons, about 13 kilodaltons, about 14 kilodaltons, about 15 kilodaltons, about 16 kilodaltons, about 17 kilodaltons, about 18 kilodaltons, about 19 or about 20 kilodaltons (kDa) or less. In some embodiments, about 60%, about 65%, or about 70% by weight of the whey protein hydrolysate has a molecular weight of about 5 kDa or less, about 4 kDa or less, about 3 kDa or less, about 2 kDa or less, or about 1 kDa or less. In some embodiments, about 40%, about 45%, or about 50% by weight of the whey protein hydrolysate has a molecular weight of about 2 kDa or less, about 2.5 Da or less, or about 1 kDa or less. The molecular weight of the protein and / or whey protein hydrolysate can be determined using methods known in the art, such as chromatographic methods (e.g., high performance liquid chromatography). In some embodiments, the whey protein hydrolysates present in and / or used to prepare the complexes of the invention may have a molecular weight profile wherein less than 5% of the whey protein hydrolysates have a molecular weight greater than 20 kDa, less than 10% of the whey protein hydrolysates have a molecular weight in the range of 10 kDa to 20 kDa, about 5% to about 15% have a molecular weight in the range of 5 kDa to 10 kDa, about 20% to about 30% of the whey protein hydrolysates have a molecular weight in the range of 2 kDa to 5 kDa, about 25% to about 35% of the whey protein hydrolysates have a molecular weight in the range of 1 kDa to 2 kDa, about 15% to about 25% of the whey protein hydrolysates have a molecular weight in the range of 500 Da to 1 kDa, and / or about 5% to about 15% of the whey protein hydrolysates have a molecular weight of 500 Da or less, optionally as determined by high performance liquid chromatography.In some embodiments, the whey protein hydrolysates present in and / or used to prepare the complexes of the invention may have a molecular weight profile wherein more than 85% of the whey protein hydrolysates have a molecular weight of less than 10 kDa, more than 70% of the whey protein hydrolysates have a molecular weight of less than 5 kDa, and more than 50% of the whey protein hydrolysates have a molecular weight of less than 2 kDa, as can optionally be determined by high performance liquid chromatography.

[0043] In some embodiments, whey protein hydrolysates present in and / or used to prepare the complexes of the invention have a particle size (e.g., diameter) in at least one dimension ranging from about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm to about 1 micron, about 1.1 micron, about 1.2 micron, about 1.3 micron, about 1.4 micron, about 1.5 micron, about 1.6 micron, about 1.7 micron, about 1.8 micron, about 1.9 micron, or about 2 microns. In some embodiments, whey protein hydrolysates present in and / or used to prepare the complexes of the invention may have a particle size (e.g., diameter) in at least one dimension of about 300 nm, about 250 nm, about 200 nm, about 150 nm, or about 100 nm, or smaller.

[0044] In some embodiments, the lactoferrin and / or whey protein hydrolysate present in and / or used to prepare the complexes of the present invention are each food-grade ingredients. As used herein, "food-grade ingredients" refers to ingredients (e.g., compounds, components, lactoferrin, whey protein hydrolysate, etc.) that are safe for consumption by animals (e.g., humans) and / or intended to be ingested by animals (e.g., humans). In some embodiments, the lactoferrin and whey protein hydrolysate of the present invention are each different food-grade ingredients present in the complexes of the present invention. In some embodiments, the lactoferrin and whey protein hydrolysate in the complexes of the present invention are each obtained from and / or derived from natural products (e.g., foods, plants, animal by-products (e.g., milk), etc.).

[0045] In some embodiments, the complexes of the present invention are interpolymeric complexes. As used herein, "interpolymer complex" refers to a co-precipitate or aggregate comprising lactoferrin and whey protein hydrolysate formed through electrostatic interactions. In some embodiments, the complexes of the present invention are amorphous interpolymeric complex structures. In some embodiments, whey protein hydrolysate encapsulates and / or surrounds (e.g., partially or completely) lactoferrin in the complexes of the present invention. In some embodiments, the complexes of the present invention have a loose matrix structure. In some embodiments, the complexes of the present invention have a rod-like structure. In some embodiments, the complexes of the present invention have a cube-like structure. In some embodiments, each complex of a plurality of complexes of the present invention has the same or different structure selected from a loose matrix structure, a rod-like structure, and / or a cube-like structure when present in a composition (e.g., a liquid such as water, a buffer, milk (e.g., skim milk), and / or an acidic whey drink). In some embodiments, the complexes of the present invention do not contain cationic and / or anionic biopolymers that are not lactoferrin or whey protein hydrolysate. In some embodiments, the complexes of the present invention are interpolymer complexes formed from clusters of two or more smaller complexes comprising lactoferrin and whey protein hydrolysate, where optionally, one or both of the lactoferrin and whey protein hydrolysate molecules are nanoparticles and / or have at least one dimension of less than 200 nm or less than 100 nm. In some embodiments, the complexes of the present invention are interpolymer complexes formed from clusters of two or more smaller complexes comprising lactoferrin and whey protein hydrolysate, where one or both of the lactoferrin and whey protein hydrolysate molecules are nanoparticles and / or have an average particle size of about 100 nm or less (e.g., less than 100 nm).

[0046] The complexes of the present invention can be particles. In some embodiments, the complexes are nanoparticles. In some embodiments, the complexes are microparticles. The complexes of the invention optionally have a size (e.g., diameter) in at least one dimension of about 300 nm or greater, about 350 nm or greater, about 400 nm or greater, about 450 nm or greater, about 500 nm or greater, about 550 nm or greater, about 600 nm or greater, about 650 nm or greater, about 700 nm or greater, about 750 nm or greater, about 800 nm or greater, about 850 nm or greater, about 900 nm or greater, about 1000 nm or greater, about 2000 nm or greater, about 3000 nm or greater, about 4000 nm or greater, about 5000 nm or greater, or about 6000 nm or greater, as may be measured using microscopy (e.g., light microscopy, confocal microscopy, scanning electron microscopy (SEM) and / or transmission electron microscopy (TEM)) and / or dynamic light scattering (DLS). In some embodiments, the particles have a size (e.g., diameter) in at least one dimension of about 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, about 900 nm, about 1000 nm, or about 1500 nm to about 2000 nm, about 2500 nm, about 3000 nm, about 3500 nm, or about 4000 nm or higher. In some embodiments, the complexes of the present invention in a liquid composition (e.g., an aqueous composition) have an average size (e.g., diameter) in at least one dimension of about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, or about 1500 nm, up to about 2000 nm, about 2500 nm, about 3000 nm, about 3500 nm, or about 4000 nm or higher.In some embodiments, the lactoferrin and whey protein hydrolysate in the complexes of the present invention are nanoparticles and / or have at least one dimension of less than 200 nm or less than 100 nm. In some embodiments, the lactoferrin and whey protein hydrolysate in the complexes of the present invention have at least one dimension of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm, or any range thereof. In some embodiments, the lactoferrin and / or whey protein hydrolysate in the complexes of the present invention have an average particle size of about 100 nm or less (e.g., less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm). In some embodiments, the lactoferrin and / or whey protein hydrolysate in the complexes of the present invention have an average particle size of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm, or any range thereof.

[0047] In some embodiments, the complexes of the present invention, when present in a composition (e.g., a liquid, e.g., water, buffer, milk (e.g., skim milk) and / or an acidic whey drink), can be optionally visualized by microscopy (e.g., light microscopy, confocal microscopy, scanning electron microscopy (SEM) and / or transmission electron microscopy (TEM) and / or dynamic light scattering (DLS)). In some embodiments, the complexes of the present invention have a particle size distribution of from about 100 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 3,000 nm, about 4,000 nm, or about 5,000 nm to about 6,000 nm, about 7,000 nm, about 8,000 nm, about 9,000 nm, or about 10,000 nm, as can be measured using ion scattering. In some embodiments, the complexes of the present invention are dispersed in a composition (e.g., a liquid, e.g., water, a buffer, milk (e.g., skim milk), and / or an acid). When present in a food product (e.g., a food-grade whey beverage), the granules optionally have an average particle size (e.g., average particle diameter) of from about 500 nm, about 1,000 nm, about 1,200 nm, or about 1,300 nm to about 1,400 nm, about 1,500 nm, about 2000 nm, about 3000 nm, or about 4000 nm, as can be measured using microscopy (e.g., light microscopy, confocal microscopy, scanning electron microscopy (SEM) and / or transmission electron microscopy (TEM) and / or dynamic light scattering (DLS)).

