Complex of protein and biopolymer, and method for producing and using the same

Abstract

A complex comprising a protein, an anionic biopolymer, and a cationic biopolymer, as well as methods for producing and using such a complex are described herein. The complex may include lactoferrin, gelatin, and a polysaccharide.

Description

Technical Field

[0001] Statement Regarding the Electronic Submission of Array Lists The array list in XML text format entitled 1213-3WO_ST26.xml (size 7,184 bytes, generated on April 25, 2023, and submitted together with this specification) is hereby incorporated by reference in its entirety into this specification.

[0002] The present invention relates to a complex comprising a protein, an anionic biopolymer, and a cationic biopolymer, and to methods for producing and using such complexes.

Background Art

[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, antiviral properties, anti-inflammatory properties, and anticancer properties. LF promotes cell growth, detoxifies harmful free radicals, and has antibacterial, antiviral, anti-inflammatory, and anticancer properties. Due to the multiple biological functions of LF, LF is incorporated into many commercial products, such as products including infant formula, nutritional supplements, therapeutic beverages, and cosmetics. However, LF is sensitive to denaturation induced by heat treatment, particularly heat treatment under neutral pH conditions, which causes structural changes and loss of biological functionality.

Summary of the Invention

Means for Solving the Problems

[0004] A first aspect of the present invention is a complex, the complex comprising a protein; an anionic biopolymer; and a cationic biopolymer, wherein the protein, anionic biopolymer, and cationic biopolymer are associated via electrostatic interactions and are directed to the above complex.

[0005] The second aspect of the present invention is directed to a composition comprising the complex of the present invention. In some embodiments, the composition is an aqueous composition.

[0006] A further aspect of the present invention is a method for preparing a complex, the method comprising preparing a composition comprising a protein, an anionic biopolymer, and a cationic biopolymer at a pH in the range of from about 3, about 3.5 or about 4 to about 4.5 or about 5; and mixing the composition to thereby provide the complex.

[0007] A further aspect of the present invention is directed to an article comprising the complex of the present invention and / or the composition of the present invention. In some embodiments, the article is a food (e.g., infant formula, dairy products, etc.), a dietary supplement, a therapeutic beverage, and / or a cosmetic.

[0008] It should be noted that the aspects described with respect to a particular embodiment may be incorporated within different embodiments even though not particularly described in relation thereto. That is, all embodiments and / or features of any embodiment can be combined in any manner and / or combination. The applicant reserves the right to amend the originally filed claims and / or to file new claims accordingly, which includes the right to amend the originally filed claims to depend on and / or incorporate features of any other claim or claims, even though 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 understood by those skilled in the art upon reading the accompanying drawings and the detailed description of the preferred embodiments that follow, which description is merely illustrative of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

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Mode for Carrying Out the Invention

[0010] Here, the present invention is described in the following specification with reference to the accompanying drawings and examples in which embodiments of the present invention are shown. This description is not intended to be a catalog of all different ways in which the present invention can be implemented or of all the features that can 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 deleted from that embodiment. Accordingly, the present invention contemplates that, in some embodiments of the present invention, any one feature or combination of features described herein may be excluded or omitted. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those of ordinary skill in the art in light of the present disclosure, but they do not depart from the present invention. Accordingly, the following description is intended to illustrate some particular embodiments of the present invention and is not intended to specifically identify all permutations, combinations, and variations thereof.

[0011] 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 limit the present invention.

[0012] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the passages and / or paragraphs to which the reference is made.

[0013] Unless the context otherwise indicates, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. By way of example, when the specification states that a composition contains components A, B, and C, it is specifically intended that any one of A, B, or C, or combinations thereof, can be omitted and excluded.

[0014] As used in the detailed description of the invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0015] Also, as used herein, "and / or" shall be interpreted to include any and all possible combinations of one or more of the recited related items, as well as the absence of combinations when interpreted in the alternative ("or").

[0016] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, etc., is meant to include not only the specified value but also variations of ±10%, ±5%, ±1%, ±0.5%, and even ±0.1% of the specified value. For example, "about X" means X and variations of ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of X when X is a measurable value. The ranges of measurable values provided herein can include any other range and / or individual values within that range.

[0017] 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 - Y" mean "about X - about Y".

[0018] The recitation of a range of values herein is, unless otherwise indicated herein, intended to be merely a shorthand way of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0019] As used herein, the terms "comprises" and "comprising" identify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0020] As used herein, the transitional phrase "consisting essentially of" when applied to the compositions of the present invention is to be construed to mean that the claim scope is to include those that do not materially affect the basic and novel one or more features of the claimed invention by the specific materials or steps recited in the claim. Accordingly, it is not intended that the term "consisting essentially of" as used in the claims of the present invention should be construed as equivalent to "comprising".

[0021] As used herein, the terms "increase", "increasing", "enhance", "enhancing", "improve" and "improving" (and their grammatical variations) mean 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 increase as compared to another measurable property or quantity (e.g., a control value).

[0022] As used herein, the terms "reduce", "reduced", "reducing", "decrease" and "decreasing" (and their grammatical variations) mean, for example, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100% decrease as compared to another measurable property or quantity (e.g., a control value). In some embodiments, the decrease can result in no detectable activity or amount, or essentially none at all (i.e., a negligible amount, e.g., less than about 10% or even less than 5%).

[0023] 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., a reduction of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues) relative to each of a reference nucleotide sequence or a reference polypeptide, which nucleotide sequence or polypeptide comprises, consists essentially of, and / or consists of contiguous residues that are identical or substantially 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) to the reference nucleotide sequence or reference polypeptide, respectively.

[0024] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant through a window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods including, but not limited to, the methods described in the following references: Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0025] As used herein, the term "percent sequence identity" or the term "percent identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule compared to a test ("subject") polynucleotide molecule (or its complementary 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 when compared to a reference polypeptide.

[0026] As used herein, in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, the phrase "substantially identical" or the phrase "substantial identity" refers to at least about 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% nucleotide or amino acid residue identity, as measured using one of the following sequence comparison algorithms or by visual inspection, when compared and aligned to obtain maximal correspondence. In some embodiments of the invention, the substantial identity exists over a region of contiguous nucleotides of the nucleotide sequences of the invention, which is in the length of about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides, or more nucleotides, and any range thereof up to the full length of the sequence. In some embodiments, the nucleotide sequence can be substantially identical over at least about 20 nucleotides (e.g., about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40 nucleotides).In some embodiments, substantially identical nucleotide or protein sequences perform substantially the same function as the nucleotides (or encoded protein sequences) to which they are substantially identical.

[0027] In the case of sequence comparison, typically, one sequence functions as a reference sequence and is compared to a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and optionally, subsequence coordinates are specified and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity for one or more test sequences relative to the reference sequence based on the specified program parameters.

[0028] Optimal alignment of sequences for aligning a comparison window is well known to those of skill 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 these algorithms, such as GCG 登録商標 Wisconsin Package 登録商標It may be carried out by computerized implementations of GAP, BESTFIT, FASTA, and TFASTA, which are available as part of (Accelrys Inc., San Diego, CA). The "identity fraction" of an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (e.g., the whole of the reference sequence or a smaller defined part of the reference sequence). The percent sequence identity is expressed as the identity fraction multiplied by 100. Comparison of one or more polynucleotide sequences can be done against the full-length polynucleotide sequence or a portion thereof, or against a longer polynucleotide sequence. For purposes of the present invention, the "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0029] Complexes comprising a protein, an anionic biopolymer, and a cationic biopolymer are provided in accordance with embodiments of the present invention. The complexes of the present invention can be ternary complexes in that the complexes comprise three different components, e.g., a protein, an anionic biopolymer, and a cationic biopolymer, each different from one another (e.g., different in chemical structure). In some embodiments, the complexes of the present invention comprise at least three different components, e.g., a protein, an anionic biopolymer, and a cationic biopolymer, each different from one another (e.g., different in chemical structure).

[0030] The complex of the present invention may comprise one or more proteins, one or more anionic biopolymers and one or more cationic biopolymers, which may associate with each other via electrostatic interactions. The complex of the present invention may optionally have a zeta potential of from about -5 mV, about -4 mV, about -3 mV, about -2 mV, about -1 mV or 0 mV to about +1 mV, about +2 mV, about +3 mV, about +4 mV or about +5 mV when present in a composition (e.g., water and / or buffer) having a pH of from about 3, about 3.5 or about 4 to about 4.5, about 5, about 5.5, about 6, about 6.5, about 7 or about 7.5. In some embodiments, the complex of the present invention may optionally have a zeta potential of from about -5 mV, about -4 mV, about -3 mV, about -2 mV, about -1, about 0, about +1, about +2, about +3, about +4 or +5 mV when present in a composition (e.g., water and / or buffer) having a pH of from about 3, about 3.5 or about 4 to about 4.5, about 5, about 5.5, about 6, about 6.5, about 7 or about 7.5. In some embodiments, the complex of the present invention may optionally have a zeta potential of about 0 mV when present in a composition (e.g., water and / or buffer) having a pH of from about 3, about 3.5 or about 4 to about 4.5, about 5, about 5.5, about 6, about 6.5, about 7 or about 7.5. The complex of the present invention may have a net negative charge at a pH of from about 6.5 to about 7.5, and may optionally have a net negative charge at a pH of about 6.5, about 7 or about 7.5.

[0031] The complex of the present invention may contain a protein, an anionic biopolymer and / or a cationic biopolymer 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% or about 85 w / w% of the complex, or more than these. In some embodiments, the protein, anionic biopolymer and / or cationic biopolymer are present in the complex of the present invention 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%, or about 85 w / w% of the complex. In some embodiments, the complex of the present invention contains a protein in an amount of about 30 w / w%, about 35 w / w% or about 40% 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%, about 75 w / w%, about 80 w / w% or about 85 w / w%, a cationic biopolymer in an amount of about 1 w / w%, about 5 w / w% or about 10% to about 15 w / w%, about 20 w / w%, about 25 w / w%, about 30 w / w%, about 35 w / w% or about 40 w / w%, and an anionic biopolymer in an amount of about 1 w / w%, about 5 w / w% or about 10% to about 15 w / w%, about 20 w / w%, about 25 w / w%, about 30 w / w%, about 35 w / w% or about 40 w / w%. In some embodiments, the complex contains a protein in an amount of about 40 w / w% to about 75 w / w%, a cationic biopolymer in an amount of about 10 w / w% to about 30 w / w%, and an anionic biopolymer in an amount of about 10 w / w% to about 30 w / w%.

[0032] One or more (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50, or more) proteins can be present in the complex of the present invention. In some embodiments, the complex of the present invention may contain two or more proteins that may be identical to or different from each other. In some embodiments, the complex of the present invention contains one or more protein molecules (e.g., individual proteins and / or protein monomers) that are identical. The proteins present in the complex of the present invention and / or used to prepare the complex of the present invention can optionally have a net positive charge at a pH of about 3, about 3.5, or about 4 to about 4.5, about 5, about 6, about 7, or about 8. In some embodiments, when the proteins present in the complex of the present invention and / or used to prepare the complex of the present invention are present in a composition (e.g., water and / or buffer) having a pH of about 3, about 3.5, or about 4 to about 4.5 or about 5, they can have a zeta potential of greater than about +10 mV, e.g., about +11, about +12, about +13, about +14, about +15, about +16, about +17, about +18, about +19, about +20, about +21, about +22, about +23, about +24, about +25, about +26, about +27, about +28, about +29, or about +30 mV, or greater.

[0033] The proteins present in the complex of the present invention and / or used to prepare the complex of the present invention may have a globular structure. In some embodiments, the proteins present in the complex of the present invention and / or used to prepare the complex of the present invention are soluble in water at a pH of less than about 8, such as 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 or about 8, optionally at a solubility of 5 mg / L or more, 10 mg / L or more, 15 mg / L or more or 20 mg / L or more in water at a pH of less than about 8 and at about 25°C. In some embodiments, the proteins present in the complex of the present invention and / or used to prepare the complex of the present invention are dairy proteins. As used herein, "dairy protein" refers to a protein naturally found in a dairy product and / or milk, and / or a protein derived from such a naturally occurring protein and 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 to have an amino acid sequence with at least 70% sequence identity to the amino acid sequence of the naturally occurring protein.

[0034] Exemplary proteins that may be present in the complex of the present invention and / or may be used to prepare the complex of the present invention include, but are not limited to, lactoferrin, alpha-lactalbumin, lysozyme, and / or osteopontin. In some embodiments, the complex of the present invention comprises lactoferrin. The protein of the present invention may be derived from any source (e.g., plants, animals, etc.). In some embodiments, the protein is obtained from and / or derived from animals, such as mammals (e.g., cows, goats, sheep, or humans). In some embodiments, the protein present in the complex of the present invention has an amino acid sequence having about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to one or more of SEQ ID NOs: 1-5. In some embodiments, the protein present in the complex of the present invention has an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to one or more of SEQ ID NOs: 1-5. In some embodiments, the protein present in the complex of the present invention has an amino acid sequence having about 100% sequence identity to one or more of SEQ ID NOs: 1-5.

