TW202325314A - A water-soluble chitosan polyelectrolyte complex (pec) and its compositions and applications - Google Patents

Abstract

The present disclosure provides a water-soluble chitosan polyelectrolyte complex (PEC). Also provided is a composition comprising water-soluble chitosan polyelectrolyte complex (PEC) and the applications of the complex or the composition such as anti-microorganism and anti-virus applications, adjuvants, aids or boosters for vaccines, etc.

Description

Water-soluble chitosan polyelectrolyte complex (PEC) and its composition and application

The present application relates to water-soluble chitosan polyelectrolyte complexes (PECs) and compositions containing them and their uses, such as antimicrobial and antiviral applications, adjuvants, adjuvants or reinforcing agents for vaccines, etc.

Chitin (also known as chitin, chitosan, chitooligosaccharide, chitin or chitin, etc.) is a polymer with a high natural content, and is an important component of the shell or exoskeleton of shrimps, crabs, insects, etc. Element. Chitin is a long-chain polysaccharide polymer, which is polymerized from N-acetylglucosamine sugar as a monomer. Chitin is insoluble in water. It can be deacetylated to increase water solubility, or it can be dissolved in water with the aid of organic acids or other solvents. Chitosan can be produced from deacetylated chitin (also known as chitosan, chitosan, methyltosan, chitosan, chitosan, polyglucosamine, etc.) .

Chitin has good biocompatibility with chitosan and is also known to provide microbial inhibition. However, the prior art only knows that this effect can be produced at high concentrations (at least 1,000 ppm or higher), and because chitin and chitosan are related to immune reactions, high concentrations of reagents may not be conducive to application, Therefore, related products with higher applicability and wider range are still required.

The application provides a water-soluble chitosan polyelectrolyte complex (PEC), which comprises chitosan complexed with one or more metal ions, wherein the chitosan has at least one of the following characteristics: (a) weight an average molecular weight (M _w ) of 8,000 to 1,000,000 Daltons (Dalton, Da, or g/mol); and (b) a number average molecular weight (M _n ) of 10,000 to 900,000 Daltons (or g/mol).

The present application also provides a composition comprising the chitosan polyelectrolyte complex (PEC) and one or more amino acids.

The present application also provides a method of using the chitosan polyelectrolyte complex (PEC) or the composition for preparing an antiviral or antimicrobial agent against microbial or viral infection.

The present application also provides a method of using the chitosan or its derivatives for the preparation of antiviral or antimicrobial agents, wherein the chitosan has the following characteristics: (a) the weight average molecular weight (M _w ) is 280,000 to 1,000,000 Daltons (Dalton, Da, or g/mol); and (b) a number average molecular weight (M _n ) of 300,000 to 900,000 Daltons (or g/mol).

The present application also provides the use of the chitosan polyelectrolyte complex (PEC) or the composition for preparing an antiviral or antimicrobial agent against microbial or viral infection.

The present application also provides the use of the chitosan or its derivatives for the preparation of antiviral or antimicrobial agents, wherein the chitosan has the following characteristics: (a) the weight average molecular weight (M _w ) is 280,000 to 1,000,000 Dalton (Dalton, Da, or g/mol); and (b) a number average molecular weight (M _n ) of 300,000 to 900,000 Dalton (or g/mol).

Every aspect and every embodiment of the application disclosed herein is intended to be combined individually and in all possible combinations with all other disclosed aspects and embodiments of the invention.

Molecular weight units are stated herein in Daltons (Da) or g/mol, which are equivalent in most cases.

In this specification and the claims, unless the context clearly requires otherwise, the singular forms "a" and "the" include the plural. The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

It is to be understood that the recitation of any numerical range in this specification is intended to include all subranges subsumed therein. For example, the range from "50°C to 70°C" includes all subranges between the minimum value of 50°C and the maximum value of 70°C (such as from 58°C to 67°C, 53°C to 62°C, 60°C or 68°C) and Inclusive of these two values, that is, the range including the minimum value equal to or greater than 50°C and the maximum value equal to or less than 70°C. Because disclosed numerical ranges are continuous, they include every value between the minimum and maximum values. Unless otherwise stated, the various numerical ranges indicated in this specification are approximate values. noun definition

The word "hydrocarbyl" used herein refers to an organic group containing only carbon atoms and hydrogen atoms in the main structure, such as alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, carboaryl, etc. group. The hydrocarbyl group used herein may be unsubstituted, or optionally substituted with appropriate substituents, such as halogen, nitro, hydroxyl, cyano, alkyl and other groups.

The term "alkyl" used herein refers to a group derived from an alkane molecule of the general formula C _n H _2n+2 , and may be a straight chain or a branched chain. Examples of alkyl groups such as but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, _C5 -alkyl and isomers, C ₆ -alkyl groups and isomers, C ₇ -alkyl groups and isomers, alkyl groups having 8 or more carbon atoms and isomers thereof.

The term "cycloalkyl" as used herein refers to a group derived from a fully saturated hydrocarbon molecule having a ring in its structure. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, methylcyclobutyl, cyclohexyl, and other cycloalkyl groups containing 6 or more carbon atoms and their isomers.

The term "aryl" as used herein refers to a group derived from aromatic hydrocarbon molecules. Examples of carboaryl groups can be monocyclic, such as phenyl; bicyclic, such as biphenyl, naphthyl; or polycyclic, such as anthracenyl, phenanthrenyl, and the like.

"Yene" or "alkylene" as used herein refers to a hydrocarbon group that is divalently linked to other structures. For example, "alkylene" refers to a divalent group derived from an alkane molecule.