[0048] In some embodiments, the complexes of the present invention have a radius of gyration (Rg) of about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, or about 15 nm to about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm, optionally as measured by small-angle X-ray scattering (SAXS). In some embodiments, the complexes of the present invention have a radius of gyration (Rg) of about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm, optionally as measured by small-angle X-ray scattering (SAXS). In some embodiments, the complexes of the present invention have a radius of gyration (Rg) of about 13.2 nm, optionally as measured by small-angle X-ray scattering (SAXS). In some embodiments, the complexes of the present invention have a radius of gyration (Rg) of about 18.8 nm, optionally as measured by small-angle X-ray scattering (SAXS). In some embodiments, the complexes of the present invention, when present in the compositions of the present invention, have an intermolecular distance (Dmax) of about 30 nm, about 35 nm, about 40 nm, or about 45 nm to about 50 nm, about 55 nm, about 60 nm, about 65 nm, or about 70 nm, optionally as measured by small-angle X-ray scattering (SAXS). In some embodiments, the complexes of the present invention have an intermolecular distance (Dmax) of about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, or about 70 nm, optionally as measured by small-angle X-ray scattering (SAXS). In some embodiments, the complexes of the present invention, when present in a composition of the present invention having a lactoferrin:whey protein hydrolysate ratio of 2:1, have an intermolecular distance (Dmax), optionally measured by small-angle X-ray scattering (SAXS), of about 41.5 nm. In some embodiments, the complexes of the present invention, when present in a composition of the present invention having a lactoferrin:whey protein hydrolysate ratio of 1:1, have an intermolecular distance (Dmax), optionally measured by small-angle X-ray scattering (SAXS), of about 60.9 nm.

[0049] The complexes of the present invention may be dried, optionally by freeze-drying and / or spray-drying a composition (e.g., an aqueous composition) containing the complex. In some embodiments, the dried complexes of the present invention contain water in an amount of about 0% to about 5% by weight of the dried complex. In some embodiments, the dried complexes of the present invention are water-free. In some embodiments, the complexes of the present invention are cross-linked, optionally using a cross-linking agent, such as, but not limited to, transglutaminase, glyceraldehyde, dialdehyde pectin, and / or genipin.

[0050] In some embodiments, the complexes of the present invention include an active ingredient. The active ingredient may be present within the complex (e.g., may be encapsulated and / or entrapped). In some embodiments, the active ingredient may be bound (e.g., covalently and / or non-covalently) to lactoferrin and / or whey protein hydrolysate present in the complex. Exemplary active ingredients include, but are not limited to, amino acids (e.g., tryptophan, leucine, phenylalanine, cysteine, and / or tyrosine), vitamin E, iron, vitamin A, vitamin D, and any combination thereof.

[0051] The complexes of the present invention may have improved (e.g., increased) storage stability, stability (e.g., thermal stability), activity, and / or function of the components present in the complex (e.g., lactoferrin and / or whey protein hydrolysate) compared to the storage stability, stability, activity, and / or function of the components alone (i.e., the components not present in the complexes of the present invention). In some embodiments, the complexes of the present invention provide increased stability of the components present in the complex (e.g., lactoferrin) compared to the stability of the components alone.

[0052] In some embodiments, lactoferrin present in a complex of the present invention has increased stability (e.g., reduced degradation of the lactoferrin) after exposure to temperatures ranging from about 70°C or about 75°C to about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or about 10 minutes to about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes, compared to the stability of the lactoferrin alone (i.e., lactoferrin not present in a complex of the present invention) after exposure to the same conditions (e.g., the same temperature for the same time). In some embodiments, lactoferrin present in a complex of the present invention has increased stability (e.g., reduced degradation of the lactoferrin) after exposure to temperatures from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds, compared to the stability of the lactoferrin alone (i.e., lactoferrin not present in a complex of the present invention) after exposure to the same conditions (e.g., the same temperature for the same time). In some embodiments, lactoferrin present in the complexes of the present invention has increased stability (e.g., reduced degradation of the lactoferrin) after exposure to high-temperature, short-time (HTST) treatment under oil bath conditions at about 145°C for about 2 seconds to about 60 seconds, compared to the stability of the lactoferrin alone (i.e., lactoferrin not present in the complexes of the present invention) after exposure to the same conditions (e.g., the same temperature for the same time). In some embodiments, the complexes may be present in a composition (e.g., a liquid, such as water, buffer, milk (e.g., skim milk), and / or an acidic whey drink) and may be exposed to temperature and / or HTST treatment. In some embodiments, the increased stability of the lactoferrin is determined and / or demonstrated by reduced degradation of the lactoferrin in the complexes compared to the degradation of the lactoferrin alone.

[0053] In some embodiments, after exposure to temperatures ranging from about 70°C or about 75°C to about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or about 10 minutes to about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes, the lactoferrin present in the complexes of the invention is degraded by less than 50%, e.g., about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1% or less, optionally as measured by chromatography (e.g., high performance liquid chromatography). In some embodiments, after exposure to temperatures ranging from about 70°C or 75°C to about 80°C, about 85°C, about 90°C, about 95°C or about 100°C for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or about 10 minutes to about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 60 minutes, the lactoferrin present in the complexes of the invention is degraded by less than 40%, e.g., about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1% or less, as can optionally be measured by high performance liquid chromatography. In some embodiments, after exposure to temperatures ranging from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds, the lactoferrin present in the complexes of the invention is degraded by less than about 30%, e.g., about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, or less, optionally as measured by chromatography (e.g., high performance liquid chromatography). In some embodiments, after exposure to high temperature, short time (HTST) treatment in an oil bath at about 145°C for about 2 seconds to about 60 seconds, the lactoferrin present in the complexes of the invention is degraded by less than about 30%, e.g., about 25%, about 20%, about 15%, about 10%, about 5%, about 1% or less, optionally as measured by chromatography (e.g., high performance liquid chromatography).In some embodiments, following exposure to HTST treatment at a temperature ranging from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds, and / or at about 145°C for about 2 seconds to about 60 seconds under oil bath conditions, the lactoferrin present in the complexes of the invention is degraded by less than about 20%, e.g., about 15%, about 10%, about 5%, about 1%, or less, optionally as measured by chromatography (e.g., high performance liquid chromatography).

[0054] In some embodiments, after about 30 seconds to about 2 minutes of exposure to temperatures ranging from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C, the antimicrobial potency and / or activity (e.g., antibacterial activity) of lactoferrin present in the complexes of the invention against Gram-positive and / or Gram-negative bacteria is maintained and / or enhanced (e.g., increased) compared to the antimicrobial potency and / or activity of the lactoferrin alone, optionally after exposure to the same conditions (e.g., the same temperature and time). In some embodiments, after about 30 seconds to about 2 minutes of exposure to temperatures ranging from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C, the antimicrobial potency and / or activity (e.g., antibacterial activity against Gram-positive and / or Gram-negative bacteria) of lactoferrin present in the complexes of the invention is maintained and / or enhanced compared to the antimicrobial potency and / or activity of the lactoferrin alone, optionally after exposure to the same conditions (e.g., the same temperature and time).

[0055] In some embodiments, the composition (e.g., a liquid, e.g., a soluble polymer) is formed in response to exposure to a temperature ranging from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C for about 30 seconds to about 2 minutes, or to a temperature ranging from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds. For example, the amount of a component (e.g., lactoferrin and / or whey protein hydrolysate) present in the complex of the present invention in water, buffer, milk (e.g., skim milk), and / or an acidic whey beverage, optionally as measured by chromatography (e.g., high performance liquid chromatography), remains within about ±30% of the amount of the component present in the complex before the exposure (e.g., the amount of the component present in the complex upon initial formation of the complex and / or immediately prior to the exposure). For example, the amount of a component (e.g., lactoferrin and / or whey protein hydrolysate) present in the complex of the present invention after exposure to a certain temperature for a certain period of time may be about 70% or more (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) of the amount of the same component present in the complex after exposure to the certain temperature for a certain period of time.In some embodiments, in response to exposure of the composition to a temperature ranging from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C for about 30 seconds to about 2 minutes, or exposure of the composition to a temperature ranging from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds, the composition (e.g., a liquid, e.g., The activity (e.g., bioactivity and / or antibacterial activity) and / or function (e.g., lactoferrin and / or whey protein hydrolysate) of a component present in a complex of the present invention in a solution containing a soluble or non-soluble component (e.g., water, buffer, milk (e.g., skim milk), and / or acidic whey beverage) retains within about ±30% of the activity (e.g., bioactivity) and / or function of the component present in the complex prior to the exposure (e.g., the activity and / or function of the component present in the complex upon initial formation of the complex and / or immediately prior to the exposure). For example, after exposure of a complex of the present invention to a temperature for a period of time, the activity and / or function of a component present in the complex may be about 70% or more (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) of the activity and / or function of the same component present in the complex prior to the exposure to the temperature for a period of time. In some embodiments, in response to exposure to temperatures ranging from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C for about 30 seconds to about 2 minutes, or exposure to temperatures from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds, the physicochemical properties (e.g., turbidity and / or particle size) of the complex of the present invention present in a composition (e.g., a liquid such as water, buffer, milk (e.g., skim milk), and / or acidic whey beverage) retain within about ±30% of the original physicochemical properties of the complex prior to the exposure (e.g., the physicochemical properties of the complex upon initial formation of the complex and / or immediately prior to the exposure).For example, after exposure of a complex of the present invention to a certain temperature for a certain period of time, the physicochemical properties of the complex may be about 70% or more (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) of the physicochemical properties of the complex before exposure to the certain temperature for the certain period of time. In some embodiments, the amount of a component present in a complex of the invention, the activity and / or function of a component present in a complex of the invention, and / or the physicochemical properties of the complex of the invention may not change significantly (e.g., by about 30% or less) after exposure to a temperature ranging from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C for about 30 seconds to about 2 minutes, or after exposure to a temperature ranging from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds, compared to the amount, activity and / or function and / or physicochemical properties before the exposure.