[0035] One or more (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50, or more) cationic biopolymers may be present in the complexes of the present invention. In some embodiments, the complexes of the present invention may contain two or more cationic biopolymers that may be the same as or different from each other. In some embodiments, the complexes of the present invention contain one or more identical cationic biopolymer molecules (e.g., individual biopolymer compounds). The cationic biopolymers present in and / or used to prepare the complexes of the present invention may optionally have a net positive charge at a pH of about 3, about 3.5 or about 4 to about 4.5, about 5, about 6, about 7 or about 8. In some embodiments, the cationic biopolymers present in and / or used to prepare the complexes of the present invention have a zeta potential of greater than about +10 mV, e.g., about +11, about +12, about +13, about +14, about +15, about +16, about +17, about +18, about +19, about +20, about +21, about +22, about +23, about +24, about +25, about +26, about +27, about +28, about +29 or about +30 mV, or more, when present in a composition (e.g., water and / or buffer) having a pH of about 3, about 3.5 or about 4 to about 4.5 or about 5. The cationic biopolymers present in and / or used to prepare the complexes of the present invention may have a pI and / or pKa of about 7 or greater, e.g., about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, or greater.

[0036] As used herein, "cationic biopolymer" refers to a polymer that carries or can carry a positive charge, a polymer produced by a living organism or a derivative thereof, and / or a polymer synthetically prepared to have a structure that matches a polymer produced by a living organism or a derivative thereof. In some embodiments, the cationic biopolymer has at least one free amine and / or hydroxyl group present on a majority of the monomer units of the polymer. In some embodiments, the free amine and / or hydroxyl group may be present on each of the monomer units of the polymer backbone. Exemplary cationic biopolymers include, but are not limited to, proteins, polyamino acids, and / or polysaccharides that can contain a positive charge (optionally having a net positive charge). As will be understood by those skilled in the art, the cationic biopolymer may be obtained synthetically (e.g., through laboratory synthesis) and / or obtained from nature (e.g., from a living organism or a previously living organism) and / or derived therefrom. Thus, the cationic biopolymer may be the same as a polymer found in nature (i.e., a natural cationic biopolymer) or a derivative thereof. For example, the cationic biopolymer of the present invention may be a derivative of a polymer produced by a living organism, where the derivative is brought about by synthetic methods used to obtain or isolate the biopolymer from nature. In some embodiments, the cationic biopolymer can be a polymer produced by bacteria and / or microorganisms. In some embodiments, exemplary cationic biopolymers that can be present in the complex of the present invention and / or used to prepare the complex of the present invention include, but are not limited to, gelatin, chitosan, lysozyme, and / or polyamino acids. In some embodiments, the complex of the present invention contains gelatin. The cationic biopolymer may be a biopolymer naturally found in animals, plants, and / or fungi, and / or derived from such naturally occurring biopolymers.In some embodiments, the cationic biopolymer is a biopolymer naturally found in animals, plants, and / or fungi and is isolated therefrom. In some embodiments, the cationic biopolymer is synthetically prepared based on biopolymers naturally found in animals, plants, and / or fungi. In some embodiments, the cationic biopolymer is obtained from a source (e.g., animals, plants, and / or fungi) and / or synthetically prepared based on natural biopolymers, and the obtained and / or prepared biopolymer is modified (e.g., modified to have cationic functional groups, etc.). The cationic biopolymer of the present invention can be derived from any source (e.g., plants, animals, etc.). In some embodiments, the cationic biopolymer is obtained from and / or derived from animals, such as mammals (e.g., cows, goats, sheep, or humans).

[0037] One or more (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50, or more) anionic biopolymers may be present in the complexes of the present invention. In some embodiments, the complexes of the present invention may contain two or more anionic biopolymers that may be the same as or different from each other. In some embodiments, the complexes of the present invention contain one or more anionic biopolymer molecules (e.g., individual biopolymer compounds) that are the same. The anionic biopolymers present in and / or used to prepare the complexes of the present invention may optionally have a net negative charge at a pH of about 3, about 3.5, or about 4 to about 4.5, about 5, about 6, about 7, or about 8. In some embodiments, the anionic biopolymers present in and / or used to prepare the complexes of the present invention, when present in a composition (e.g., water and / or buffer) having a pH of about 3, about 3.5, or about 4 to about 4.5 or about 5, may have a zeta potential of less than about -10 mV, e.g., 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, about -20 mV, about -21 mV, about -22 mV, about -23 mV, about -24, -25 mV, about -26 mV, about -27 mV, about -28 mV, about -29 mV, about -30 mV, about -31 mV, about -32 mV, about -33 mV, about -34 mV, about -35 mV, about -36 mV, about -37 mV, about -38 mV, about -39 mV, about -40 mV, about -41 mV, about -42 mV, about -43 mV, about -44 mV, about -45 mV, about -46 mV, about -47 mV, about -48 mV, about -49 mV, about -50 mV, about -51 mV, about -52 mV, about -53 mV, about -54, or about -55 mV, or lower. In some embodiments, the anionic biopolymers present in and / or used to prepare the complexes of the present invention, when present in a composition (e.g., water and / or buffer) having a pH of about 3, about 3.5, or about 4 to about 4.5 or about 5, may have a zeta potential in the range of about -10 mV to about -25 mV.In some embodiments, the anionic biopolymer present in and / or used to prepare the complex of the present invention has a zeta potential in the range of about -35 mV to about -55 mV when present in a composition (e.g., water and / or buffer) optionally having a pH of about 3, about 3.5 or about 4 to about 4.5 or about 5. The anionic biopolymer present in and / or used to prepare the complex of the present invention can have a pKa of about 4 or less, for example, about 4, about 3.5, about 3, about 2.5, about 2, about 1.5 or about 1, or lower than them.

[0038] As used herein, "anionic biopolymer" refers to a polymer that bears or can bear a negative charge, a polymer produced by a living organism or a derivative thereof, and / or a polymer synthetically prepared to have a structure that coincides with a polymer produced by a living organism or a derivative thereof. In some embodiments, the anionic biopolymer has at least one free amine and / or hydroxyl group present on a majority of the monomer units of the polymer. In some embodiments, the free amine and / or hydroxyl group may be present on each of the monomer units of the polymer backbone. Exemplary anionic biopolymers include, but are not limited to, proteins, polyamino acids, glycosaminoglycans, glycoproteins, and / or polysaccharides, which can contain a negative charge (optionally having a net negative charge). As will be understood by those skilled in the art, anionic biopolymers may be obtained synthetically (e.g., through laboratory synthesis), and / or obtained from nature (e.g., from living organisms or previously living organisms) and / or derived therefrom. Thus, anionic biopolymers may be the same as polymers found in nature (i.e., natural anionic biopolymers) or derivatives thereof. For example, the anionic biopolymers of the present invention may be derivatives of polymers produced by living organisms, where the derivatives are brought about by synthetic methods used to obtain or isolate the biopolymers from nature. In some embodiments, the anionic biopolymer can be a polymer produced by bacteria and / or microorganisms. In some embodiments, exemplary anionic biopolymers that can be present in the complexes of the present invention and / or used to prepare the complexes of the present invention include, but are not limited to, polysaccharides, glycosaminoglycans, glycoproteins, and / or polyamino acids. In some embodiments, the complexes of the present invention contain polysaccharides.In some embodiments, the complex of the present invention comprises a linear polysaccharide (i.e., a polysaccharide that is a linear chain of linked / attached monosaccharides, optionally where each of the monosaccharides is linked by an α-1,4-glycosidic bond or a β-1,4-glycosidic bond). In some embodiments, the complex of the present invention comprises a branched polysaccharide (e.g., a polysaccharide comprising two or more monosaccharides linked by α-1,4-glycosidic bonds and two or more monosaccharides linked by α-1,6-glycosidic bonds). The anionic biopolymer may be a biopolymer naturally found in animals, plants, and / or fungi, and / or may be derived from such naturally occurring biopolymers. In some embodiments, the anionic biopolymer is a biopolymer naturally found in animals, plants, and / or fungi and is isolated from them. In some embodiments, the anionic biopolymer is synthetically prepared based on biopolymers naturally found in animals, plants, and / or fungi. In some embodiments, the anionic biopolymer is obtained from a source (e.g., animals, plants, and / or fungi) and / or is synthetically prepared based on natural biopolymers, and the obtained and / or prepared biopolymer is modified (e.g., modified to have an anionic functional group, etc.). The anionic biopolymer of the present invention can be derived from any source (e.g., plants, animals, etc.). In some embodiments, the anionic biopolymer is obtained from and / or derived from animals, such as mammals (e.g., cows, goats, sheep, or humans).

[0039] Further exemplary anionic biopolymers present in and / or used to prepare the complexes of the present invention include, but are not limited to, gum arabic, high methyl pectin (HMP) (e.g., HMP having an esterification degree of more than about 50%), kappa-carrageenan, iota-carrageenan, dextran sulfate, sodium hyaluronate, gum acacia, xanthan gum, gellan gum, and / or plant soluble polysaccharides (e.g., soybean soluble polysaccharide and / or lupinus soluble polysaccharide). In some embodiments, further exemplary anionic biopolymers present in and / or used to prepare the complexes of the present invention include gum arabic, gum acacia, dextran sulfate, sodium hyaluronate, and / or plant soluble polysaccharides (e.g., soybean soluble polysaccharide and / or lupinus soluble polysaccharide). In some embodiments, further exemplary anionic biopolymers present in and / or used to prepare the complexes of the present invention include HMP (e.g., HMP having an esterification degree of more than about 50%), kappa-carrageenan, iota-carrageenan, xanthan gum, and / or gellan gum. In some embodiments, the anionic biopolymer (e.g., polysaccharide) present in and / or used to prepare the complexes of the present invention has a molecular weight (e.g., average molecular weight) in the range of about 50 kDa, about 100 kDa, or about 150 kDa to about 200 kDa, about 250 kDa, or about 300 kDa. In some embodiments, the anionic polymer has a molecular weight (e.g., average molecular weight) of about 50 kDa, about 100 kDa, about 150 kDa, about 200 kDa, about 250 kDa, or about 300 kDa.

[0040] In some embodiments, the proteins, cationic biopolymers, and / or anionic biopolymers present in and / or used to prepare the complexes of the present invention are food-grade components. As used herein, "food-grade component" refers to a component (e.g., a compound, ingredient, biopolymer, etc.) that is safe for ingestion by animals (e.g., humans) and / or is intended to be ingested by animals (e.g., humans). In some embodiments, the proteins, cationic biopolymers, and anionic biopolymers of the present invention are each different food-grade components present in the complexes of the present invention. In some embodiments, the proteins, cationic biopolymers, and anionic biopolymers 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.).

[0041] In some embodiments, the complexes of the present invention comprise lactoferrin, a polysaccharide (e.g., a branched or linear polysaccharide), and gelatin. In some embodiments, the complexes of the present invention comprise lactoferrin, gum arabic, and gelatin.

[0042] The complexes of the present invention can optionally be dried by lyophilizing and / or spray-drying a composition (e.g., an aqueous composition) comprising the complexes. In some embodiments, the dried complexes contain from about 0 wt% to about 5 wt% water, based on the weight of the dried complexes. In some embodiments, the dried complexes 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.

[0043] In some embodiments, the complex of the present invention is a complex coacervate in a liquid (e.g., water and / or a buffer, such as phosphate buffered saline). As used herein, "complex coacervate" refers to droplets formed by associative liquid-liquid phase separation in a mixture of polyvalent, oppositely charged molecules (e.g., oppositely charged biopolymers). The coacervate complex can be dried and / or cured to form a solid phase. In some embodiments, the complex of the present invention is a multiphase coacervate in a liquid in that the complex coacervate has two or more (e.g., 2, 3, 4, or more) phases. In some embodiments, the complex of the present invention is a multiphase coacervate in a liquid, and the complex has two phases (e.g., an inner phase and an outer phase). In some embodiments, the complex of the present invention has a coacervate-in-coacervate structure in a liquid, and the complex comprises an inner coacervate and an outer coacervate. The inner coacervate may contain a protein (e.g., lactoferrin) and an anionic biopolymer (e.g., a polysaccharide), and / or the outer coacervate may contain an anionic biopolymer (e.g., a polysaccharide) and a cationic biopolymer (e.g., gelatin). In some embodiments, the complex of the present invention is not a binary coacervate complex that is a complex coacervate formed by only two different molecules (e.g., two different biopolymers). In some embodiments, the complex of the present invention is an interpolymeric complex.As used herein, "interpolymer complex" refers to a coprecipitate or aggregate comprising a protein, a cationic biopolymer, and an anionic biopolymer, which is formed via electrostatic interactions. In some embodiments, the anionic biopolymer and / or the cationic biopolymer encapsulates the protein in the complex of the present invention.

[0044] The complex of the present invention may be a particle. In some embodiments, the complex is a nanoparticle. In some embodiments, the complex is a microparticle. The complex of the present invention has a size (e.g., diameter) in at least one dimension of from about 50 nm, about 75 nm, about 100 nm or about 125 nm to 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, about 2000 nm, about 3000 nm, about 4000 nm, about 5000 nm or about 6000 nm or larger, and optionally is measured using microscopy (e.g., optical microscopy, confocal microscopy, scanning electron microscopy (SEM) and / or transmission electron microscopy (TEM)) and / or dynamic light scattering (DLS). In some embodiments, the particle has a size (e.g., diameter) in at least one dimension of from 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, about 2000 nm, about 3000 nm, about 4000 nm, about 5000 nm or about 6000 nm or larger. In some embodiments, the complex of the present invention in a liquid composition (e.g., an aqueous composition) has an average size (e.g., diameter) of from about 50 nm or about 100 nm to about 150 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 2000 nm, about 3000 nm, about 4000 nm, about 5000 nm or about 6000 nm or larger.In some embodiments, the plurality of complexes of the present invention, the complexes prepared according to the method of the present invention, and / or the complexes present in the compositions of the present invention, when optionally measured using microscopy (e.g., SEM and / or TEM) and / or DLS, have a Dv(50) 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, about 2000 nm, about 3000 nm, about 4000 nm, about 5000 or about 6000 nm, or greater than that.

[0045] In some embodiments, the complex of the present invention contains an active ingredient. The active ingredient may be present within the complex (e.g., may be encapsulated and / or encapsulated within the complex). In some embodiments, the active ingredient can be bound (e.g., covalently and / or non-covalently) to the proteins, anionic biopolymers, and / or cationic biopolymers 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.

[0046] Compared to the preservation, stability, activity, and / or function of the biopolymer alone (i.e., the biopolymer not present in the complex of the present invention), the complex of the present invention can have improved (e.g., increased) preservation, stability (e.g., thermal stability), activity (e.g., antiviral activity and / or antibacterial activity), and / or function with respect to the biopolymers (e.g., proteins, cationic biopolymers, and / or anionic biopolymers) present in the complex of the present invention. In some embodiments, compared to the stability of the biopolymer alone, the complex of the present invention provides increased stability to the biopolymers (e.g., proteins) present in the complex of the present invention.

[0047] In some embodiments, depending on storage at about 4°C to about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months in a sealed container, the solubility of the complex of the present invention in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acidic whey beverage) remains within about 30% of its original solubility before storage (e.g., the solubility of the complex in the composition at the initial formation of the complex and / or on the first day of storage). In some embodiments, depending on storage at about 4°C to about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months in a sealed container and optionally in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acidic whey beverage), the amount of biopolymer present in the complex of the present invention, e.g., protein, cationic biopolymer and / or anionic biopolymer), remains within about 30% compared to the amount of the biopolymer present in the complex before storage (e.g., the amount of the biopolymer present in the complex at the initial formation of the complex and / or on the first day of storage), when optionally measured by chromatography (e.g., high performance liquid chromatography), assay (e.g., ELISA), and / or spectroscopy (e.g., circular dichroism and / or UV-vis).In some embodiments, in the complex of the present invention and optionally in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acid whey beverage), the activity (e.g., bioactivity, antiviral activity and / or antibacterial activity) and / or function of a biopolymer (e.g., protein, cationic biopolymer and / or anionic biopolymer) remains within about 30% of the activity (e.g., bioactivity) and / or function (e.g., the activity and / or function of the biopolymer present in the complex at the initial formation of the complex and / or on the first day of storage) of the biopolymer present in the complex before storage, depending on storage at about 4°C to about 25°C in a sealed container for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months. In some embodiments, depending on storage at about 4°C to about 25°C in a sealed container and optionally in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acid whey beverage) for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months, the original physicochemical properties of the complex before storage (e.g., the physicochemical properties of the complex at the initial formation of the complex and / or on the first day of storage) remain within about 30% thereof.

[0048] In some embodiments, the antimicrobial ability and / or antimicrobial activity (e.g., the antibacterial activity against Gram-positive bacteria and / or Gram-negative bacteria, and / or the antiviral activity) of the biopolymer (e.g., protein, cationic biopolymer, and / or anionic biopolymer) present in the complex of the present invention remains at about 30% and / or within about 30% of the antimicrobial ability and / or antimicrobial activity of the biopolymer before storage (e.g., the antibacterial activity and / or antiviral activity of the biopolymer present in the complex at the initial formation of the complex and / or on the first day of storage) depending on storage at about 4°C to about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months in a sealed container and optionally in a composition (e.g., water, buffer, milk (e.g., skim milk), and / or acidic whey beverage).

[0049] In some embodiments, the complex of the present invention is present in a composition (e.g., water, buffer, milk (e.g., skim milk), and / or acidic whey beverage) stored at about 4°C to about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months in a sealed container, and optionally, the solubility of the complex in the composition, the retention of the biopolymer (e.g., protein, cationic biopolymer, and / or anionic biopolymer) in the complex, and / or the activity (e.g., bioactivity, e.g., antibacterial activity and / or antiviral activity) and / or function of the biopolymer (e.g., protein, cationic biopolymer, and / or anionic biopolymer) present in the complex are measured at the end of the storage period.

[0050] In some embodiments, depending on storing for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months in a sealed container and optionally in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acidic whey beverage) at about 4°C to about 25°C, the size (e.g., diameter) in at least one dimension of the complex (e.g., particle) of the present invention remains within ± about 20% of its original size (the size at the initial formation of the complex and / or the size on the first day of storage). For example, at an initial time point (e.g., the start of the first day of the storage period), the complex (e.g., particle) can have a diameter of about 25 nm to about 6000 nm, and after storing for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months starting from the first day of the storage period in a sealed container at about 4°C to about 25°C, the complex can have a size increased or decreased by about 20% or less. In some embodiments, depending on storing for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months in a sealed container at about 4°C to about 25°C, the complex (e.g., particle) of the present invention has a size (e.g., diameter) in at least one dimension that has increased by less than about 20% compared to its original size. In some embodiments, the dried complex (e.g., particle) of the present invention (e.g., lyophilized particle, and / or spray-dried particle, and / or particle containing about 0 wt% to about 5 wt% water of the dried particle) is stored for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months in a sealed container at about 4°C to about 25°C, and optionally, at the end of the storage period, the size (e.g., diameter) of the dried complex is measured, and / or the dried complex is reconstituted (e.g., dissolved and / or dispersed in the composition) in a composition (e.g., water and / or buffer), and the size (e.g., diameter) of the complex in the composition is measured.In some embodiments, the complex (e.g., particle) of the present invention is present in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acidic whey beverage), and is stored in a sealed container at about 4°C to about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months, and optionally, the size (e.g., diameter) of the complex in the composition is measured at the end of the storage period.

[0051] In some embodiments, the complex of the present invention provides increased stability to the protein present in the complex after exposure at a temperature in the range of from 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, 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, as compared to the stability of the protein alone (i.e., the protein not present in the complex of the present invention) after exposure under the same conditions (e.g., the same period, the same temperature). In some embodiments, the complex can be present in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acid whey beverage) and can be exposed to the temperature. In some embodiments, the increased stability for the protein is determined and / or demonstrated by a decreased degradation of the protein in the complex as compared to the degradation of the protein alone. In some embodiments, after exposure at a temperature in the range of from about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes or 10 minutes to about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 60 minutes, 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, the protein present in the complex of the present invention is optionally degraded by less than about 30%, e.g., about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, or lower, as measured by chromatography (e.g., high performance liquid chromatography), assay (e.g., ELISA) and / or spectroscopy (e.g., circular dichroism).In some embodiments, after exposure to a temperature in the range of 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, 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 (e.g., about 2 seconds to about 60 seconds, at about 145°C, high-temperature short time (HTST) treatment or ultra-high temperature (UHT) treatment), the protein present in the complex of the present invention is decomposed by less than about 30%, such as about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, only or lower when optionally measured by chromatography (e.g., high performance liquid chromatography), assay (e.g., ELISA), and / or spectroscopy (e.g., circular dichroism). In some embodiments, after exposure to a temperature in the range of 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, 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 (e.g., about 2 seconds to about 60 seconds, at about 145°C, high-temperature short time (HTST) treatment or ultra-high temperature (UHT) treatment), the protein present in the complex of the present invention is decomposed by less than about 20%, such as about 15%, about 10%, about 5%, about 1%, only or lower than them when optionally measured by chromatography (e.g., high performance liquid chromatography), assay (e.g., ELISA), and / or spectroscopy (e.g., circular dichroism).

[0052] In some embodiments, the antimicrobial ability and / or antimicrobial activity (e.g., the antibacterial activity against Gram-positive bacteria and / or Gram-negative bacteria, and / or the antiviral activity) of the protein present in the complex of the present invention is retained and / or improved (e.g., increased) after exposure to a temperature in the range of about 30 seconds to about 2 minutes, from about 70 °C or about 75 °C to about 80 °C, about 85 °C or about 90 °C, as compared to the antimicrobial ability and / or antimicrobial activity of the protein alone after exposure to the same conditions (e.g., the same temperature and time). In some embodiments, the complex can be present in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acid whey beverage) and can be exposed to the temperature. In some embodiments, the antimicrobial ability and / or antimicrobial activity (e.g., the antibacterial activity against Gram-positive bacteria and / or Gram-negative bacteria, and / or the antiviral activity) of the protein present in the complex of the present invention is retained and / or improved after exposure to a temperature in the range of about 30 seconds to about 2 minutes, from about 70 °C or about 75 °C to about 80 °C, about 85 °C or about 90 °C, optionally as compared to the microbial ability and / or antimicrobial activity of the protein alone after exposure to the same conditions (e.g., the same temperature and time).

[0053] In some embodiments, in response to exposure to a temperature in the range of about 30 seconds to about 2 minutes, about 70 °C or about 75 °C to about 80 °C, about 85 °C or about 90 °C, or in response to exposure to a temperature in the range of 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, 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, the solubility of the complex of the present invention in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acidic whey beverage) remains within about 30% of its original solubility prior to exposure (e.g., the initial formation of the complex and / or the solubility of the complex in the composition immediately prior to exposure). In some embodiments, in response to exposure to a temperature in the range of about 30 seconds to about 2 minutes, about 70 °C or about 75 °C to about 80 °C, about 85 °C or about 90 °C, or in response to exposure to a temperature in the range of 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, 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, the amount of biopolymer (e.g., protein, cationic biopolymer and / or anionic biopolymer) present in the complex of the present invention in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acidic whey beverage) is optionally chromatographed (e.g., high performance liquid chromatography), assayed (e.g., ELISA) and / or spectrophotometrically (e.g., circular dichroism and / or UV-vis) when measured compared to the amount of the biopolymer present in the complex prior to exposure (e.g., the initial formation of the complex and / or the amount of the biopolymer present in the complex immediately prior to exposure), remains within about 30%.In some embodiments, the activity (e.g., bioactivity, antiviral activity, and / or antibacterial activity) and / or function of the biopolymer (e.g., protein, cationic biopolymer, and / or anionic biopolymer) present in the complex of the present invention in a composition (e.g., water, buffer, milk (e.g., skim milk), and / or acidic whey beverage) remains within about 30% of the activity (e.g., bioactivity) and / or function of the biopolymer present in the complex prior to exposure (e.g., initial formation of the complex and / or the activity and / or function of the biopolymer present in the complex immediately before exposure) in response to exposing the composition to a temperature in the range of about 30 seconds to about 2 minutes, about 70°C or about 75°C to about 80°C, about 85°C or about 90°C, or in response to exposing the composition to a temperature in the range of 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, 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. In some embodiments, the physicochemical properties (e.g., turbidity and / or particle size) of the complex of the present invention present in a composition (e.g., water, buffer, milk (e.g., skim milk), and / or acidic whey beverage) remain within about 30% of the original physicochemical properties of the complex prior to exposure (e.g., initial formation of the complex and / or the physicochemical properties of the complex immediately before exposure) in response to exposing the composition to a temperature in the range of about 30 seconds to about 2 minutes, about 70°C or about 75°C to about 80°C, about 85°C or about 90°C, or in response to exposing the composition to a temperature in the range of 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, 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.

[0054] In some embodiments, the antimicrobial ability and / or antimicrobial activity (e.g., the antibacterial activity against Gram-positive bacteria and / or Gram-negative bacteria, and / or the antiviral activity) of the biopolymer (e.g., protein, cationic biopolymer and / or anionic biopolymer) present in the complex of the present invention present in a composition (e.g., water, buffer, milk (e.g., skim milk) and / or acidic whey beverage) is maintained at about 30% and / or within about 30% of the antimicrobial ability and / or antimicrobial activity of the biopolymer prior to exposure (e.g., the antibacterial activity and / or antiviral activity of the biopolymer present in the complex at the initial formation of the complex and / or immediately prior to exposure) in response to exposure to a temperature in the range of about 30 seconds to about 2 minutes, from about 70 °C or about 75 °C to about 80 °C, about 85 °C or about 90 °C, or in response to exposure to a temperature in the range of 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, 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.

[0055] In some embodiments, the complex of the present invention increases the thermal stability of the biopolymer (e.g., protein, cationic biopolymer and / or anionic biopolymer) present in the complex as compared to the thermal stability of the biopolymer alone. For example, the presence of the biopolymer in the complex can reduce or avoid the denaturation of the biopolymer (e.g., thermal denaturation, such as thermal denaturation during the preparation of a food product containing the biopolymer) compared to the amount of denaturation of the biopolymer alone (i.e., when the biopolymer is not present in the complex of the present invention) under the same conditions. In some embodiments, the complex of the present invention increases the thermal stability of lactoferrin present in the complex as compared to the thermal stability of lactoferrin alone in response to exposure to a temperature in the range of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes or 10 minutes to about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or 60 minutes, and about 70°C or about 75°C to about 80°C, about 85°C, about 90°C, about 95°C or 100°C for a certain period of time. In some embodiments, the complex of the present invention increases the stability (e.g., thermal stability), structure, activity and / or function of the biopolymer (e.g., lactoferrin) present in the complex as compared to the stability, structure, activity and / or function of the biopolymer alone in response to exposure to a pH in the range of about 6.5 to about 7.5 under the same conditions (e.g., the same pH).