[0056] In some embodiments, the composition (e.g., a liquid, such as water, a buffer, milk (e.g., starch, or the like)) is exposed to a temperature ranging from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C for about 30 seconds to about 2 minutes, or to a temperature ranging from about 100°C, about 105°C, about 110°C, about 115°C, or about 120°C to about 125°C, about 130°C, about 135°C, about 140°C, or about 145°C for about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, or about 30 seconds to about 40 seconds, about 50 seconds, or about 60 seconds. The antimicrobial capacity and / or activity (e.g., antibacterial activity against Gram-positive and / or Gram-negative bacteria) of the components (e.g., lactoferrin and / or whey protein hydrolysate) present in the complex of the present invention present in kibble milk, kibble milk, and / or acidic whey beverages is maintained within about ±30% of the antimicrobial capacity and / or activity of the components prior to the exposure (e.g., the antibacterial activity of the components present in the complex upon initial formation of the complex and / or immediately prior to the exposure). For example, after exposure of the complex of the present invention to a temperature for a period of time, the antimicrobial capacity and / or activity of the components present in the complex may be about 70% or more (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) of the antimicrobial capacity and / or activity of the components present in the complex prior to the exposure to the temperature for a period of time.

[0057] In some embodiments, the complexes of the present invention increase the thermal stability of a component (e.g., lactoferrin and / or whey protein hydrolysate) present in the complex compared to the thermal stability of the component alone. For example, the presence of the component in the complex may reduce or avoid denaturation of the component (e.g., thermal denaturation, e.g., during preparation of a food product containing the component) compared to the amount of denaturation of the component alone (i.e., the component not present in the complexes of the present invention) under the same conditions. In some embodiments, the complexes of the present invention increase the thermal stability of lactoferrin present in the complex upon exposure to a temperature for a period of time (e.g., from about 70°C or about 75°C to about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or about 10 minutes to about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes), compared to the thermal stability of lactoferrin alone upon exposure to the same conditions (e.g., the same temperature and time). In some embodiments, the complexes of the present invention increase the stability (e.g., thermal stability), structure, activity, and / or function of a component (e.g., lactoferrin) present in the complex upon exposure to the same conditions (e.g., the same pH) at a pH of about 5 or about 5.5 to about 6, about 6.5, or about 7, compared to the stability, structure, activity, and / or function of the component alone.

[0058] The activity and / or function of a component (e.g., lactoferrin and / or whey protein hydrolysate) present in a complex of the present invention may be increased compared to the activity and / or function of the component alone. For example, after heating the complex (e.g., at a temperature ranging from about 70°C or about 75°C to about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or about 10 minutes to about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes), the component (e.g., lactoferrin) present in the complex may have increased activity and / or function compared to the activity and / or function of the component (e.g., lactoferrin) alone after the same heating conditions. In some embodiments, after storing a complex of the present invention for a period of time (e.g., storing a dried complex in a sealed container at a temperature ranging from about 20°C to about 30°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months), a component (e.g., lactoferrin) present in the complex may have increased activity and / or function compared to the activity and / or function of the component (e.g., lactoferrin) alone after the same storage and / or heating conditions. In some embodiments, a complex comprising lactoferrin and whey protein hydrolysate may provide increased activity and / or function (e.g., increased antimicrobial activity) after heating and / or storage of the complex compared to the activity and / or function of lactoferrin after the same heating and / or storage conditions. In some embodiments, after exposing a complex of the present invention to temperatures of 70°C to about 80°C, about 90°C, or about 100°C for about 1 minute to about 60 minutes, the activity of lactoferrin is increased compared to the activity of lactoferrin alone after the same exposure conditions. In some embodiments, other than whey protein hydrolysate, the complexes of the present invention do not comprise any agent configured to preserve and / or stabilize the activity, function and / or stability (e.g., thermal stability) of lactoferrin present in the complex.

[0059] In some embodiments, the complexes of the present invention may provide increased bioavailability of a component (e.g., lactoferrin and / or whey protein hydrolysate) present in the complexes of the present invention compared to the bioavailability of the component alone. Bioavailability may be determined after administering the complex to a subject, optionally wherein administering includes ingesting the complex by the subject. In some embodiments, the bioavailability of the lactoferrin after ingesting the complex by the subject is increased compared to the bioavailability after ingesting the lactoferrin by the subject. In some embodiments, a component (e.g., lactoferrin) present in the complexes of the present invention has increased bioavailability in the intestinal tract of a subject compared to the bioavailability of the component alone. In some embodiments, a component (e.g., lactoferrin) present in the complexes of the present invention has reduced enzymatic hydrolysis (e.g., reduced enzymatic hydrolysis in the gastric phase of digestion in a subject) compared to the amount of enzymatic hydrolysis of the component alone.

[0060] According to some embodiments, compositions comprising the conjugates of the present invention are provided, and / or articles comprising the conjugates of the present invention are provided. In some embodiments, the compositions and / or articles comprise a plurality of conjugates of the present invention. In some embodiments, the conjugates are in the form of particles, optionally wherein the particles are freeze-dried particles, spray-dried particles, and / or the like. In some embodiments, the compositions and / or articles comprise a plurality of particles, optionally wherein the compositions and / or articles are in the form of a powder (e.g., a dry powder). In some embodiments, the compositions of the present invention are aqueous compositions, and the conjugates of the present invention can be dissolved and / or suspended in the composition.

[0061] As used herein, "free lactoferrin" refers to lactoferrin that is not associated with whey protein hydrolysate via electrostatic interactions. In some embodiments, the complexes of the present invention and / or particles comprising the complexes can be prepared and / or isolated from a solution containing less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% w / v free lactoferrin. In some embodiments, the complexes of the present invention and / or particles comprising the complexes can be prepared and / or isolated from a solution containing less than 1% w / v free lactoferrin. In some embodiments, the compositions of the present invention, articles of the present invention, and particles of the present invention can contain less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% wt% free lactoferrin. In some embodiments, the compositions, articles, and / or particles can contain less than 1% wt% free lactoferrin.

[0062] In some embodiments, compositions and / or articles of the present invention comprise a complex of the present invention and a carrier. The carrier can be a liquid, such as, but not limited to, water, buffer, milk, food, and / or oil. In some embodiments, the composition of the present invention is an aqueous composition, and the complex is dissolved and / or suspended in the composition. In some embodiments, the composition of the present invention is a suspension, optionally wherein the complex of the present invention is suspended in the composition. In some embodiments, the complex of the present invention stabilizes a composition comprising the complex, optionally wherein the composition is an emulsion. In some embodiments, the carrier is a solid (e.g., particles and / or powder), and the complexes of the present invention may be present together with the solid, optionally above, below, in combination with, and / or in a mixture with the solid. In some embodiments, the carrier is a food-grade ingredient, such as, but not limited to, milk, a milk drink, infant formula, and / or an instant drink powder. One or more additives may be present in the compositions of the present invention, such as, but not limited to, gum arabic, sodium caseinate, and / or maltodextrin.

[0063] In some embodiments, the compositions and / or articles of the present invention are foods, dietary supplements, therapeutic beverages, and / or cosmetics. In some embodiments, the complexes of the present invention can be present in foods. In some embodiments, the foods are dairy products (e.g., milk, yogurt, etc.). In some embodiments, the compositions are infant formulas and / or dietary supplements (optionally, beverages).

[0064] According to some embodiments of the present invention, methods for preparing the complexes of the present invention are provided. In some embodiments, the methods include preparing a composition comprising lactoferrin and whey protein hydrolysate at a pH ranging from about 5, about 5.5, or about 5.8 to about 6, about 6.5, or about 7; and mixing the composition to thereby prepare the complexes. The composition comprising lactoferrin and whey protein hydrolysate may be an aqueous composition, optionally containing a buffer. In some embodiments, the composition used to prepare the complexes of the present invention has a pH of about 5, about 5.5, about 5.8, about 6, about 6.5, or about 7. In some embodiments, the composition used to prepare the complexes of the present invention includes a salt, optionally in an amount from about 0.1 mM, about 0.5 mM, about 1 mM, or about 5 mM to about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM. In some embodiments, the composition used to prepare the complexes of the invention comprises salt in an amount of about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM.

[0065] The compositions used to prepare the complexes of the present invention may contain lactoferrin and whey protein hydrolysate, each independently present in the composition in an amount of about 0.01%, about 0.1%, about 0.5%, or about 1% by weight to about 2% or about 3% by weight of the composition. In some embodiments, the compositions used to prepare the complexes of the present invention may contain lactoferrin and whey protein hydrolysate, each independently present in an amount of about 0.01%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, or about 3% by weight of the composition. In some embodiments, the compositions used to prepare the complexes of the present invention contain lactoferrin and whey protein hydrolysate in a weight ratio of about 10:1 to about 1:10 (lactoferrin:whey protein hydrolysate), for example, a weight ratio of about 1:1 or about 2:1 (lactoferrin:whey protein hydrolysate). In some embodiments, the composition used to prepare the complex of the present invention has a total concentration of lactoferrin and whey protein hydrolysate in the composition in an amount of about 5% or less by weight of the composition, for example, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% by weight of the composition.