[0056] The activity and / or function of the biopolymer (e.g., protein, cationic biopolymer and / or anionic biopolymer) present in the complex of the present invention can be increased as compared with the activity and / or function of the biopolymer alone. For example, the complex of the present invention is stored for a certain period (e.g., at a temperature in the range of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months or about 6 months, about 20 °C to about 30 °C, in a sealed container, storing the dried complex) and / or the complex is heated (e.g., 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, at a temperature in the range of about 70 °C or about 75 °C to about 80 °C, about 85 °C, about 90 °C, about 95 °C or about 100 °C). After that, the biopolymer (e.g., protein) present in the complex can have increased activity and / or function as compared with the activity and / or function of the biopolymer alone under the same storage conditions and / or heating conditions. For example, a complex containing lactoferrin, an anionic biopolymer and a cationic biopolymer can provide increased activity and / or function (e.g., increased antimicrobial activity) as compared with the activity and / or function of lactoferrin under the same storage conditions and / or heating conditions after storage and / or heating. In some embodiments, in addition to the anionic biopolymer and / or cationic biopolymer, the complex of the present invention does not contain an agent configured to maintain and / or stabilize the activity, function and / or stability (e.g., thermal stability) of the protein (e.g., lactoferrin) present in the complex.

[0057] In some embodiments, the complex of the present invention can provide increased bioavailability for a biopolymer (e.g., protein, cationic biopolymer, and / or anionic biopolymer) present in the complex of the present invention, as compared to the bioavailability of the biopolymer alone. The bioavailability can be determined after administration of the complex to a subject, and optionally, where the administration includes ingestion of the complex by the subject. In some embodiments, the biopolymer present in the complex of the present invention has increased bioavailability in the intestinal tract of the subject as compared to the bioavailability of the biopolymer alone. In some embodiments, the biopolymer present in the complex of the present invention has decreased enzymatic hydrolysis (e.g., decreased enzymatic hydrolysis in the gastric phase of digestion in a subject) as compared to the amount of enzymatic hydrolysis of the biopolymer alone.

[0058] According to some embodiments, there is provided a composition comprising the complex of the present invention and / or an article comprising the complex of the present invention. In some embodiments, the composition and / or article comprises a plurality of complexes of the present invention. In some embodiments, the composition and / or article comprises the complex of the present invention and a carrier. The carrier may be a liquid, such as, but not limited to, water and / or oil. In some embodiments, the composition of the present invention is an aqueous composition. In some embodiments, the composition of the present invention is a suspension, optionally where the complex of the present invention is suspended in the composition. In some embodiments, the complex of the present invention stabilizes the composition comprising the complex, optionally where the composition is an emulsion. In some embodiments, the carrier is a solid (e.g., particulate and / or powder), and a plurality of complexes of the present invention may be present with the solid, optionally on the solid, under the solid, incorporated with the solid, and / or mixed with the solid. In some embodiments, the carrier is a food-grade component, such as, but not limited to, milk, dairy beverage, infant formula, and / or instant beverage powder. One or more excipients, such as, but not limited to, gum arabic, sodium caseinate, maltodextrin, may be present in the composition of the present invention.

[0059] In some embodiments, the composition and / or article of the present invention is a food, a dietary supplement, a therapeutic beverage, and / or a cosmetic. In some embodiments, the complex of the present invention may be present in a food. In some embodiments, the food is a dairy product (e.g., milk, yogurt, etc.). In some embodiments, the composition is an infant formula and / or a dietary supplement (optionally a beverage).

[0060] In some embodiments, depending on storage in a sealed container at about 4°C to about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months, the turbidity of the composition of the present invention, when optionally measured by spectroscopy, such as ultraviolet-visible (UV-vis) spectroscopy, remains within about 30% of the original turbidity of the composition before storage (e.g., the initial formation of the composition and / or the turbidity on the first day of storage). In some embodiments, after storage in a sealed container at about 4°C to about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months, the composition of the present invention does not have undesirable sensory properties (e.g., undesirable taste (e.g., sourness and / or bitterness), undesirable color, undesirable appearance, and / or undesirable texture), or has minimal undesirable sensory properties (e.g., undesirable taste (e.g., sourness and / or bitterness), undesirable color, undesirable appearance, and / or undesirable texture).

[0061] Methods for preparing the complexes of the present invention are provided according to several embodiments of the present invention. In some embodiments, the method comprises preparing a composition comprising a protein, an anionic biopolymer, and a cationic biopolymer at a pH in the range of from about 3, about 3.5, or about 4 to about 4.5 or about 5; and mixing the composition, thereby providing the complex. The composition comprising a protein, an anionic biopolymer, and a cationic biopolymer may be an aqueous composition optionally comprising a buffer. In some embodiments, the composition used to prepare the complexes of the present invention has a pH of about 3, about 3.5, about 4, about 4.5, or about 5. In some embodiments, the composition used to prepare the complexes of the present invention comprises a salt, optionally, wherein the composition comprises a salt in an amount of 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 present invention comprises a 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.

[0062] The composition used to prepare the complex of the present invention may independently contain protein, anionic biopolymer, and / or cationic biopolymer in an amount of about 0.01 wt%, about 0.1 wt%, about 0.5 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about or 5 wt% to about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt% or about 10 wt% of the composition. In some embodiments, the composition used to prepare the complex of the present invention independently contains protein, anionic biopolymer, and / or cationic biopolymer in an amount of about 0.01 wt%, about 0.1 wt%, about 0.5 wt%, about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt% or about 10 wt% of the composition. In some embodiments, the composition used to prepare the complex of the present invention may independently contain protein, anionic biopolymer, and / or cationic biopolymer in an amount of about 0.01 wt%, about 0.1 wt%, about 0.5 wt%, about 1 wt%, about 2 wt% or about 3 wt% of the composition. In some embodiments, the composition used to prepare the complex of the present invention contains anionic biopolymer and protein in a weight ratio of about 0.5:1 to about 1:5 (anionic biopolymer: protein), for example, a weight ratio of about 0.5:1, 1:1, 1:2, 1:3, 1:4 or 1:5 (anionic biopolymer: protein). In some embodiments, the composition used to prepare the complex of the present invention contains protein and cationic biopolymer in a weight ratio of about 1:1 to about 10:1 (protein: cationic biopolymer), for example, a weight ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10 (protein: cationic biopolymer).In some embodiments, the composition used to prepare the complex of the present invention comprises an anionic biopolymer, a protein, and a cationic biopolymer in a weight ratio of about 1:3:1, about 4:5:1 or about 3:6:1 (anionic biopolymer:protein:cationic biopolymer). In some embodiments, the composition used to prepare the complex of the present invention has the total concentration of the protein, anionic biopolymer and cationic biopolymer in the composition in an amount of about 10% by weight or less of the composition, for example, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1% by weight, about 1.5% by weight, about 2% by weight, about 2.5% by weight, about 3% by weight, about 3.5% by weight, about 4% by weight, about 4.5% by weight, about 5% by weight, about 5.5% by weight, about 6% by weight, about 6.5% by weight, about 7% by weight, about 7.5% by weight, about 8% by weight, about 8.5% by weight, about 9% by weight, about 9.5% by weight or about 10% by weight of the composition. In some embodiments, the composition used to prepare the complex of the present invention has the total concentration of the protein, anionic biopolymer and cationic biopolymer in the composition in an amount of about 5% by weight or less of the composition, for example, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1% by weight, about 1.5% by weight, about 2% by weight, about 2.5% by weight, about 3% by weight, about 3.5% by weight, about 4% by weight, about 4.5% by weight, or about 5% by weight of the composition.

[0063] Mixing the composition used to prepare the complex of the present invention can be carried out using methods known in the art. In some embodiments, mixing the composition used to prepare the complex of the present invention comprises mixing the composition at a temperature of 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 of 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.

[0064] In some embodiments, the method of the present invention includes forming an intermediate composition comprising a protein and an anionic biopolymer, optionally wherein the intermediate composition is an aqueous composition. In some embodiments, the method of the present invention includes combining an anionic biopolymer and a protein to provide an intermediate composition (optionally an aqueous composition), and mixing the intermediate composition to optionally form an intermediate complex comprising the anionic biopolymer and the protein. In some embodiments, mixing the intermediate composition can be carried out at a temperature in the range of 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 in the range of about 20°C, about 25°C, about 25°C or about 60°C. The method can further include adding a cationic biopolymer to the intermediate composition to provide a composition comprising the anionic biopolymer, the protein (optionally in the form of an intermediate complex), and further the cationic biopolymer, and mixing the composition to thereby form the complex.

[0065] The method of the present invention can further include curing the complex of the present invention. In some embodiments, curing the complex includes adjusting the temperature of the composition comprising the complex 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 6 hours.

[0066] In some embodiments, the method of the present invention includes isolating and / or obtaining the complex of the present invention from the composition. Isolating and / or obtaining the complex of the present invention from the composition may include centrifuging, drying, lyophilizing, filtering, and / or spray drying the composition, thereby including isolating and / or obtaining the complex. In some embodiments, the isolated and / or obtained complex is a dried complex, and optionally, wherein the dried complex contains water in an amount of about 0 wt% to about 5 wt% of the dried complex. The dried complex may be in the form of particulates 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, such as a size (e.g., diameter) of less than about 1 mm. In some embodiments, the complex of the present invention can be crosslinked using a crosslinking agent such as, but not limited to, transglutaminase, glyceraldehyde, dialdehyde pectin, and / or genipin. Proteins, cationic biopolymers, and / or anionic biopolymers can be crosslinked in the complex of the present invention.

[0067] In some embodiments, the method of the present invention includes combining the complex of the present invention with a carrier, and optionally, wherein the carrier is liquid or solid. In some embodiments, the complex of the present invention is added to food-grade ingredients and / or foods (e.g., beverages or powder formulations). Combining the complex of the present invention with a carrier may include mixing the isolated and / or obtained complex into the carrier, and / or mixing the complex of the present invention present in a composition (e.g., an aqueous composition) into the carrier. In some embodiments, the complex of the present invention is optionally dispersed in the carrier by mixing, stirring, homogenizing, etc. at a temperature in the range of 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.

[0068] The method of the present invention may 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 method may include administering (e.g., orally) to the subject the complex of the present invention and / or the composition of the present invention, optionally, wherein administering includes the subject ingesting the complex and / or composition.

[0069] In some embodiments, the method of the present invention includes administering to a subject a therapeutically effective amount of the complex of the present invention and / or the composition of the present invention. As used herein, the term "therapeutically effective amount" refers to the amount of the complex of the present invention and / or the composition that elicits a therapeutically useful response in a subject. One of ordinary skill in the art will understand that the therapeutic effect need not be complete or curative so long as some benefit is provided to the subject.

[0070] As used herein, "treating," "being treated," or "treatment" (and grammatical variations thereof) refers to any type of treatment that confers a benefit to a subject, and includes that the severity of the subject's condition is reduced, at least partially improved, or improved, and / or that some alleviation, reduction, or decrease in at least one clinical symptom associated with the subject's condition is achieved, and / or that a delay in the progression of symptoms occurs. In some embodiments, the severity of symptoms associated with iron deficiency can be reduced in a subject as compared to the severity of symptoms in the absence of the method of the present invention. In some embodiments, the complex of the present invention and / or the composition of the present invention is administered to a subject to improve iron delivery and / or uptake in the subject and / or to treat a disease and / or its symptoms.

[0071] In some embodiments, the complex of the present invention and / or the composition of the present invention can be administered in a treatment effective amount. As used herein, a "treatment effective" amount is an amount sufficient to treat a subject (as defined herein). One of ordinary skill 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 complex and / or composition of the present invention to a subject, optionally, where administering includes the subject ingesting the complex and / or composition.

[0072] As used herein, the terms "prevent," "preventing," and "prevention" (and their grammatical variations) refer to the avoidance, reduction, and / or delay of the onset of symptoms associated with a disease, disorder, or condition, and / or a reduction in the severity of the onset of symptoms associated with a disease, disorder, or condition as compared to what would occur in the absence of the method of the present 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 symptoms in the subject is less than what would occur in the absence of the method of the present invention. In some embodiments, the complex of the present invention and / or the composition of the present invention are administered to a subject to prevent a disease, disorder, or condition.

[0073] In some embodiments, the complex and / or composition of the present invention can be administered in a prevention effective amount. As used herein, a "prevention effective" amount is an amount sufficient to prevent (as defined herein) the symptoms associated with a disease, disorder or condition in a subject. One of ordinary skill in the art will understand that the level of prevention need not be complete so long as some benefit is provided to the subject. In some embodiments, a prevention effective amount can be achieved by administering the complex and / or composition of the present invention to a subject, optionally, where administering includes the subject ingesting the complex and / or composition.

[0074] The present invention finds use in both veterinary and medical applications. Suitable subjects to be treated by 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), etc., and mammalian fetuses in utero. Any mammalian subject in need of treatment according to the present invention is suitable. Human subjects of both sexes and of any developmental stage (i.e., neonate, infant, juvenile, adolescent, adult) can be treated according to the present invention. In some embodiments of the present invention, the subject is a mammal, and in certain embodiments, the subject is a human. Human subjects include both males and females of all ages, including fetal subjects, neonatal subjects, infant subjects, juvenile subjects, adolescent subjects, adult subjects, and geriatric subjects, as well as pregnant subjects. In certain embodiments of the present invention, the subject is a human adolescent and / or adult.

[0075] The method of the present invention may be carried out for veterinary purposes and / or for drug screening and drug development purposes on an animal subject, particularly a mammalian subject, such as a mouse, rat, dog, cat, livestock, and horse.

[0076] In some embodiments, the subject "needs" or "is in need of" the method of the present invention, and the subject has findings 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.

[0077] Here, the present invention will be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claims of the present invention, but rather are intended to illustrate specific 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.

[0078] Examples

[0079] Example 1:

[0080] The effective delivery of the bioactive protein lactoferrin (LF) is sensitive to environmental changes and easily denatures during heating, thus still remaining an issue as its application to functional foods is restricted. To overcome these issues, polyelectrolyte ternary complexes of LF with gelatin (G) and negatively charged polysaccharides were formulated. Linear, highly charged polysaccharides formed interpolymeric complexes between LF and G, while branched polysaccharides formed coacervates. A unique multiphase coacervate was observed in the gum arabic GA-LF-G complex, where a special coacervate-in-coacervate structure was found. Ternary complexes made with GA and soy soluble polysaccharide (SSP) or high methoxyl pectin (HMP) retained the protein structure and demonstrated the enhanced thermal stability of LF. The GA-LF-G complex, in particular, remained stable, retaining more than 90% of native LF after being treated in a water bath at 90 °C for 2 minutes or at 145 °C for 30 seconds, while the LF control had only about 7% of undenatured LF under both conditions. Compared to untreated LF, LF in the ternary complex retained significant antibacterial activity against both Gram-positive and Gram-negative bacteria even after heat treatment. These ternary complexes of LF maintained the desired functionality, thermal stability and antibacterial activity of LF in the final product. The ternary complex structure, especially the multiphase coacervate, could serve as a template for the encapsulation and stabilization of other bioactive substances and peptides.

[0081] 1. Introduction

[0082] It is reported herein that a positively charged ternary complex of LF and G was formulated with five negatively charged polysaccharides to investigate whether this complex could improve the structural stability and antibacterial activity of LF during heat treatment. As described below, complexes formed with polysaccharides having various charge densities and chain flexibilities exhibited different structures and thermal stabilities. The physicochemical properties of these ternary complexes were investigated using turbidity, zeta potential, particle size, and microscopic analysis. Next, the structural changes and retention ratio of LF after heat treatment were evaluated through intrinsic fluorescence, circular dichroism (CD) spectroscopy, SDS-PAGE, and HPLC.

[0083] 2. Materials and Methods

[0084] 2.1. Materials

[0085] Bovine lactoferrin (LF) (Bioferrin 2000, iron content > 15 mg / 100 g) was obtained from Glanbia Nationals, Inc. (Fitchburg, WI, USA). Soy soluble polysaccharide (SSP) was obtained from Fuji Oil Co., Ltd. (Izumisano City, Japan). High methoxyl pectin (HMP), Kappa carrageenan (Kappa), and Iota carrageenan (Iota) were provided by Tic gum (Riverside, MD, USA). Gum arabic (GA) was provided by Colony Gum (Monroe, NC, USA). Gelatin (Knox unflavored gelatin) was purchased from a local grocery store (Target, Ithaca, NY, USA). Fluorescein isothiocyanate isomer I (FITC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid, acetonitrile (HPLC grade), hydrochloric acid, sodium hydroxide, dimethyl sulfoxide (DSMO) were purchased from Fisher Scientific (Hampton, NH, USA). Reagents for SDS-PAGE were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Bicinchoninic acid (BCA) Assay Kit II was purchased from BioVision (Waltham, MA, USA). Coomassie Brilliant Bule G-250 was purchased from bioWORLD (Dublin, OH, USA). Luria broth (LB) and LB agar were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and reagents were purchased from Fisher Scientific (Hampton, NH, USA).

[0086] 2.2 Preparation of ternary complex

[0087] The LF, SSP, and GA solutions were prepared at 1% or 0.2% (w / v) by dissolving the biopolymer in Milli-Q water and mixing for 2 h at room temperature (25 °C). The HMP, kappa, and iota solutions were prepared at 0.2% (w / v) by dissolving the biopolymer in Milli-Q water, heating to at least 60 °C, and mixing for 2 h. The gelatin (G) solution was prepared at 1% or 0.2% (w / v) by dissolving the biopolymer in Milli-Q water and mixing for 2 h at 45 °C. All solutions were cooled to 4 °C and left to stand overnight to be fully hydrated.

[0088] Figure 1 shows an exemplary schematic diagram of the ternary complex formation procedure using a branched polysaccharide (Figure 1, panel a) and a linear polysaccharide (Figure 1, panel b). The ternary complex was prepared at a total concentration of 1% or 0.2% (w / v) by proportionally adding biopolymer solutions at the same concentration. The ratio of the added biopolymers was designed according to a ternary plot with a ratio interval of 10% (mass ratio of total biopolymers). All biopolymer solutions were first adjusted to the target pH at pH 4 or pH 7. These pH levels were selected to represent the pH when LF has a high positive charge (pH 4) and a low positive charge (pH 7). Next, different polysaccharide solutions were first mixed with the LF solution at room temperature (25 °C) for 30 minutes to form an intermediate complex. Then, the mixture was heated to 45 °C and, at 45 °C, further mixed with the preheated gelatin solution for another 30 minutes. After completely mixing all biopolymers, the solution was cured under the condition of 10 °C for 2 hours. To isolate the complex, the ternary mixtures were centrifuged at 10,000 g for 25 minutes at 4 °C. The pellet was recovered and then frozen overnight (-20 °C), and then freeze-dried using a freeze dryer (Labcono, Kansas, MO, USA) at a vacuum pressure of -0.175 mBar and a moisture collector temperature of -53 °C for 48 - 36 hours. The supernatant was recovered and stored at 4 °C for complexation efficiency measurement.

[0089] 2.3 Characteristics of the complex

[0090] 2.3.1 Turbidity measurements

[0091] The turbidity of the ternary mixture was measured using a UV-Vis light spectrophotometer (UV-2600, SHIMADZU Co., Japan). The transmittance at a wavelength of 600 nm was measured at room temperature using quartz cuvettes with an optical 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

[0092] 2.3.2 Particle Size Measurement

[0093] The average diameter and particle size distribution of the ternary mixture were analyzed using a dynamic light scattering device (Zetasizer Nano-ZS, Malvern, Germany). All analyses were performed at 25 °C using a 1 cm optical path length cuvette at a wavelength of 633 nm and a backscattering angle of 173 o . The refractive index of the dispersion medium was set to 1.33 and the refractive index of the material was set to 1.45. The analysis was performed 3 times, and each measurement was performed at least 11 times.

[0094] 2.3.3 Zeta Potential Measurement

[0095] The zeta potential of the ternary mixture was measured using Nano-ZS (Malvern, Germany) in Smouluchwski mode. The software was able to determine the appropriate measurement type after acquiring the conductivity of the sample using a voltage of approximately 150 V. The sample was measured 3 times, and each measurement was performed 10 times.

[0096] 2.3.5 Morphological Characteristics

[0097] 2.3.5.1 Confocal microscopy analysis

[0098] LF was labeled with FITC (Fluorescein isothiocyanate) for imaging purposes using the following procedure. LF was dissolved at 5 w / v% in 10 mM carbonate buffer (pH 10.0). FITC was dissolved at 10 w / v% in DMSO. The LF solution was mixed with FITC at a volume ratio of 20:1 in the dark at room temperature for 3 - 4 hours. The mixture was washed through a gel filtration column (packed with Sephadex G - 25) to remove excess FITC. The yellow - green solution was recovered from the column, and the labeled protein was confirmed by UV - vis spectroscopy. The resulting FTIC - labeled LF solution was lyophilized for later use in ternary complex and confocal microscopy analysis.

[0099] Confocal microscopy images of the ternary complex made with FTIC - labeled LF were obtained using a Zeiss LSM 710 confocal laser scanning microscope connected to an Inverted Axio Observer.Z1 microscope with a 40× water immersion objective (NA 1.2). A small aliquot of the complex solution was transferred to a glass microscope slide and covered with a glass coverslip. Using an excitation argon laser (488 nm) and detection light 500 - 559 nm, green (confocal) images were made to visualize LF in the complex. Normal optical images were taken with the laser off and normal white polarized light on. Images were analyzed by instrument software (EZ CS1 version 3.8, Niko, Melville, NY).

[0100] 2.3.5.2 Scanning electron microscopy analysis

[0101] The microstructure of the LF ternary complex was visualized by the method described by (Lin et al., 2022) using a field-emission scanning electron microscope (SEM) (Zeiss Gemini 500, Jena, Germany). Before taking the microscope images, a fresh ternary mixture sample (about 10 μL) was dried under vacuum overnight and then coated with Au / Pd in a sputter coater (Denton Desk V, NJ, USA).

[0102] 2.3.6 Measurement of the complexation efficiency and loading ratio of LF

[0103] The supernatant obtained after centrifuging the ternary mixture was appropriately diluted and used to quantify free LF according to the Bradford method (Bradford, 1976). The complexation efficiency was calculated according to the following formula (Formula 2):

Number

[0104] Total LF was the theoretical concentration (w / v%) of LF contained in the ternary mixture, and free LF was the measured concentration (w / v%) of LF in the supernatant.

[0105] The freeze-dried complex sample was weighed for yield measurement, which was calculated according to the following formula (Formula 3).

Number

[0106] The loading rate (mass ratio) of LF in the freeze-dried composite sample was quantified using the BCA assay at a concentration of 0.2 w / v% for the composite sample redispersed in PBS buffer pH 7. The loading rate of LF in the freeze-dried composite was calculated according to the following formula (Formula 4).

Number

[0107] 2.3.7 Thermal stability test of LF in the ternary composite

[0108] Samples containing pure LF and rehydrated ternary composite solutions in water or PBS buffer (10 mM; 0.2 w / v%) at pH 7 were placed in glass tubes (2 mL), and then placed in a water bath at different temperatures of 75 °C or 90 °C for 2 minutes, and then immersed in an ice water bath and cooled to ambient temperature (T = 25 °C) for further analysis. Samples were also treated at 145 °C using an oil bath and treated at this temperature for 2 seconds, 10 seconds, 30 seconds, 60 seconds. Optical images, turbidity, and particle size of the ternary composite solution before and after heat treatment were obtained. Intrinsic fluorescence and circular dichroism spectroscopy were analyzed to understand the protein structural changes during heating. Electrophoresis analysis and HPLC analysis were used to quantify the LF retention rate after heat treatment.

[0109] 2.3.8 Electrophoresis analysis

[0110] Unheated and heated LF and ternary complex samples were analyzed using sodium dodecyl sulfate (SDS)-PAGE in a vertical mini gel electrophoresis system (Mini-PRO-TEAN Tetra cell, Bio-Rad, USA). A pre-mixed TGA fast Cast acrylamide starter kit was used for the preparation of the PAGE gel. 20 μL of the diluted sample (2 mg / mL protein) was mixed with 2X Laemmli buffer in a 1:1 ratio and then heated in a boiling water bath for 5 minutes. Next, 20 μL of the mixture was loaded onto the gel and electrophoresis (200 V) was performed for approximately 30 - 45 minutes. The gel was stained in a 0.15% (w / v) Coomassie Brilliant R-250 solution consisting of 50% (v / v) methanol and 10% (v / v) acetic acid for 30 minutes. Next, the gel was decolorized with a decolorizing solution (20% (v / v) methanol and 10% (v / v) acetic acid) for 24 hours. The gel was decolorized in a decolorizing solution (20% (v / v) methanol and 10% (v / v) acetic acid) for 24 hours.

[0111] 2.3.9 HPLC analysis

[0112] HPLC analysis was developed to quantify native LF in the complex solution after heat treatment. All samples were top liquid solutions free of protein aggregates and filtered through a 0.45 μm filter. Reverse-phase HPLC was performed on an Agilent 1100 / 1200 series HPLC system (Agilent Technologies, CA, USA) equipped with a diode array detector and ChemStation data acquisition program. Detection was carried out at 214 nm. LF separation was performed at 40 °C using a BioZen Intact XB-C8 column (150 × 4.6 mm, 3.6 m; Phenomenex, Torrance CA, USA). The following gradient elution was carried out at a mobile phase flow rate of 1.0 mL / min using 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B): 0 - 5 min, 5% B; 5 - 20 min, 5 - 20% B; 20 - 25 min, 50 - 5% B. The injection volume was 10 μL. The concentration of native LF remaining in the sample solution can be measured and quantified according to the standard curve of raw LF at a concentration of 0 - 0.2 w / v% (R 2 >99%). The LF retention rate indicates how much LF remains intact after heat treatment in the complex solution and was calculated using the following formula.

Equation

[0113] 2.3.10 Analysis by Circular Dichroism (CD) Spectroscopy

[0114] The secondary structure of lactoferrin in pure LF and ternary complex solutions before and after heat treatment was measured using CD spectroscopy. The CD spectra of LF and the redispersed complex were measured at 25 °C in the far-UV region (190 - 260 nm) using an AVIV-202-01 spectrophotometer (Lakewood, NJ, USA). To reduce the gain, the samples were diluted to 0.02% (w / w) LF before measurement. The samples were analyzed in quartz cells with a 1 mm optical path length. The obtained data were converted to molar ellipticity, [θ] (deg cm 2 dmol -1 ) using the DichroWeb online processing platform. Pure gelatin did not show a secondary structure according to its limited CD spectral signal. Therefore, the CD spectrum of the ternary complex is mainly referred to as the secondary structure of LF.