[0066] Mixing the compositions used to prepare the complexes of the present invention can be carried out using methods known in the art. In some embodiments, mixing the compositions used to prepare the complexes of the present invention comprises mixing the compositions for about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes to about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes, optionally at a temperature ranging from about 20°C, about 25°C, or about 30°C to about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C.

[0067] The method of the present invention may further comprise curing the composite of the present invention. In some embodiments, curing the composite comprises adjusting the temperature of a composition comprising the composite of the present invention to a temperature in the range of about 5° C. to about 10° C. and exposing the composition to a temperature in the range of about 5° C. to about 10° C. for about 1 hour or about 2 hours to about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

[0068] In some embodiments, the methods of the present invention include isolating and / or obtaining a complex of the present invention from a composition. Separating and / or obtaining a complex of the present invention from a composition may include centrifuging, drying, freeze-drying, filtering, and / or spray-drying the composition, thereby isolating and / or obtaining the complex. In some embodiments, the isolated and / or obtained complex is a dried complex, optionally comprising water in an amount of about 0% to about 5% by weight of the dried complex. The dried complex may be in the form of particles and / or powder. In some embodiments, the isolated and / or obtained complex is milled, ground, and / or micronized to provide particles having a desired size, e.g., a size (e.g., diameter) of less than about 1 mm. In some embodiments, the complexes of the present invention may be cross-linked using a cross-linking agent, such as, but not limited to, transglutaminase, glyceraldehyde, dialdehyde pectin, and / or genipin. Lactoferrin and whey protein hydrolysate may be cross-linked in the complexes of the present invention.

[0069] In some embodiments, the methods of the present invention include combining the complexes of the present invention with a carrier, optionally wherein the carrier is liquid or solid. In some embodiments, the complexes of the present invention are added to food-grade ingredients and / or foods (e.g., beverages or powder formulations). Combining the complexes of the present invention with a carrier may include mixing the isolated and / or obtained complexes into the carrier and / or mixing the complexes of the present invention present in a composition (e.g., an aqueous composition) with the carrier. In some embodiments, the complexes of the present invention are dispersed in the carrier, optionally by mixing, stirring, homogenizing, and / or the like, at a temperature ranging from about 20°C, about 25°C, or about 30°C to about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C.

[0070] The methods of the present invention can include providing a therapeutic effect and / or benefit to a subject and / or treating and / or preventing a disease, disorder and / or condition in a subject. The methods can include administering (e.g., orally administering) a conjugate and / or a composition of the present invention to a subject, and optionally, the administering includes ingesting the conjugate and / or composition by the subject.

[0071] In some embodiments, the methods of the present invention comprise administering a therapeutically effective amount of a conjugate of the present invention and / or a composition of the present invention to a subject. As used herein, the term "therapeutically effective amount" refers to an amount of a conjugate and / or composition of the present invention that elicits a therapeutically useful response in a subject. Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0072] As used herein, "treat," "treating," or "treatment" (and grammatical variations thereof) refers to any type of treatment that confers benefit to a subject and can mean that the severity of the subject's condition is alleviated, at least partially improved, or ameliorated, 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 symptoms occurs. In some embodiments, the severity of symptoms associated with iron deficiency in a subject can be reduced compared to the severity of the symptoms in the absence of the methods of the present invention. In some embodiments, the complexes and / or compositions of the present invention are administered to a subject to improve iron delivery and / or absorption in the subject and / or to treat the disease and / or symptoms thereof.

[0073] In some embodiments, the conjugates and / or compositions of the present invention can be administered in a treatment-effective amount. As used herein, a "therapeutically effective" amount is an amount sufficient to treat a subject (as defined herein). One skilled in the art will understand that the therapeutic effect need not 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 conjugates and / or compositions of the present invention to a subject, optionally wherein the administering can include the subject ingesting the conjugates and / or compositions.

[0074] As used herein, the terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to avoiding, alleviating, and / or delaying the onset of symptoms associated with a disease, disorder, or condition, and / or reducing the severity of the onset of symptoms associated with a disease, disorder, or condition compared to that which would occur in the absence of the methods of the invention. Prevention can be complete, e.g., the complete absence of symptoms. The prevention can also be partial, such that the occurrence and / or severity of the onset of symptoms in a subject is less than that which would occur in the absence of the methods of the invention. In some embodiments, the complexes of the invention and / or compositions of the invention are administered to a subject to prevent a disease, disorder, or condition.

[0075] In some embodiments, the complexes and / or compositions of the present invention may be administered in a prophylactically 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. One of skill in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject. In some embodiments, a prophylactically effective amount may be achieved by administering the complexes and / or compositions of the present invention, optionally wherein the administering may include ingesting the complexes and / or compositions by the subject.

[0076] 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 mammals in utero. Any mammal in need of treatment according to the present invention is suitable. Human subjects of both genders and at any stage of development (i.e., neonates, infants, juveniles, adolescents, and adults) can be treated according to the present invention. In some embodiments of the present invention, the subject is a mammal, and in some embodiments, the subject is a human.Human subjects include both males and females of all ages, including fetal subjects, newborn subjects, infant subjects, young subjects, adolescent subjects, adult subjects, and elderly subjects, as well as pregnant subjects.In certain embodiments of the present invention, the subject is a human adolescent and / or adult.

[0077] The methods of the invention may be carried out on animal subjects, particularly mammalian subjects, such as mice, rats, dogs, cats, livestock and horses, for veterinary purposes and / or for drug screening and drug development purposes.

[0078] In some embodiments, the subject is "in need of" or "in need of" a method 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.

[0079] 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 as claimed, but rather to illustrate particular 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.

[0080] Example

[0081] Example 1

[0082] 1. Introduction The biological function of lactoferrin (LF) is highly related to its native protein structure and conformation. Commercial heat treatment procedures have been shown to significantly denature LF, altering its native structure and affecting its biological function. Because the thermal denaturation temperature of native LF is close to the pasteurization conditions of most foods, the loss of functionality of LF during heat treatment limits its ultimate use and bioactivity in the final commercial product.

[0083] The present inventors developed an LF-whey protein hydrolysate (WPH) complex that improves the heat stability of LF.

[0084] 2. Materials and Methods

[0085] 2.1 Materials

[0086] Lactoferrin (LF) powder (natural bovine LF, Bioferrin 2000, Iron >15 mg / 100 g) was provided by Glanbia Nationals, Inc., Fitchburg, WI. Whey protein hydrolysate (WPH) powder (BIOZATE) 登録商標 9, hydrolysis rate 10–11% was provided by Agropur USA. Hydrochloric acid and sodium hydroxide were purchased from Fisher Scientific (Hampton, NH, USA).

[0087] 2.2 Experimental Approach

[0088] Lactoferrin (LF) solution (1 w / v%) and whey protein hydrolysate (WPH) solution (1 w / v%) were prepared by dissolving LF or WPH powder in Milli-Q water for 1 hour using a magnetic stirrer and then storing in a refrigerator at -4°C overnight to allow complete dissolution.

[0089] Figure 1 shows a schematic diagram of an exemplary experimental design for the formation of LF / WPH complexes. The pH of the LF or WPH solution was adjusted to the target pH (5.8, 6, 6.2, or 6.5) using 0.1 M NaOH or HCl. Next, LF and WPH solutions under the same pH conditions were mixed at a fixed ratio at a total protein concentration of 1 w / v% using a magnetic stir bar at 25 °C. After 30 min of mixing, the turbidity, zeta potential, and particle size of the mixed solution were measured. Next, the LF / WPH mixed solution was centrifuged at 10,000 g for 20 min at 4 °C, and the biopolymer-rich phase (pellet) was collected, lyophilized, weighed, and used for characterization and thermal studies of the complexes.

[0090] The effects of total concentration (1 w / v%, 3 w / v%, 5 w / v% and 10 w / v%) and mixing temperature (45°C) and time (0–30 min) on complex formation were also investigated by comparing the turbidity, zeta potential and particle size of the mixed solutions.

[0091] 2.3 Characterization

[0092] 2.3.1 Turbidity measurements

[0093] The turbidity of the LF / WPH mixture was measured using a UV-Vis light spectrophotometer (UV-2600, SHIMADZU Co., Japan). The transmittance was measured at room temperature at a wavelength of 600 nm using quartz cuvettes with a path length of 1 cm. Milli-Q water was used as a blank (transmittance 100%). The turbidity (T) was calculated as follows (Equation 1 below):

number

[0094] 2.3.2 Particle size measurement

[0095] The mean diameter and particle size distribution of the LF / WPH mixture were analyzed using a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern, Germany). All analyses were performed at 25°C using a 1 cm path-length cuvette at a wavelength of 633 nm and a backscattering angle of 173°. The refractive index of the dispersion medium was set to 1.33, and that of the material was set to 1.45. The analyses were performed in triplicate, with at least 11 replicates for each measurement.

[0096] 2.3.3 Zeta potential measurement

[0097] The zeta potential of the LF- / WPH mixture was measured using a Nano-ZS (Malvern, Germany) in Smouluchwski mode. The software was able to determine the appropriate measurement type after acquiring the sample conductivity using a voltage of approximately 150 V. Samples were measured in triplicate, and each measurement was run 10 times.