[0115] 2.3.11 Intrinsic Fluorescence Analysis

[0116] The intrinsic fluorescence of LF samples and ternary complex samples before and after heat treatment was measured using a Shimadzu RF-6000 spectrophotometer (Shimadzu Corporation, Japan). The samples were measured in 1 cm quartz cuvettes at an excitation wavelength of 280 nm, and the emission was monitored over the range of 310 - 400 nm. The excitation / emission slit widths were both set to 10 nm.

[0117] 2.3.12 Antimicrobial Activity Analysis

[0118] A Staphylococcus aureus strain and an Escherichia coli strain were used in this test as the target Gram-positive and Gram-negative bacteria, respectively. The Staphylococcus aureus strain was isolated from bovine feces by the Animal Health Diagnostic Center of Cornell University (AHDC). The Escherichia coli (k12 Mg1655) strain was obtained from the American Type Culture Collection (ACTT) (Freddolino et al., 2012). The frozen bacteria were first activated in Luria broth (LB) agar medium. Next, a loop of pure colonies was transferred and incubated in fresh LB medium at 37 °C for 24 h, and the bacterial activity was further measured.

[0119] Antibacterial activity was measured using the UV absorbance method on a 96-well microtiter plate. First, the bacteria Staphylococcus aureus or Escherichia coli were diluted 1000-fold in LB broth so that the absorbance of 100 μl of the bacterial broth was less than 0.04 at 625 nm. The increase in absorbance at 625 nm (OD 625nm) was used to indicate the growth of bacteria. In the case of the MIC (minimal inhibitory concentration) test, LF at different diluted concentrations (0.1 - 1 w / v%) was used. 100 μL of the diluted bacterial broth and 100 μL of the LF solution were added to each well. Next, 100 μL of the bacterial broth with 100 μL of PBS buffer added 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 used as the concentration for further antibacterial tests of the LF ternary complex. Similarly, 100 μL of the diluted bacterial broth and 100 μL of the unheated and heated LF complex solutions at a concentration (0.2%, w / v) selected based on the MIC test were added to each well. The microtiter plate was incubated at 37 °C, and OD625nm was read after shaking for 10 seconds and measured at 0 hours, 24 hours, and 48 hours of incubation to monitor the growth of bacteria.

[0120] 2.4 Data analysis

[0121] The data obtained were presented as the mean and standard deviation of duplicate or triplicate measurements and analyzed using Analysis of Variance (ANOVA). The differences between the means were evaluated using the Tukey HSD comparison test (P < 0.05). All statistical analyses were performed using JMP Pro15 (SAS Institute, USA) and plotted using GraphPad Prism9 (GraphPad Software Inc.). Ternary plots were drawn in MATLAB (登録商標) (R2022a, MathWorks, Natick, MA, USA).

[0122] 3. Results and Discussion

[0123] 3.1 Effects of Biopolymer Ratio, PS, and Concentration on the Formation of Ternary Complexes

[0124] Figure 2 shows the effects of the ratios of various polysaccharides to biopolymers on the formation of ternary complexes between LF and gelatin at pH 4. The charge density, structural characteristics, and approximate molecular weights of five polysaccharides are tabulated below for reference (Table 1). Generally, soy soluble polysaccharide (SSP) and gum arabic (GA) are branched polysaccharides with a charge density of less than 0.3, and high methoxyl pectin (HMP), kappa-carrageenan (kappa), and iota-carrageenan (iota) are linear polysaccharides with charge densities of 0.3 - 0.6, 0.5, and 1, respectively. The total biopolymer concentration was the same (0.2 w / v%) for each ternary system.

[0125] Ternary plots were used to design the mixing ratios of the three biopolymers (Figure 2). The scales on the three axes indicate the mass ratios of the biopolymers added to the ternary mixture. The darker gray of the ternary mixture at a certain biopolymer ratio indicates a higher turbidity (3 - 7) of the mixture, which further indicates stronger interactions and complex formation.

[0126] [Table 1]

[0127] As shown in Figure 2, different PS systems showed different maximum turbidities (T max ). Complex formation was indicated when the turbidity was found to be greater than 2. The maximum turbidity represents the maximum extent of complexation and the strength of the electrostatic interactions in the system, which are related to the relative amounts of oppositely charged groups in the solution, respectively. Generally, PSs with branched chains / structures (GA and SSP) had smaller T maxwas shown. The latter system also showed a larger complex formation region (when T>3), probably due to their larger negative charge and higher charge density. Considering the relatively low complex formation at 0.2% in the GA and SSP systems, a high concentration (1%) was applied as shown in Figure 3.

[0128] In the case of the GA-LF-G system, complexes were formed in the case of a mixture consisting of 60 - 80% LF and 20 - 40% GA (Figure 2), while the gelatin concentration seemed to have little effect. Generally, the SSP-LF-G mixture showed very limited complex formation under all tested conditions, with a relatively low turbidity (less than 3) over the range of mass ratios (Figure 2). In the case of the HMP-LF-G system, complexes were formed in the case of a mixture consisting of 30 - 90% LF, 30 - 90% HMP, and 40 - 50% gelatin (Figure 2). In the case of the mixture of kappa-LF-G and iota-LF-G, the complex formation range shifted to 50 - 90% LF, 50 - 90% kappa or iota, and less than 50% gelatin (Figure 2). The higher the gelatin concentration (above 50%), the less favorable the complex formation seemed to be when linear polysaccharides were involved, showing a lower turbidity. Without wishing to be bound by any particular theory, the higher charge density and more negative charges retained by HMP, kappa, and iota may contribute to the increased ability of these polysaccharides to form complexes with LF at a higher mass ratio compared to SSP and GA (Table 1 and Table 2).

[0129]

Table 2

[0130] Considering the relatively low turbidity in the 0.2 w / v% SSP and GA systems, a higher concentration (1 w / v%) of biopolymers was further tested in these two ternary systems (Figure 3). The higher concentration, especially in the GA ternary system, led to interactions and T maxwas enhanced. As the concentration is higher, due to the enhanced density of the counter charges and the short intermolecular distance in the solution, a high degree of complexation is expected to be promoted. On the other hand, the degree of complexation is much more significant than expected in the GA-LF-G system and less obvious than expected in the SSP-LF-G system. The zeta potential representing the charge density of GA and SSP was very similar at concentrations of 0.2 w / v% or 1 w / v% (Table 2). Therefore, while not wishing to be bound by any particular theory, the difference in chain flexibility between GA and SSP may be the cause of the observed differences in the complex formation between LF and gelatin.

[0131] Ternary complexes formed at pH 7 were also investigated (data not shown). However, all samples at any biopolymer ratio showed very low turbidity (<2), indicating limited complex formation. Since LF and gelatin had limited positive charges at pH 7, electrostatic interactions were low at all biopolymer ratios. In total, the final selected concentration was 1% in the ternary system of GA and SSP, and 0.2% in the ternary systems of HMP, kappa, and iota. The pH condition was fixed at pH 4 where all biopolymers obtained relatively high charges. According to these triangular turbidity plots, the biopolymer ratios with the highest turbidity were selected as the conditions for forming complexes because they showed a higher level of complex formation in each system. Specifically, the selected biopolymer ratios were GA-LF-G 2-6-2, SSP-LF-G 4-5-1, HMP-LF-G 4-5-1, kappa-LF-G 3-6-1, and iota-LF-G 2-6-2, respectively.

[0132] 3.2 Zeta Potential and Particle Size of Ternary Complexes

[0133] The zeta potential of the ternary mixture formed at pH 4 was selectively evaluated as a proof of concept. Typically, a ternary mixture with a fixed ratio of gelatin and LF was selected to investigate the effect of the addition of PS or LF on the zeta potential of the ternary mixture. As shown in Figure 4, with the addition of negatively charged PS, the zeta potential of the ternary mixture gradually decreased from positive charge to zero and then further decreased to a negative charge. Most importantly, the ratio when the overall zeta potential was close to zero was also the condition showing high turbidity as shown in Figures 2 and 3. This is because it means that when the overall zeta potential reached zero, all the biopolymers had similar opposite charges on the surface. They tended to form complexes by electrostatic interactions, and the repulsive interactions between the biopolymers decreased as the system had the minimum charge, further promoting the formation of larger complexes. The particle sizes of the GA and iota ternary mixtures at different ratios and pH conditions were also selectively measured and compared. As shown in Table 3, the mixture sample of GA-LF-G 2-6-2 showed a large average particle size as the complex was formed, but at pH 7, it showed a small average particle size indicating limited complex formation even at the same ratio. Similarly, the sample of iota-LF-G 2-6-2 showed an even larger size than that obtained in the GA-LF-G system, probably because of the high charge density of iota even though the total concentration of the iota system was lower than that of the GA system.

[0134]

Table 3

[0135] 3.3 Effect of salts on complex formation

[0136] Figure 5 shows the turbidity of the ternary complex formed at various salt concentrations (up to 500 mM) and pH 4, and the ratios obtained from the above tests. Since salts can weaken or select the electrostatic interactions between these polymers, salts generally showed a negative effect on the formation of the ternary complex, especially at high concentrations (above 100 mM). An interesting finding was that the turbidity of the GA-LF-G ternary complex increased at low salt concentrations (0 - 50 mM) and then gradually decreased with increasing salt concentration. Generally, the ternary complex formed by SSP and HMP is more sensitive to salts, where the turbidity approaches zero when the salt concentration is higher than 100 mM (which is also called the critical salt concentration). The complex formed by GA is less sensitive to salts and has a critical salt concentration of approximately 200 mM. The complex formed by kappa-carrageenan and iota-carrageenan showed the lowest sensitivity to salts as they could maintain a turbidity of more than 1 even at a salt concentration of 500 mM. Overall, most of the ternary complex can be retained at salt concentrations lower than 50 mM, preferably lower than 30 mM.

[0137] 3.4 Complex Yield, Complexation Efficiency and Loading Rate of LF in the Ternary Complex

[0138] After obtaining the desired conditions for forming the ternary complex, the ternary mixture solution was centrifuged, and the supernatant containing the uncomplexed biopolymer was removed. Next, the resulting pellet containing the complexed biopolymer was lyophilized. The average yield of LF, complexation efficiency, and final mass loading in the lyophilized complex were measured (Table 4). Ternary mixtures formed with linear and highly charged PS kappa-carrageenan and iota-carrageenan showed the highest complex yields (70 - 80%) and had the highest complexation efficiency (96%). In contrast, mixtures formed with linear and low-charged PS HMP provided moderate complex yields (about 40%) and moderate complexation efficiency (52%). Ternary mixtures formed with branched and even lower-charged PS, GA, and SSP showed low yields (40% and 17%) and moderate to low complexation efficiencies (50% and 20%). The high complexation efficiency (96%) in the kappa-LF-G and iota-LF-G mixtures indicated that almost all of the LF present in the mixture was incorporated into the complex, while the GA / HMP-LF-G mixture incorporated only about 50% of the available LF. Generally, complexes formed with linear and more highly charged PS, kappa, and iota showed higher yields and complexation efficiencies than those formed with more neutral PS, HMP, and GA. This result is consistent with the turbidity results showing that kappa-carrageenan and iota-carrageenan, which are highly charged polysaccharides, exhibited higher turbidity and complex formation than LF and gelatin, indicating stronger interactions in these ternary complexes.

[0139]

Table 4

[0140] The final mass ratio of LF in the freeze-dried complex was quantified using the following three different methods: Bradford assay, Bicinchoninic acid (BCA) assay, and HPLC. Based on these three methods, the mass ratio of LF in the complex containing GA, SSP, or HMP was in the range of 40 - 56% (w / w). Due to the interaction between the Bradford reagent and the sulfate groups of carrageenan, the mass ratio of LF in the kappa-carrageenan / iota-carrageenan ternary complex was underestimated by the Bradford assay. HPLC analysis was also limited in obtaining the LF peak from the kappa-carrageenan / iota-carrageenan ternary complex due to the strong negative charge from the sulfate groups of kappa-carrageenan and iota-carrageenan. Therefore, the mass ratio of LF in the kappa-carrageenan / iota-carrageenan ternary complex was more reliable when measured by the BCA method, which was about 68 - about 75% (w / w). The LF loading rate in the GA complex was about 52%, and the loading rate in the HMP complex was about 40 - about 42%, while the LF loading rate in the kappa-carrageenan and iota-carrageenan complex was approximately 54 - approximately 68% (Table 4). The relative mass ratio of LF in the carrageenan ternary complex can be expected to be higher because these two polysaccharides form a complex with LF through electrostatic interaction and thus carry a higher negative charge.

[0141] 3.5 Microscopic Observation of the Formed Complex

[0142] The optical microscopic observation of the ternary complex under the selected conditions (pH, concentration, and ratio) was investigated and is shown in Fig. 6. Interestingly, different complex structures were formed in these ternary systems. The GA system and the SSP system showed a somewhat spherical structure, while the HMP system, the kappa system, and the iota system showed an irregular polymer network structure. The former complexes showed the formation of coacervate complexes, while the latter complexes showed the formation of inter-polymer complexes. Generally speaking, the formation of coacervates and complexes follows the same initial pathway. First, soluble intra-polymer complexes are formed at specific pH, ratio, and ionic strength, and then, as the biopolymer ratio continues to approach the condition where the overall charge reaches zero, the soluble complexes begin to interact with each other, forming inter-polymer complexes, and then bulk phase separation occurs. Generally, coacervates are formed when PS or proteins have a low charge density or a very flexible backbone such as gelatin, gum acacia, and gum arabic. On the other hand, inter-polymer complexes, also called co-precipitates, are formed when the PS or proteins in the system are highly charged and / or have a very rigid linear structure such as carrageenan, gellan gum, or xanthan gum. The formation of the intra-polymer complexes may be related to a greater binding affinity between biopolymers that results in strong interactions.

[0143] Confocal microscopy images of ternary systems of SSP, GA, HMP, kappa and iota with LF (pale grey) labeled with FITC are shown in Fig. 7, and SEM images are shown in panels A1–E1 and A2–E2 of Fig. 17. Interestingly, the GA-LF-G ternary complex was shown as a multiphase coacervate droplet, which means that multiple coacervate droplets are within a larger coacervate droplet. Moreover, LF (FTIC labeled green) appeared to be located within the inner phase coacervate. SEM of the GA-LF-G complex after overnight vacuum drying (panels A1 and A2 of Fig. 17) showed that the GA-LF-G complex was flattened particles compressed due to evaporation of moisture, showing properties like that of coacervate liquid. In confocal images, scattering of SSP-LF-G nano-sized coacervate particles could be seen, indicating the low yield (8%) of the formed SSP-LF-G complex, while the HMP-LF-G, kappa-LF-G and iota-LF-G complexes all showed characteristic morphologies associated with a high-density polymer inter-network structure (Fig. 7). From the SEM images, the inter-polymer complex structures of the ternary complexes using these three linear polysaccharides, HMP, kappa and iota, were confirmed (panels C1 and C2; D1 and D2; and E1 and E2 of Fig. 17).

[0144] Multiphase coacervate droplets have been recently reported in biological systems, but to the best of the inventors' knowledge, this type of phenomenon has been observed for the first time in food-grade biopolymers, particularly in protein-polysaccharide systems. While not wishing to be bound by any particular theory, the main driving forces for the formation of coacervate complexes in multiphase droplets were considered to be different critical salt concentrations and densities. The SSP-LF-G ternary complex was shown as a conventional single-phase coacervate droplet. For the ternary systems of HMP and Iota, the confocal microscopy images were similar to the optical microscopy images, showing the inter-polymer complex structure of the ternary complex containing linear polysaccharides.

[0145] 3.6 Thermal stability of LF in the ternary complex

[0146] 3.6.1 Optical Images, Turbidity and Particle Size of the LF Ternary Complex after Heat Treatment

[0147] LF is known to readily denature under heat treatment conditions at neutral pH. Therefore, to test the thermal stability of LF in the complexed samples, the lyophilized complex samples were redispersed at 0.2% (m / v) in 10 mM PBS buffer at pH 7 and then heat-treated under pasteurization conditions (75 °C / 2 min and 90 °C / 2 min). Pure LF was readily denatured and aggregated in PBS buffer at pH 7. Moreover, pure LF tended to aggregate more highly at 90 °C / 2 min than at 75 °C / 2 min, showing a more turbid solution under the former condition. Compared with pure LF, any type of complex solution showed a more transparent solution after heat treatment. This can be observed more directly in the turbidity chart in Figure 8, where pure LF showed a large increase in turbidity from <0.5 to >4 in PBS buffer after heating, while the increase in turbidity of LF in the complex samples was very limited (<1). Figure 9 shows the change in particle size for LF and the redispersed complex solution after heat treatment. Due to the aggregation of the LF protein, the particle size increased significantly from <100 nm to >2000 nm, while the complex solution showed only a slight change in particle size. The kappa and iota complexes even showed a decrease in particle size, which is thought to be due to the heating promoting the solubilization of the formed complexes by breaking down large complexes into smaller soluble complexes. As pointed out earlier, the complexes formed by kappa-carrageenan and iota-carrageenan showed strong interactions between LF and G, whereby they were able to form larger particles and thus showed low solubility in solution. Without wishing to be bound by any particular theory, it is speculated that the heating process can break down large ternary complexes into smaller complex particles, improve their solubility, and reduce their overall particle size. However, since their particle size was still larger than that of native LF, which was measured to be about 60 nm, they were still considered complexes.

[0148] 3.6.2 SDS-PAGE of LF and ternary complex after heat treatment

[0149] SDS-PAGE of pure LF and LF in the ternary complex after heat treatment in PBS buffer was analyzed and is shown in Figures 10 - 11. In pure native LF, a distinct band was seen at approximately 75 kDa, which was the band shown by the LF protein. After heating at 75 °C / 2 min and 90 °C / 2 min, due to thermal decomposition, the density of the LF band became significantly thinner compared to unheated LF. This result is consistent with the changes in the CD signals and the changes in the internal fluorescence peak intensity of pure LF described later. In the complex sample, the density of the LF band also decreased, but to a much smaller extent compared to pure LF. The GA-LF-G complex showed negligible changes in the LF band after heat treatment. Compared to the GA-LF-G complex, slightly more decomposition of LF was seen in the other PS ternary complexes. In the case of the kappa complex and the iota complex, significant band tailing was evident in the SDA-PAGE. While not wishing to be bound by any particular theory, this is probably because the binding interaction between LF and the negative sulfate groups of carrageenan is strong and the structure of the complex is more rigid, thus affecting the separation of LF molecules in SDS-PAGE.

[0150] 3.6.3 LF retention percentage in the ternary complex after heat treatment by HPLC analysis Rate

[0151] To mathematically quantify the amount or ratio of LF decomposed or retained in the composite after heat treatment, an HPLC method for LF analysis was developed and applied. Since the strong binding interaction between the applied LF and kappa-carrageenan and iota-carrageenan interferes with the method, the ternary complexes of GA-LF-G, SSP-LF-G, and HMP-LF-G were focused on before and after heat treatment. First, the percentage of LF retention in pure LF at different concentrations was analyzed. The LF retention percentage in the ternary complex was measured and compared with the corresponding binary complex. As shown in Table 5, LF at a concentration of 0.05 - 0.2% (w / v) (i.e., 0.5 - 2 mg / mL) in PBS buffer (pH 7) retained only half of the LF after heating at 75 °C for 2 minutes. After heating at 90 °C for 2 minutes, almost all of the LF was denatured and less than 10% of the LF was retained at all tested concentrations.

[0152] A comparison was also made between the LF ternary complex and the general binary complexes (LF with GA, SSP, and HMP). The binary complexes showed an increase in LF retention percentage of 70 - 80% and 50 - 60% respectively upon heating at 75 °C for 2 minutes and 90 °C for 2 minutes. Compared to the binary complexes, the ternary complex demonstrated enhanced LF stability during heat treatment. LF in the ternary complex was retained at 80 - 100% after heat treatment at 75 °C for 2 minutes and was only slightly decomposed. Even when heated at 90 °C for 2 minutes, the retention rate of LF was 70 - 99%, which is 7 - 9 times higher than that of the pure LF sample and almost 1 time higher than that of the binary complex. In the GA - LF - G ternary complex, LF was hardly decomposed under any heating conditions and showed the highest LF thermal stability among all the ternary complex samples. Without wishing to be bound by any particular theory, the enhanced thermal stability of LF in the ternary complexes described herein is thought to be due to additional complexation interactions from the additional biopolymers. In addition to the interaction between LF and PS, without wishing to be bound by any particular theory, the interaction between gelatin and PS is thought to enhance the polymer - polymer interactions within the complex, strengthen the protection around LF, and suppress the structural changes of the protein during heat treatment.

[0153]

Table 5

[0154] 3.6.4 Structural Changes of LF in the Ternary Complex after Heat Treatment

[0155] CD spectroscopy of LF ternary before and after heat treatment was also measured to investigate the change in the secondary structure of LF during heat treatment. As shown in Figure 12, pure native LF had a positive peak at 196 nm and a negative peak at 210 nm, indicating the beta and alpha structures of the LF protein. LF was reported to consist of 16 - 20% alpha - helix, 33 - 42% beta - strand, 10 - 12% beta - turn, and 30 - 34% unordered structure (Lin et al., Wang et al., 2017). After heating, pure LF showed a significant decrease in peak intensity at 210 nm, indicating the loss of the alpha - helix structure. The higher the heating temperature, the greater the decrease in peak intensity. Especially after heating at 90 °C for 2 minutes, the CD spectroscopy signal and peak intensity of LF were very low, indicating a high degree of decomposition and loss of the alpha - helix of LF. However, CD spectroscopy for all complex solutions hardly changed even after heating up to 90 °C for 2 minutes, demonstrating that the secondary structure of LF was well - preserved in the complex solution during the heating process (Panels B - F in Figure 12). In particular, the CD spectrum of the GA - LF - G complex solution hardly changed after heat treatment, indicating a high degree of preservation of the native structure of LF (Panel B in Figure 12). Our previous studies also found improved thermal stability of the secondary structure of LF by complexation with negatively charged soluble soybean polysaccharides (Lin et al., 2022). However, such limited changes in the secondary structure of LF demonstrated in the current GA - LF - G samples have not been reported so far.

[0156] To further understand the structural changes of LF in the system after heat treatment, as shown in Figures 13 and 14, the internal fluorescence intensities of the non-heated sample and the heated sample were examined. The fluorescence emission maximum peak of pure LF showed a red shift from 333 nm to 339 nm in the fluorescence spectrum, which is typical of tryptophan residues in unfolded proteins. Therefore, it indicated the unfolding of LF folding during the heating process. Moreover, the peak intensity of LF increased by 29% after heating. This indicated that the quenching to tryptophan decreased in response to the unfolding of LF folding during the heating process (Figure 14). After heat treatment, the emission peak of the complex sample had less red shift, which indicated less unfolding of the protein structure (Figure 14). The increase in peak intensity in the complex sample after heating was also smaller than that of pure LF (4.8 - 18.7%) except for SSP (Figure 14). Overall, the results of internal fluorescence showed that, compared with pure LF, LF in the ternary complex (especially the GA system, HMP system, kappa system or iota system) had less unfolding after heat treatment and retained a more native protein structure.

[0157]

Table 6

[0158] Considering the common use of ultra-high temperature (UHT) treatment in industrial food processing, the thermal stability of LF at higher heating temperatures of about 145 °C for 0 - 60 seconds was investigated (Table 6). The LF solution began to show observable denaturation or loss of native LF with a retention rate of 16.5% after being heated in an oil bath for 30 seconds and decreased to only 7.8% retention rate by 60 seconds according to HPLC. The GA-LF-G ternary complex showed little change after heating at 145 °C for 30 seconds, while the HMP-LF-G ternary complex had low resilience and remained intact for only 10 seconds. Both complexes showed a significant decrease in LF retention rate after 60 seconds of UHT treatment (84.1% for GA-LF-G and 80.0% for HMP-LF-G). Changes in turbidity and particle size of the LF solution and ternary complex solutions were analyzed, showing results consistent with the LF retention rate analysis (Figure 18). Specifically, the LF solution showed a significant increase in turbidity (panel A in Figure 18) and average particle size (panel B in Figure 18) after 30 seconds of treatment, while the ternary complex showed negligible changes in turbidity and particle size after treatment at 145 °C for 60 seconds. The ternary complex maintained the stability of LF even under high-temperature conditions.

[0159] 3.7 Antimicrobial properties of LF in ternary complexes before and after heat treatment

[0160] LF is known to exhibit antibacterial ability against both Gram-positive and Gram-negative bacteria, and it is desirable to maintain these properties while enhancing thermal stability. The mechanism of the antimicrobial activity of LF is not currently fully understood. The antimicrobial properties of LF were evaluated in pure LF samples and composite LF samples before and after heat treatment. The purpose of this test was to investigate whether complexation affects the functionality of LF and whether complexation can retain this ability even after heat treatment. Antimicrobial ability was selected as a representative biological functionality of LF because it can be easily and relatively safely performed in most biological laboratories.

[0161] Considering that it is one of the most common pathogenic bacteria in dairy products, Staphylococcus aureus (S. aureus) was selected as the target Gram-positive bacterium. The MIC (minimal inhibitory concentration) test of LF against S. aureus was first carried out, which is defined as the minimum concentration of LF that can inhibit half of the bacterial growth compared to the control group (Matijasic et al., 2020). As shown in Panel A of Figure 15, the MIC of LF against S. aureus is 0.1%. This is because the OD625nm value in 0.1% LF is only half of the value in the control sample (which means that 0.1% of LF is sufficient to inhibit 50% of bacterial growth). In the redispersed ternary complex solution (0.2% w / v), the concentration of LF was about 0.1 - 0.14%, and thus it was suitable for antibacterial tests. Panels B - D of Figure 15 show the OD625nm values of non-heated samples or heated samples after 75 °C / 2 minutes or 90 °C / 2 minutes. Pure polysaccharide and gelatin showed no antibacterial effect, but rather promoted bacterial growth (data not shown). The initial ternary complex of the present inventors showed an OD625 similar to that of natural LF, and it was confirmed that the function of LF was sufficiently retained even after the ternary complex was formed before heat treatment.

[0162] In the case of the non-heated sample (Panel B of Figure 15), after 24 hours of incubation, LF showed an antibacterial activity with the OD625nm value being halved compared to the control. The ternary complex sample also showed an inhibitory effect. However, the effect was lower compared to pure LF. Without wishing to be bound by any particular theory, it is thought that this is because LF is complexed / encapsulated in the ternary complex, which affects the interaction between LF and bacteria and further affects the inhibitory effect to some extent.