[0098] 2.3. Microstructure analysis

[0099] A small aliquot of the binary complex solution was transferred to a glass microscope slip and covered with a glass cover slip. Light microscopy images were observed under a 20x lens (Leica HI ​​Plan, Buffalo Grove, IL, USA) with a light microscope (Leica DM IL LED, Buffalo Grove, IL, USA) equipped with a camera (Vision Research, AMETEK, Miro Lab 3a10). Images were analyzed using Image J software (version 1.52a, NIH, USA).

[0100] Scanning electron microscopy images of the composites were taken using a field-emission scanning electron microscope (SEM) (Zeiss Gemini 500, Jena, Germany). An aliquot (approximately 10 μL) of the solution was dropped onto a pin stub with carbon tape and then vacuum dried overnight in a desiccator. The sample was coated with Au / Pd in ​​a sputter coater (Denton Desk V, NJ, USA) and scanned and photographed by a high-efficiency secondary electron detector with a 20.0 μm aperture. The accelerating voltage was 1 kV.

[0101] 2.3.5 Quantification of LF by HPLC

[0102] An HPLC method was developed to quantify LF in binary complex solutions or after heat treatment. Reversed-phase HPLC was performed on an Agilent 1100 / 1200 Series HPLC system (Agilent Technologies, CA, USA) equipped with a diode array detector and a ChemStation data acquisition program. Detection was at 214 nm. LF separation was performed at 40 °C using a BioZen Intact XB-C8 column (150 × 4.6 mm, 3.6 μm; Phenomenex, Torrance, CA, USA). The mobile phase A was 0.1% trifluoroacetic acid (TFA) in water and the mobile phase B was 0.1% trifluoroacetic acid in acetonitrile. The mobile phase flow rate was 1.0 mL / min, with the following gradient elution: 0–5 min, 5% B; 5–20 min, 5–20% B; 20–25 min, 50–5% B. The injection volume was 10 μL. The residual natural LF concentration in the sample solution was 0 to 0.2 w / v% (R 2 >99%) can be measured and quantified according to a standard curve of native LF.

[0103] 2.3.6 Complexation efficiency, LF loading ratio and LF retention measurement

[0104] The supernatant obtained after centrifugation of the mixture was appropriately diluted and used to quantify the free LF concentration by HPLC analysis. The conjugation efficiency was calculated according to the following formula (Equation 2):

number

[0105] Total LF was the theoretical concentration of LF contained in the mixture (w / v), and free LF was the measured concentration of LF in the supernatant (w / v).

[0106] The loading rate (i.e., mass ratio) of LF in the lyophilized conjugate samples was quantified using HPLC analysis of the conjugate samples redispersed in phosphate buffer, pH 7, at a concentration of 0.2 w / v%. The loading rate of LF in the lyophilized conjugate was calculated according to the following formula (Equation 3):

number

[0107] The LF retention ratio indicates how native the LF is after heat treatment and was calculated using the following formula (Equation 4):

number

[0108] 2.3.7 Electrophoretic analysis

[0109] Unheated and heated pure LF solutions or LF / WPH complex solutions were analyzed using sodium dodecyl sulfate (SDS)-PAGE in a vertical minigel electrophoresis system (Mini-PRO-TEAN Tetra cell, Bio-Rad, USA). A premixed TGA fast Cast acrylamide starter kit was used for PAGE gel preparation. 20 μL of diluted sample (2 mg / mL protein) was mixed 1:1 with 2X Laemmli buffer and then incubated at room temperature for 3 to 4 hours. Next, 20 μL of the mixture was loaded onto the gel for electrophoresis, and electrophoresis (200 V) was performed for approximately 30 to 45 minutes. The gel was stained for 30 minutes in 0.15 w / v% Coomassie Brilliant R-250 solution consisting of 50 v / v% methanol and 10 v / v% acetic acid. The gel was then destained in a destaining solution (20 v / v % methanol and 10 v / v % acetic acid) for 24 hours.

[0110] 2.3.8 Thermal stability test of LF in the complex

[0111] The thermal stability of LF was tested and compared between pure LF and a LF / WPH complex solution redispersed in 10 mM phosphate buffer (0.2 w / v%) at pH 7. Solution samples (2 mL) were placed in glass tubes and placed in water baths at different temperatures (75 and 90 °C) for 2 min. The samples were then immersed in an ice-water bath for several minutes until cooled to ambient temperature (T = 25 °C) and further analyzed. The optical images, turbidity, and particle size of the complex solutions before and after heat treatment were measured. HPLC analysis was used to quantify the loading rate of L and LF retention after heat treatment. SDS-PAGE images of the LF samples and the LF binary complex samples before and after heating were compared.

[0112] 2.3.9 Antimicrobial activity analysis

[0113] Staphylococcus aureus and Escherichia coli strains were used as target Gram-positive and Gram-negative bacteria in this study, respectively. LB broth and agar medium were prepared and autoclaved at 121°C for 15 minutes. S. aureus or E. coli were activated by culturing on LB agar medium for 24 hours. A loop of a pure colony was transferred into 10 mL of fresh LB medium and incubated at 37°C for 24 hours. Generally, the antibacterial activity of LF and the LF binary complex against bacteria was measured using UV absorbance in a 96-well microtiter plate. First, the bacteria were diluted 1000-fold with LB broth, and the absorbance of 100 μL of bacterial broth was confirmed to be less than 0.04 at 625 nm. The increase in absorbance at 625 nm (OD625nm) was used to indicate bacterial growth.

[0114] For the MIC (minimal inhibitory concentration) test, LF at different diluted concentrations (0.1–1 w / v%) was used. 100 μL of diluted S. aureus broth and 100 μL of LF solution were added to each well. Next, 100 μL of bacterial broth supplemented with 100 μL of PBS buffer was applied as a control. The minimum concentration of LF required to inhibit 50% of bacterial growth (i.e., 50% of the OD value in the control) was referred to as the MIC of LF that inhibited the bacteria and was used as the LF concentration for further antibacterial testing in the LF-WPH complex test. Similarly, 100 μL of diluted bacterial broth and 100 μL of unheated and heated LF complex solutions at selected concentrations (0.2 w / v%) were added to each well. The microtiter plates were incubated at 37°C and the OD625nm was measured with shaking for 10 seconds before reading to monitor bacterial growth at 0, 24 and 48 hours of incubation.

[0115] 2.4 Data analysis

[0116] The data obtained are presented as the mean and standard deviation of duplicate or triplicate replicates and were analyzed using analysis of variance (ANOVA). Differences between means were assessed using Tukey's HSD comparison test (P < 0.05). All statistical analyses were performed using JMP Pro 15 (SAS Institute, USA), and plots were generated using GraphPad Prism 9 (GraphPad Software Inc.).

[0117] 3. Results and Discussion

[0118] 3.1 Effect of pH and ratio on the formation of LF / WPH complexes

[0119] Turbidity is an important indicator for determining whether complexes are formed. Figures 2-5 show the effect of different LF to WPH mixing ratios on the turbidity of LF / WPH mixtures at different pH conditions. Solutions with higher turbidity generally indicate a higher level of complex formation. As the pH increases, the turbidity of LF / WPH mixtures decreases, and a higher LF ratio is required to form a more turbid solution. As shown in Figure 2, at pH 5.8, a 1:1 LF / WPH mixture has the highest turbidity; as shown in Figure 3, at pH 6.0, a 2:1 LF / WPH mixture has the highest turbidity; as shown in Figure 4, at pH 6.2, a 3:1 LF / WPH mixture has the highest turbidity; and as shown in Figure 5, at pH 6.5, a 6:1 LF / WPH mixture has the highest turbidity. In addition, Figures 2 to 5 show that the LF / WPH complex can be most significantly formed at optimal ratios of 1:1 and 2:1 at pH 5.8 and pH 6.0, respectively.

[0120] Figures 6 to 9 show the effects of different pH conditions and different mixing ratios of LF and WPH on the average particle size distribution of LF / WPH mixed solutions, and the results are consistent with the turbidity tests in Figures 2 to 5. The size of the LF / WPH complexes increased with decreasing pH. As shown in Figure 6, the average size of the LF is approximately 10 nm, and that of the WPH is approximately 40 nm. Changes in pH do not affect the average sizes of the LF and WPH. As a result, the rapid increase in particle size (e.g., diameter) of the LF / WPH mixtures under optimal conditions (i.e., pH 5.8 at LF / WPH ratios of 1:1 and 2:1 [Figure 6], and pH 6 at LF / WPH ratios of 1:1 and 2:1 [Figure 7]) can be explained by the formation of complexes.

[0121] The particle size distributions of LF, WPH, and LF / WPH mixed solutions at pH 5.8 are further illustrated in Figures 10-15. LF and WPH showed monopeaks in the particle size ranges of 1-100 nm (Figure 10) and 10-500 nm (Figure 11), respectively. As shown in Figure 12, LF / WPH mixtures formed at pH 5.8 and a 2:1 ratio (LF:WPH) showed two peaks corresponding to free LF biopolymer (5-100 nm) and LF / WPH complexes (1000-10,000 nm). In contrast, LF / WPH mixtures at pH 5.8 and a 1:1 ratio (LF:WPH) showed almost exclusively complexes with a monopeak in the 1000-10,000 nm range (Figure 13). LF / WPH mixtures formed at pH 5.8 and a 1:2 (Figure 14) and 1:3 (Figure 15) ratios showed multiple peaks due to the excess free biopolymer in the system.