[0163] After heat treatment at 75 °C for 2 minutes (Panel C in Fig. 15), pure LF lost its inhibitory effect against bacteria and even promoted bacterial growth. This is probably because bacteria can utilize the decomposed LF fragments as an energy source. However, the heat-treated ternary complex showed a significant inhibitory effect with an OD625nm value approximately half that of the control. This effect persisted up to 48 hours of incubation. As shown in Fig. 15, the heat-treated generated ternary complex exhibited an increased antibacterial effect. Without wishing to be bound by any particular theory, the improved antibacterial effect after heat treatment at 75 °C is attributed to the decomposition of the ternary complex structure and the partial exposure or release of LF during the heating process, thereby promoting the interaction between LF and bacteria (Panel C in Fig. 15).

[0164] Nevertheless, at 90 °C, partial denaturation of LF led to a significant decrease in the antibacterial activity of the ternary complex (Panel D in Fig. 15). The formed ternary complex demonstrated the temperature-responsive release ability of LF. During heat treatment, LF can be gradually released from the complex while maintaining its native structure and antibacterial ability. In the sample heat-treated at 90 °C for 2 minutes (Panel D in Fig. 15), the loss of the inhibitory effect of LF and the presence of bacterial growth were more significant as it showed a higher OD625nm value compared to the sample treated at 75 °C for 2 minutes. However, the ternary complex, especially the ternary complex of GA, SSP, and HMP, still showed an approximately 50% inhibitory effect against bacterial growth with an OD625nm value half that of the control sample. The ternary complexes formed by kappa-carrageenan and iota-carrageenan showed lower antibacterial ability (i.e., higher OD values) compared to the other three PSs, which is probably due to their strong binding to LF, limiting the release of LF for exerting the antibacterial effect. Overall, pure LF showed a significantly decreased antibacterial ability after heating, while the antibacterial ability was well retained in the tested ternary complexes.

[0165] Escherichia coli (E. coli) is a common spoilage Gram-negative bacterium in food, and thus, the antibacterial activity of LF against E. coli and the ternary complex of the inventors was also tested. Within 24 hours of incubation, the OD625 of E. coli in the control sample was slightly higher than that of Staphylococcus aureus, indicating that the bacterial growth rate of E. coli is generally faster than that of Staphylococcus aureus (Panel A of Figure 15 and Panel A of Figure 16). The MIC of LF against E. coli was approximately the same as that of LF against Staphylococcus aureus, at approximately 0.1 w / v%, but since all E. coli samples showed high OD625 values after 24 hours of incubation, the antibacterial effect of LF against E. coli was weaker than that against Staphylococcus aureus. The higher environmental tolerance of Gram-positive bacteria compared to Gram-negative bacteria is due to the protective and impenetrable cell wall of Gram-negative bacteria surrounded by an outer membrane that Gram-positive bacteria do not have (Breijyeh et al., 2020). In the case of non-heated samples, both LF and the LF ternary complex demonstrated antibacterial effects against E. coli after 24 hours of incubation (Panel B of Figure 16). Similar to the results for Staphylococcus aureus, the heated LF sample lost its antibacterial effect against E. coli after heat treatment due to the heat denaturation of LF (Panels C and D of Figure 16), while the ternary complex retained its antibacterial effect and showed significantly lower OD625 values than the control.

[0166] Overall, the conditions (ratios, pH, and concentrations) for forming ternary complexes of LF, gelatin, and various polysaccharides were identified for each ternary system according to turbidity plots. Branched, less charged gum arabic and SSP formed coacervate complexes with LF and gelatin at a total concentration of 1%. Considering the relatively high solubility and low viscosity of GA and SSP, even higher concentrations can also be applied in both systems. Linear, highly charged HMP, kappa, and iota formed polymer - polymer complexes with LF and gelatin at a total concentration of 0.2%. The yield of the formed complexes can be increased by appropriate curing and / or cross - linking procedures. Furthermore, by freeze - drying and / or spray - drying processes, coacervate complexes can be produced within microcapsules. The formed complex samples were shown to retain almost the native LF structure even after being heated up to 90 °C / 2 min, based on HPLC analysis and CD spectroscopy. Also, ternary complexes containing GA, SSP, or HMP were shown to maintain the antimicrobial ability of LF after pasteurization conditions (75 °C / 2 min and 90 °C / 2 min) at neutral pH (for example, as indicated by the value of OD625nm, the growth of bacteria could be suppressed by about half compared to the control).

[0167] Example 2

[0168] In this study, we prepared thermostable lactoferrin (LF) ternary complexes through electrostatic interactions under laboratory - scale and industrial - scale processes.

[0169] The formed LF complexes showed improved thermal stability compared to individual LF in different matrices, such as water, skim milk, and acidic whey beverages, under high - temperature short - time (HTST) / ultra - high - temperature (UHT) treatments. The retention rate of LF after heat treatment was quantified by HPLC and ELISA analyses, and the secondary structure of the protein was further confirmed through circular dichroism spectroscopy. The bioactivities, including the antibacterial activity (Escherichia coli and Staphylococcus aureus) and antiviral activity (coronavirus) of LF, were well retained in the complex after heat treatment.

[0170] During storage, the LF complex components demonstrated good solubility, LF retention rate, and bioactivity at 25 °C for 12 months. The LF complexes formed in various food models (water, skim milk, and acid whey beverage) showed stable physicochemical properties (e.g., turbidity and particle size) under storage conditions of 4 °C for 12 months and 25 °C for 6 months, and undesirable sensory properties were minimized.

[0171] The above is illustrative of the present invention and is not to be construed as limiting the present invention. The present invention is defined by the following claims, and equivalents of the claims are included therein.

Claims

1. It is a complex, protein; Anionic biopolymers; and, Cationic biopolymers Includes, Here, the protein, anionic biopolymer, and cationic biopolymer associate via electrostatic interactions. The aforementioned composite.

2. The composite according to claim 1, wherein the composite has a zeta potential of about -5mV to about +5mV.

3. The complex according to claim 1, wherein the complex comprises about 30% to about 85 w / w% of the protein, about 5% to about 40 w / w% of the cationic biopolymer, and about 5% to about 40 w / w% of the anionic biopolymer.

4. The complex according to claim 1, wherein the protein has a net positive charge at a pH of about 3 to about 8.

5. The complex according to claim 1, wherein the protein has a globular structure and / or is a dairy protein.

6. The complex according to claim 1, wherein the protein is soluble in water at a pH of less than approximately 8.

7. The complex according to claim 1, wherein the protein is lactoferrin, alpha-lactalbumin, lysozyme, and / or osteopontin.

8. The composite according to claim 1, wherein the cationic biopolymer has a net positive charge at a pH of about 3 to about 5.

9. The composite according to claim 1, wherein the cationic biopolymer has a pI and / or pKa of about 7 or more.

10. The composite according to claim 1, wherein the cationic biopolymer is selected from gelatin, chitosan, lysozyme, and / or polyamino acids.

11. The composite according to claim 1, wherein the anionic biopolymer has a net negative charge at a pH of about 3 to about 5.

12. The composite according to claim 1, wherein the anionic biopolymer at a pH of about 3 to about 5 has a zeta potential in the range of about -10 mV to about -55 mV.

13. The composite according to claim 1, wherein the anionic biopolymer is a polysaccharide or a glycosaminoglycan.

14. The composite according to claim 1, wherein the anionic biopolymer is gum arabic, high methyl pectin (HMP), copper-carrageenan, iota-carrageenan, dextran sulfate, sodium hyaluronate, acacia gum, xanthan gum, gellan gum, and / or plant-soluble polysaccharides.

15. The composite according to claim 1, wherein the anionic biopolymer has a pKa of about 4 or less.

16. The composite according to claim 1, wherein the protein, cationic biopolymer, and anionic biopolymer are each food-grade components.

17. The complex according to claim 1, wherein the protein is lactoferrin, the anionic biopolymer is a polysaccharide, and the cationic biopolymer is gelatin.

18. The composite according to claim 1, wherein the composite is a composite coacervate in a liquid.

19. The composite according to claim 1, wherein the composite has a coacervate-in-coacervate structure in a liquid and comprises an inner coacervate and an outer coacervate.

20. The composite according to claim 1, wherein the composite is an interpolymer composite.

21. The complex according to claim 1, wherein the protein has increased stability after being exposed to a temperature of approximately 70°C to approximately 100°C for approximately 1 minute to approximately 60 minutes, compared to the stability of the protein alone after exposure to the same temperature for the same amount of time.

22. The complex according to claim 1, wherein, after exposure to a temperature of approximately 70°C to approximately 100°C for approximately 1 minute to approximately 60 minutes, the protein is degraded by less than approximately 30% as measured by high-performance liquid chromatography.

23. The complex according to claim 1, wherein the activity of the protein increases after storage under the same conditions compared to the activity of the protein alone.

24. The composite according to claim 1, wherein the solubility of the composite in the composition remains within approximately 30% of its original solubility before the storage and / or exposure, in response to storage in a sealed container at approximately 4°C to approximately 25°C for less than 12 months, and / or exposure to a temperature in the range of approximately 70°C to approximately 90°C for approximately 30 seconds to approximately 2 minutes, or to a temperature in the range of approximately 100°C to approximately 145°C for approximately 2 seconds to approximately 60 seconds.

25. The composite according to claim 1, wherein, in response to being stored in a sealed container at approximately 4°C to approximately 25°C for less than 12 months, and / or being exposed to a temperature in the range of approximately 70°C to approximately 90°C for approximately 30 seconds to approximately 2 minutes, or to a temperature in the range of approximately 100°C to approximately 145°C for approximately 2 seconds to approximately 60 seconds, the amount of biopolymer present in the composite remains within approximately 30% of the amount of biopolymer present in the composite before the storage and / or exposure.

26. The complex according to claim 1, wherein, in response to being stored in a sealed container at approximately 4°C to approximately 25°C for less than 12 months, and / or being exposed to a temperature in the range of approximately 70°C to approximately 90°C for approximately 30 seconds to approximately 2 minutes, or to a temperature in the range of approximately 100°C to approximately 145°C for approximately 2 seconds to approximately 60 seconds, the antimicrobial capacity and / or antimicrobial activity of the biopolymer present in the complex is maintained at approximately 30% and / or within approximately 30% compared to the antimicrobial capacity and / or antimicrobial activity of the biopolymer before the storage and / or exposure.

27. The complex according to claim 1, wherein the bioavailability of the protein after ingestion by the subject is increased compared to the bioavailability of the protein alone after ingestion by the subject.

28. The complex according to claim 1, wherein the complex is a particle, and the anionic biopolymer and / or cationic biopolymer encapsulates the protein.

29. The composite according to claim 1, wherein the composite in the liquid composition has an average size of about 50 nm to about 6000 nm.

30. The composite according to claim 1, wherein the size of the composite remains within ± approximately 20% of its original size when stored in a sealed container at approximately 4°C to approximately 25°C for less than 12 months, and / or when exposed to temperatures in the range of approximately 70°C to approximately 90°C for approximately 30 seconds to approximately 2 minutes, or to temperatures in the range of approximately 100°C to approximately 145°C for approximately 2 seconds to approximately 60 seconds.

31. The complex according to claim 1, wherein, after exposure to a temperature of approximately 145°C for approximately 2 to 60 seconds, the protein is degraded by less than 30% as measured by high-performance liquid chromatography.

32. The complex according to claim 1, wherein, after exposure to a temperature of approximately 70°C to approximately 90°C for approximately 30 seconds to approximately 2 minutes, the antimicrobial capacity and / or antimicrobial activity of the protein is maintained and / or improved compared to the antimicrobial capacity and / or antimicrobial activity of the protein alone.

33. A composition comprising the composite according to any one of claims 1 to 32, wherein the composition is an aqueous composition.

34. The composition according to claim 33, wherein the composition is a suspension.

35. A method for preparing a complex, A composition containing a protein, anionic biopolymer, and cationic biopolymer is prepared at a pH in the range of approximately 3 to approximately 5; and, The composition is mixed to provide the composite. The method, including the method described above.

36. The method according to claim 35, wherein the protein, anionic biopolymer, and cationic biopolymer are each independently present in the composition in an amount of about 0.01% to about 10% by weight.

37. The method according to claim 35 or 36, wherein the composition comprises the anionic biopolymer and the protein in a weight ratio of about 0.5:1 to about 1:5, and / or the composition comprises the protein and the cationic biopolymer in a weight ratio of about 1:1 to about 10:1 (protein:cationic biopolymer).

38. The method according to claim 35 or 36, wherein the composition comprises the anionic biopolymer, protein, and cationic biopolymer in a weight ratio of about 1:3:1, about 4:5:1, or about 3:6:1 (anionic biopolymer:protein:cationic biopolymer).

39. The method according to claim 35 or 36, wherein the mixing of the compositions is carried out for about 15 minutes to about 60 minutes.

40. The method according to claim 35 or 36, wherein preparing the composition comprises forming an intermediate composition containing the protein and the anionic biopolymer, and adding the cationic biopolymer to the intermediate composition to provide the composition.

41. The method according to claim 35 or 36, further comprising curing the composite in the method.

42. The method according to claim 35 or 36, further comprising separating the complex from the composition.

43. The method according to claim 35 or 36, wherein the total concentration of the protein, anionic biopolymer, and cationic biopolymer in the composition is about 10% by weight or less of the composition.

44. An article comprising the composite according to claim 1 or 2, the composition according to claim 33, and / or a composite prepared according to the method described in claim 35 or 36.

45. The article according to claim 44, wherein the article is a food, a nutritional supplement, a therapeutic beverage, and / or a cosmetic.

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