[0122] Figures 16 to 19 show the zeta potential of LF / WPH mixtures under different pH conditions and different mixing ratios of LF and WPH. The pI of LF is approximately 8.2, thus carrying a net positive charge in an acidic environment, while the pI of WPH is approximately 4.5, thus carrying a negative charge at pH levels higher than 4.5. When two polymers have opposite charges, they can form complexes through electrostatic interactions. For LF and WPH, the optimal pH range for LF / WPH complex formation is between pH 5.8 and pH 6.5. When the zeta potential of the LF / WPH mixed solution is close to zero, ratios of 1:1 and 2:1 result in the formation of a large amount of LF / WPH complex with the highest turbidity. This confirms the hypothesis that electrostatic interactions are the primary driving force for complexation between LF and WPH.

[0123] As shown in Figures 20-25, the effects of total concentration (1%-10%), mixing temperature, and time on complex formation at selected mixing ratios of 1:1 and 1:2 (LF:WPH) were further investigated. Upon mixing for 30 minutes at room temperature, the 1% mixtures, regardless of the LF:WPH ratio, exhibited significant complexation, as indicated by high turbidity (<6) and large average particle size (>6,000 nm). However, as shown in Figures 20-23, when the concentration increased to 3%, 5%, and 10%, the turbidity of the mixtures decreased (0-4), and the mixtures exhibited smaller average particle sizes (<200 nm). Without wishing to be bound by any particular theory, an increase in concentration may increase the distance between identical molecules (LF and LF, or WPH and WPH) and decrease the distance between different molecules (LF and PH), thus reducing the intermolecular interactions between LF and WPH. Such a decrease in interaction and complexation at high concentrations can be further explained by the zeta potential charge. As shown in Figures 24-25, the absolute charge of both LF and WPH decreased with increasing concentration. This indicates that the surface charge of the biopolymers decreased, which may result in a decrease in electrostatic interaction.

[0124] To further investigate whether higher mixing temperatures promote complexation, the mixed solutions were incubated in a 45°C water bath for 10, 20, and 30 minutes. The effect of incubation was minimal in the 1% sample, as large complexes had already formed without incubation. At higher concentrations, incubation at 45°C promoted more complexation, as the mixtures showed increased turbidity and particle size. Furthermore, the longer the incubation time, the more complexation was induced. Notably, after 30 minutes of incubation, the 1:1 and 1:2 (LF:WPH) mixtures at 3% concentration reached a turbidity of 6, similar to that of the 1% mixture (Figures 20 and 21). The mean particle sizes of the 1:1 and 1:2 (LF:WPH) mixtures at 3% concentration after 30 minutes of incubation were 384 nm and 2,431 nm, respectively (Figures 22 and 23). Although the 5% and 10% mixtures also showed increased turbidity and particle size after 30 minutes of mixing, such effects were less significant when compared to the 3% sample. Therefore, the complexation conditions selected were 1% with 30 minutes of mixing at room temperature and 3% with 30 minutes of mixing at 45°C. Given the high complexation and turbidity formed at 1% concentration, these samples were further studied with respect to yield, complexation efficiency, structure, and thermal stability.

[0125] 3.2 Yield and complexation of LF / WPH complex

[0126] The yield, complexation efficiency, and loading ratio of the LF / WPH complex formed at pH 5.8 were calculated (Table 1). Based on the turbidity (Figures 2–5) and particle size (Figures 6–9) measurements, freeze-dried LF / WPH complexes were prepared at a ratio that would form LF / WPH complexes at certain pH conditions after the centrifugation step. Table 1 shows that a high yield of approximately 40% could be achieved by mixing LF and WPH at a 1:1 ratio at pH 5.8 across all test conditions. Higher complexation efficiencies were observed at pH 5.8 with LF / WPH ratios of 1:1 and 1:2. This result is consistent with the turbidity and particle size measurements. Generally, the loading ratio of LF in the final freeze-dried sample corresponds to the mass ratio of LF used to prepare the mixed solution. For example, as shown in Table 1 for a 1:1 ratio, LF has a final loading ratio of 48%, which is close to the mass ratio of LF in the initial mixed sample (50%).

[0127] [Table 1]

[0128] As shown in Figures 33 and 34, optical micrographs of LF / WPH mixtures formed at a 2:1 (LF:WPH) ratio at pH 5.8 (Figure 33) and LF / WPH mixtures formed at a 1:1 (LF:WPH) ratio at pH 5.8 (Figure 34) showed that a large amount of protein-peptide complexes formed and then sedimented to the bottom of the mixture after standing overnight at 4°C, indicating an amorphous interpolymer complex structure. Optical and scanning electron micrographs of LF-WPH mixtures at pH 5.8 and different LF:WPH ratios are shown in Figure 26. Ratios of 1:1 (LF:WPH; Figure 26, panels A3 and B3) and 1:2 (LF:WPH; Figure 26, panel A4) showed more and larger complex particles compared to the other ratio conditions. Generally, the protein-peptide complexes were exhibited as amorphous interpolymer complex structures. After air-drying, the composite particles demonstrated different morphologies and sizes depending on the mixing ratio. Due to limited complexation at a 3:1 ratio, only small complexes with sizes of approximately 100–200 nm were formed (Figure 26, Panels B1 and C1). Interestingly, in the 1:1 ratio sample, the composites were classified into three shapes: loose matrix, rod-like, and cube-like (Figure 26, Panels B2 and C2). The loose matrix particles were likely uncomplexed individual free biopolymers, while the rod- and cube-like particles were composites with different densities. However, when the mixing ratio was shifted to 1:3, only small cube-like particles with sizes of 100–200 nm appeared, indicating exclusively nanocomposites (Figure 26, Panels A5, B3, and C3). At a ratio of 1:3, the negative charge may prevent further aggregation of small complexes into large complexes, as occurred at a ratio of 1:1.

[0129] Small angle X-ray scattering (SAXS) was further applied to provide molecular structure and conformational information of the individual biopolymers and the complexes (Figures 27-32). The obtained SAXS data of the LFs were comparable to previous studies (Figure 27). The pair-distance distribution functions (PDDFs) of all samples are shown in Figures 28 and 29. The individual LFs exhibited a radius of gyration of approximately 5.7 nm, which is slightly larger (R ) than that reported in other studies. g = 4.2 nm). This difference may be related to the pH conditions of the measurements. In this study, LF was measured at pH 5.8, resulting in a more open structure compared to that measured at pH 7 reported in other studies. The maximum intermolecular distance (Dmax) was 21.2 nm (Figure 29). The shape of the PDDF graph indicated that LF was not a globular structure but rather an elongated structure consisting of two globular lobes. WPH as a hydrolyzed peptide had a smaller radius of gyration and Dmax than that of LF (Figure 30). Although composed of a mixture of different peptide chains, WPH appeared to exist primarily as a globular structure. In the case of the LF-WPH complex at pH 5.8, the one formed at a 2:1 ratio showed slightly larger Rg and Dmax than the one formed at a 1:1 ratio, indicating a larger complex size (Figures 31 and 32).

[0130] 3.3 Effect of LF / WPH complex formation on the thermal stability of LF

[0131] LF is susceptible to denaturation during heat treatment, especially in a neutral pH environment where the pH is close to the pI of LF. To test whether the formation of LF / WPH complexes can improve the thermal stability of LF, both centrifuged and lyophilized LF / WPH complexes and direct lyophilized (DF) samples formed at the optimal pH (5.8) in ratios of 2:1 to 1:3 were dissolved at 0.2 w / v% in 10 mM phosphate buffer at pH 7 and then heated in a water bath at 75°C for 2 min and 90°C for 2 min. Because denatured proteins aggregate and form large particles, stability can be qualitatively indicated by an increase in both turbidity and particle size. It can also be quantitatively measured by calculating the concentration of native LF retained in the heat-treated samples using HPLC analysis.

[0132] Figure 35 shows optical images of a pure LF sample, a centrifuged and lyophilized LF / WPH complex sample, and a LF / WPH complex sample directly lyophilized without centrifugation in buffer (10 mM, pH 7) before and after heat treatment. Figure 35 shows that pure LF solutions tend to aggregate and show increased turbidity after heating. However, all LF / WPH complexes lyophilized by centrifugation remained clear solutions with low turbidity even after heating at 90°C for 2 minutes. In the case of samples directly lyophilized without centrifugation, the solutions became cloudy after heat treatment at increasing LF ratios, especially at ratios of 2:1 and 1:1. This can be expected because free, uncomplexed LF remains in the samples that are not protected from thermal aggregation in the dried samples directly lyophilized without centrifugation.

[0133] The turbidity and particle size of the unheated and heated samples were further quantitatively measured to confirm whether protein aggregation occurred during heating. As shown in Figures 36-39, pure LF solutions showed significant increases in turbidity and average particle size at both 0.1% and 0.05% concentrations. However, centrifuged and lyophilized LF / WPH complexes showed limited changes in turbidity and average particle size after heat treatment, regardless of the LF / WPH ratio, as shown in Figure 36. This indicates a significant improvement in the thermal stability of LF when LF is present in the centrifuged and lyophilized LF / WPH complexes. As shown in Figure 38, only the 2:1 ratio of non-centrifuged directly lyophilized LF / WPH samples (LF / WPH DF samples) showed a large increase in turbidity and average particle size due to the large proportion of uncomplexed LF present in the samples. However, no changes in turbidity or average particle size were detected in the other LF / WPH DF sample ratios, indicating good protection of LF during heat treatment. These results are consistent with the appearance of the LF / WPH DF sample (see Figure 35).

[0134] To further confirm and quantify the extent to which LF retained its native conformation after heat treatment, HPLC analysis was used to measure the LF concentration in the sample solutions before and after heating. Figures 40–43 show the LF loading and retention in phosphate buffer (10 mM, pH 7) before and after heat treatment for pure LF, the centrifuged and lyophilized LF / WPH complex, and the LF / WPH (DF) sample directly lyophilized without centrifugation. As expected, with a decrease in the LF mass ratio (from 2:1 to 1:3), the loading rate of LF in the complex decreased from approximately 70% to approximately 20% in both the centrifuged and lyophilized LF / WPH complex and the LF / WPH DF sample, as shown in Figures 40 and 41. The loading rates were comparable to the initial mass ratios of the prepared samples. In the case of pure LF solution, the loading of LF after 90°C / 2 min treatment significantly decreased from 25% to 2% and from 54% to 5% at concentrations of 0.1% and 0.05%, respectively.

[0135] As shown in Figures 42 and 43, the retention of pure LF was approximately 40% after 75°C / 2 min and only 8% after 90°C / 2 min. After forming the complexes with WPH, the LF retention was 85–90% after 75°C / 2 min and 50–80% after 90°C / 2 min at different LF / WPH ratios, which was significantly higher than the LF retention in pure LF solution. Overall, the centrifuged and lyophilized LF / WPH complexes showed slightly higher LF retention than the LF / WPH (DF) samples directly lyophilized without centrifugation. Samples with a 1:3 ratio showed the highest overall LF retention, but the initial LF mass ratio in the prepared samples was the lowest (20–24%). Considering both LF loading and retention, a 1:1 ratio was the optimal condition, with a relatively high LF mass ratio (35–50%) and high LF retention (60–87%) in both the unheated and heated samples.

[0136] The high LF retention after heat treatment was also confirmed by SDS-PAGE analysis. As shown in Figures 44-45, SDS-PAGE analysis was performed on unheated and heated samples (75°C / 2 min or 90°C / 2 min) of pure LF, redispersed LF / WPH complex (Figure 44), and LF / WPH direct mixture (Figure 45) in phosphate buffer (10 mM, pH 7). As shown in Figure 44, the LF in the formed LF / WPH complex showed negligible change in band density (around 75 kDa) during heat treatment, indicating good LF retention and thermal stability in the LF / WPH complex sample. However, as shown in Figure 45, the LF in the direct mixture of LF and WPH showed a fainter band, indicating poor LF retention and thermal stability.

[0137] The thermal stability of the LF, LF / WPH complex samples, and non-centrifuged LF / WPH complex samples was further tested under oil bath conditions at 145 °C for 0 to 60 seconds to mimic high-temperature, short-time (HTST) treatment. The optical images, turbidity, and particle size changes of all samples before and after oil bath heating are shown in Figures 46-50. The pure LF sample began to show protein aggregation and a cloudy appearance after 10 seconds of oil bath heating, and after 30 seconds of oil bath heating, the turbidity and particle size increased significantly. However, all LF / WPH complex samples showed limited turbidity and particle size changes even after 60 seconds of heat treatment, indicating enhanced thermal stability (Figures 46-50). In the case of non-centrifuged complex samples, those formed at 2:1 and 1:1 ratios showed increased turbidity and lower thermal stability than the 1:2 and 1:3 ratio samples (Figures 48 and 50). This is due to the higher amount of unconjugated LF in the 2:1 and 1:1 ratio samples. HPLC analysis of LF retention further confirmed that all conjugated samples, whether centrifuged or not, showed high LF retention (>80%) when treated for up to 30 seconds under oil bath heating conditions, while pure LF only retained 16.5% of the native protein (Figures 51-54).

[0138] 3.3 Antibacterial activity of LF / WPH complex

[0139] The antibacterial properties of LF were evaluated in pure and complexed LF samples before and after heat treatment. Part of this study aimed to determine whether complexation affects the functionality of LF and whether this ability is maintained after heat treatment. Antimicrobial performance was chosen as a representative biological functionality of LF because it can be easily performed in most biology laboratories and is relatively safe. Staphylococcus aureus and Escherichia coli were tested as representatives of Gram-positive and Gram-negative bacteria, respectively.

[0140] As shown in Figures 55 and 56, after 24 hours of incubation, both pure LF and the LF / WPH complex inhibited bacterial growth by half, while WPH did not significantly inhibit bacterial growth, indicating that the antibacterial effect of the LF / WPH complex was primarily due to LF, not WPH. Figures 57–60 show that after heating at 75°C for 2 minutes and 90°C for 2 minutes, pure LF exhibited similar OD625nm values ​​to the control and lost its antibacterial properties against both S. aureus and E. coli. In the case of the LF / WPH complex, the OD625nm values ​​increased slightly after 75°C for 2 minutes (Figures 57 and 58) and 90°C for 2 minutes (Figures 59 and 60) compared to the unheated LF / WPH complex. However, all of the LF / WPH complex samples still demonstrated significant antibacterial effects compared to the control, even after heating to 90°C for 2 minutes. This result indicates that the antibacterial activity of LF is fully retained even after heat treatment in the LF / WPH complex.

[0141] Example 2

[0142] Complex formation between LF and chymotrypsin-hydrolyzed whey protein isolate (WPI) was examined. Specifically, whey protein isolate (WPI) was enzymatically hydrolyzed with chymotrypsin at a concentration of 5% by weight of WPI at 50°C for 24 hours to yield a degree of hydrolysis of 15% to 20%. LF and the commercially available whey protein hydrolysate BIOZATE were used. 登録商標 Complexation with 9 (available from Agropur) was also tested. The complexes were formed as described in Example 1. The results for turbidity, zeta potential, and mean particle size of the resulting complexes at various ratios of LF:WPH are given in Figure 61 (LF-WPH hydrolyzed with chymotrypsin) and Figure 62 (LF-BIOZATE). 登録商標 9). Without wishing to be bound by any particular theory, the different complexation results between these two types of WPH may be related to the difference in zeta potential (WPH hydrolyzed with chymotrypsin has a higher zeta potential than BIOZATE). 登録商標9 (e.g., less than -10 mV)), differences in particle size (chymotrypsin-hydrolyzed WPH has an average particle size of more than 1,000 nm, while BIOZATE 登録商標 9 has an average particle size of less than 100 nm), and differences in molecular weight (BIOZATE 登録商標 The molecular weight of 9 is generally lower than that of WPH hydrolyzed with chymotrypsin (e.g., BIOZATE with >85% 登録商標 9 are less than 10 kDa, over 70% are less than 5 kDa, and over 50% are less than 2 kDa). Each of these differences may affect electrostatic interactions with lactoferrin and therefore complex formation.

[0143] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The present invention is defined by the appended claims, including equivalents of the claims. All publications, patent applications, patents, patent publications, 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 presented.

Claims

1. A complex, the complex comprising: Lactoferrin; and whey protein hydrolysate Including, wherein the lactoferrin and the whey protein hydrolysate are associated via electrostatic interactions; The complex.

2. 2. The complex of claim 1, wherein the complex has a zeta potential of from about -10, about -5, about -2, or about -1 mV to about +1, about +2, about +5, about +10, or about +15 mV, optionally wherein the complex has a zeta potential of about 0 at a pH of about 5.8 to about 6.

5.

3. 3. The complex of claim 1, wherein the complex comprises about 25 w / w% to about 75 w / w% or about 80 w / w% of the lactoferrin and about 25 w / w% to about 75 w / w% of the whey protein hydrolysate, and optionally, the complex comprises about 50 w / w% or about 65 w / w% to about 75 w / w% of the lactoferrin and about 25 w / w% to about 50 w / w% of the whey protein hydrolysate, or the complex comprises about 50 w / w% of the lactoferrin and about 50 w / w% of the whey protein hydrolysate.

4. 4. The complex of any one of claims 1 to 3, wherein the lactoferrin has a net positive charge at a pH of less than 4, less than 5, less than 6, or less than 7, optionally wherein the lactoferrin has a zeta potential of greater than about +1 mV to about +20 mV at a pH of from about 4, about 4.5, or about 5 to about 6, about 6.5, or about 7.

5. 5. The complex of claim 1, wherein the lactoferrin is bovine lactoferrin or human lactoferrin and / or the lactoferrin has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 1 and / or SEQ ID NO:

2.

6. 6. The complex of claim 1, wherein the lactoferrin is soluble in water at a pH below 8 and / or above 9.

7. The complex of any one of claims 1 to 6, wherein the lactoferrin has a pI of about pH 8.

8. 8. The complex of any one of claims 1 to 7, wherein the whey protein hydrolysate has a net negative charge at a pH of less than 5, less than 6, less than 7 or less than 8, optionally wherein the whey protein hydrolysate has a zeta potential of about -20mV to about -1mV at a pH of from about 5, about 5.5 or about 6 to about 6.5, about 7, about 7.5 or about 8.

9. 9. The complex of claim 1, wherein the whey protein hydrolysate is soluble in water at a pH above 5 and below 8.

10. 10. The complex of claim 1, wherein the whey protein hydrolysate has a degree of hydrolysis of from about 8%, about 9%, or about 10% to about 11%, about 12%, or about 13%.

11. 11. The complex of any one of claims 1 to 10, wherein the whey protein hydrolysate comprises a mixture of peptides and / or amino acids, and at least about 75%, at least about 80% or at least about 85% by weight of the mixture has a molecular weight of less than 10 kilodaltons (kDa) or less than 20 kilodaltons (kDa), optionally wherein at least about 60%, at least about 65% or at least about 70% by weight of the mixture has a molecular weight of less than 5 kDa, and / or at least about 40%, at least about 45% or at least about 50% by weight of the mixture has a molecular weight of less than 2 kDa.

12. 12. The complex of any one of claims 1 to 11, wherein the whey protein hydrolysate has a particle size (e.g. diameter) ranging from about 1 nm, about 50 nm, or about 100 nm to about 1 or 2 microns, and / or wherein the whey protein hydrolysate has a particle size (e.g. diameter) of less than 300 nm, less than 200 nm, or less than 100 nm.

13. 13. The complex of any one of claims 1 to 12, wherein the whey protein hydrolysate has a pI of about 3 to about 5, optionally wherein the whey protein hydrolysate has a pI of about pH 4.

5.

14. 14. The complex of any one of claims 1 to 13, wherein the lactoferrin and the whey protein hydrolysate are each food-grade ingredients, optionally wherein the lactoferrin and / or the whey protein hydrolysate are obtained from and / or derived from natural products (e.g., foods, plants, animal by-products (e.g., milk), etc.).

15. 15. The composite of any one of claims 1 to 14, wherein the composite is a polymer-polymer composite, optionally wherein the composite is an amorphous polymer-polymer composite structure.

16. 16. The complex of any one of claims 1 to 15, wherein the lactoferrin has increased stability (e.g., reduced degradation of the lactoferrin) after exposure to temperatures of from about 70°C to about 80°C, about 90°C, or about 100°C for about 1 minute to about 60 minutes, compared to the stability of the lactoferrin alone (i.e., not present in the complex) after exposure at the same temperature for the same period of time.

17. 17. The complex of any one of claims 1 to 16, wherein the lactoferrin is degraded by less than 50% when measured by high performance liquid chromatography after exposure to temperatures from about 70°C to about 80°C, about 90°C, or about 100°C for about 1 minute to about 60 minutes, optionally wherein the lactoferrin is degraded by less than 40% when measured by high performance liquid chromatography after exposure to temperatures from about 70°C to about 80°C, about 90°C, or about 100°C for about 1 minute to about 60 minutes.

18. 18. The complex of any one of claims 1 to 17, wherein after exposing the complex to a temperature of about 70°C to about 80°C, about 90°C or about 100°C for about 1 minute to about 60 minutes, the activity of the lactoferrin is increased compared to the activity of the lactoferrin alone after the same exposure conditions.

19. 19. The complex of any one of claims 1 to 18, wherein the lactoferrin has increased stability after exposure of the complex to a temperature of about 145°C for about 2 seconds to about 60 seconds (e.g., high temperature, short time (HTST) treatment at about 145°C for about 2 seconds to about 60 seconds under oil bath conditions) compared to the stability of the lactoferrin alone (i.e., not present in the complex) after exposure to the same conditions (e.g., the same temperature for the same time).

20. 20. The complex of any one of claims 1 to 19, wherein after exposure of the complex to a temperature of about 145°C for about 2 seconds to about 60 seconds (e.g., high temperature, short time (HTST) treatment at about 145°C for about 2 seconds to about 60 seconds under oil bath conditions), the lactoferrin is degraded by less than 30%, optionally by less than 20%, optionally as measured by high performance liquid chromatography.

21. 21. The complex of any one of claims 1 to 20, wherein the antimicrobial capacity and / or activity of the lactoferrin (e.g., the antibacterial activity of the lactoferrin against gram-positive and / or gram-negative bacteria) is maintained and / or improved (e.g., increased) after exposure to a temperature of about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C for about 30 seconds to about 2 minutes, optionally compared to the antimicrobial capacity and / or activity of the lactoferrin alone (e.g., the antibacterial activity of the lactoferrin against gram-positive and / or gram-negative bacteria) after exposure to the same conditions (e.g., the same temperature and temperature exposure time).

22. 22. The complex of any one of claims 1 to 21, wherein the bioavailability of the lactoferrin is increased after ingestion of the complex by a subject compared to the bioavailability of the lactoferrin after ingestion by a subject.

23. 23. The complex of any one of claims 1 to 22, wherein the complex, optionally in a liquid composition, has an average size (e.g., diameter) of from about 300, about 500, about 1000, or about 1500 nm to about 2000, about 2500, about 3000, about 3500, or about 4000 nm.

24. 24. The composite of any one of claims 1 to 23, wherein the composite has a loose matrix structure, a rod-like structure and / or a cube-like structure.

25. 25. The complex of any one of claims 1 to 24, wherein the complex is devoid of cationic and / or anionic biopolymers (i.e., the complex is devoid of cationic and / or anionic biopolymers that are not lactoferrin or whey protein hydrolysate).

26. A composition comprising the complex of any one of claims 1 to 25.

27. 27. The composition of claim 26, wherein the complex is in the form of particles, optionally the particles being freeze-dried particles, spray-dried particles, and / or the like.

28. 28. The composition of claim 26 or 27, wherein the composition is a plurality of particles, optionally wherein the composition is in the form of a powder (e.g., a dry powder).

29. The composition according to any one of claims 26 to 28, wherein the composition is an aqueous composition and the complex is dissolved or suspended in the composition.

30. 30. A composition according to any one of claims 26 to 29, wherein the particles are prepared and / or isolated from a solution containing less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% w / v of free lactoferrin (i.e. lactoferrin that is not associated with whey protein hydrolysate via electrostatic interactions).

31. 31. The composition of any one of claims 26 to 30, wherein the composition and / or plurality of particles comprises less than 5% by weight of free lactoferrin, optionally wherein the composition and / or plurality of particles comprises less than 1% by weight of free lactoferrin.

32. 32. The composition of any one of claims 26 to 31, wherein the physicochemical properties (e.g., turbidity and / or particle size) of the complex are maintained within about ±30% of the original physicochemical properties of the complex prior to exposure (e.g., the physicochemical properties of the complex at the time of initial formation of the complex and / or immediately prior to the exposure) after exposure of the composition to a temperature of from about 70°C or about 75°C to about 80°C, about 85°C, or about 90°C for about 30 seconds to about 2 minutes.

33. 33. The composition of any one of claims 26 to 32, wherein the physicochemical properties (e.g., turbidity and / or particle size) of the complex are maintained within about ±30% of the original physicochemical properties of the complex prior to exposure (e.g., the physicochemical properties of the complex at the time of initial formation of the complex and / or immediately prior to the exposure) after exposure of the composition to a temperature of about 145°C for about 2 seconds to about 60 seconds (e.g., high temperature, short time (HTST) treatment at about 145°C for about 2 seconds to about 60 seconds under oil bath conditions).

34. 1. A method for preparing a complex, comprising: providing a composition comprising lactoferrin and whey protein hydrolysate at a pH ranging from about 5, about 5.5, or about 5.8 to about 6, about 6.5, or about 7; and mixing said compositions, thereby providing said composite; The method comprising:

35. 35. The method of claim 34, wherein the lactoferrin and the whey protein hydrolysate are each independently present in the composition in an amount of from about 0.01%, 0.1%, 0.5%, or 1% to about 2% or about 3% by weight of the composition.

36. 36. The method of claim 34 or 35, wherein the composition comprises the lactoferrin and the whey protein hydrolysate in a weight ratio of about 10:1 to about 1:10 (lactoferrin:whey protein hydrolysate), optionally wherein the composition comprises the lactoferrin and the whey protein hydrolysate in a weight ratio of about 1:1 or about 2:1 (lactoferrin:whey protein hydrolysate).

37. 37. The method of any one of claims 34 to 36, wherein mixing the composition is carried out for about 1 minute, about 5 minutes, about 10 minutes, or about 15 minutes to about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes, optionally at a temperature of about 20°C to about 60°C.

38. 38. The method of any one of claims 34 to 37, further comprising separating the complex from the composition, optionally wherein separating the complex from the composition comprises centrifuging, drying, freeze-drying and / or spray-drying the composition.

39. 39. The method of any one of claims 34 to 38, wherein the combined concentration of the lactoferrin and the whey protein hydrolysate in the composition is about 6% or less by weight of the composition (e.g., about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% by weight of the composition).

40. An article comprising a composite according to any one of claims 1 to 25, a composition according to any one of claims 26 to 33, and / or a composite prepared according to a method according to any one of claims 34 to 39.

41. 41. The article of claim 40, wherein the article is a food product (e.g., infant formula, dairy product, etc.), a nutritional supplement, a therapeutic beverage, and / or a cosmetic.

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