The term "amine" as used herein refers to a molecule having at least one -NR'R" group in its molecular structure, wherein R' and R" can independently be hydrogen or a hydrocarbon group. The term "polyamine" refers to a molecule that contains two or more -NR'R" groups in its structure. For the convenience of expression, terms such as "diamine" and "triamine" are also used to specifically refer to molecules The number of -NR'R" groups contained in .

The term "alcohol" as used herein refers to a molecule having at least one -OH group in its molecular structure. The term "polyol" refers to a molecule containing two or more -OH groups in its structure. For the convenience of expression, terms such as "dihydric alcohol" and "trihydric alcohol" are also used to specifically refer to the number of -OH groups contained in the molecule.

The term "polyacid" used herein refers to a molecule with at least 2 -COOH groups in its molecular structure, or a structure derived from -COOH in its molecular structure that can produce at least 2 Molecules of -COOH; such derived structures are, for example, esters or acid anhydrides. For the convenience of expression, terms such as "dibasic acid" and "tribasic acid" are also used to specifically refer to the number of -OH groups contained in the molecule.

If there are any conflicts between the definitions of groups or molecules mentioned above, they are named according to the important order of functional groups; the naming rules can also refer to the regulations promulgated by the International Union of Pure and Applied Chemistry (IUPAC).

The term "microorganism" used herein includes, but is not limited to, microorganisms such as Gram-negative bacteria, Gram-positive bacteria, molds, spores, anaerobic bacteria, and parasites.

The term "virus" used herein includes, but is not limited to, viruses such as coronaviruses, enveloped viruses, non-enveloped viruses, DNA viruses, RNA viruses, and retroviruses.

In one aspect of the present application, a water-soluble chitosan polyelectrolyte complex (PEC) is provided, comprising chitosan complexed with one or more metal ions, wherein the chitosan has at least A characteristic of: (a) a weight average molecular weight ( _Mw ) of 8,000 to 1,000,000 Daltons (Dalton, Da, or g/mol); and (b) a number average molecular weight ( _Mn ) of 10,000 to 900,000 Daltons (or g/mol).

In another aspect of the present application, a method of using the chitosan or its derivatives for the preparation of antiviral or antimicrobial agents is provided, wherein the chitosan has the following characteristics: (a) weight average a molecular weight (M _w ) of 280,000 to 1,000,000 Daltons (Dalton, Da); and (b) a number average molecular weight (M _n ) of 300,000 to 900,000 Daltons.

In another aspect of the present application, the use of the chitosan or its derivatives for the preparation of antiviral or antimicrobial agents is provided, wherein the chitosan has the following characteristics: (a) weight average molecular weight (M _w ) is 280,000 to 1,000,000 Daltons (Dalton, Da); and (b) the number average molecular weight (M _n ) is 300,000 to 900,000 Daltons.

Chitosan (Chitosan) (also known as Chitosamine, Metosan, Chitosan, Chitosan and Polyglucosamine) is composed of glucosamine (deacetyl unit) and N- Acetylglucosamine sugars (acetyl units) are randomly distributed and combined through glycosidic bonds. Glycosidic bonds can be α(1→4) glycosidic bonds (poly(α-glucosamine)), β(1→4) glycosidic bonds (poly(β-glucosamine)) or γ(1→4) glycosidic bonds (poly(gamma-glucosamine)). As used herein, chitosan includes derivatives thereof, such as oligomers, oligomers, modified with functional groups, and the like.

In one embodiment, the _Mw (weight average molecular weight) of chitosan is at least 8,000 g/mol, preferably at least 10,000 g/mol, at least 15,000 g/mol, at least 20,000 g/mol, at least 25,000 g/mol mol, at least 30,000 g/mol, at least 35,000 g/mol, at least 40,000 g/mol, at least 50,000 g/mol, at least 60,000 g/mol, at least 70,000 g/mol, at least 80,000 g/mol, at least 90,000 g/mol, At least 100,000 g/mol, at least 110,000 g/mol, at least 120,000 g/mol, at least 130,000 g/mol, at least 140,000 g/mol, at least 150,000 g/mol, at least 160,000 g/mol, at least 170,000 g/mol, at least 180,000 g/mol, at least 190,000 g/mol, at least 200,000 g/mol, at least 250,000, preferably at least 280,000, more preferably at least 300,000. In one embodiment, the chitosan has a weight average molecular weight of at most about 1,000,000, preferably at most about 800,000, more preferably at most about 600,000. In one embodiment, the chitosan has a weight average molecular weight within a reasonable range consisting of the aforementioned endpoints, such as about 8,000 to 1,000,000 g/mol; about 8,000 to 800,000 g/mol; about 8,000 to 700,000 g/mol mol; about 8,000 to 650,000 g/mol; about 8,000 to 600,000 g/mol; about 8,000 to 550,000 g/mol; about 8,000 to 500,000 g/mol; about 8,000 to 450,000 g/mol; about 8,000 to 350,000 g/mol; about 8,000 to 300,000 g/mol; about 8,000 to 250,000 g/mol; about 8,000 to 200,000 g/mol; about 8,000 to 150,000 g/mol; to 80,000 g/mol; about 8,000 to 75,000 g/mol; about 8,000 to 70,000 g/mol; about 8,000 to 65,000 g/mol; about 8,000 to 60,000 g/mol; g/mol; about 8,000 to 45,000 g/mol; about 8,000 to 40,000 g/mol; about 8,000 to 35,000 g/mol; about 8,000 to 30,000 g/mol; about 8,000 to 15,000 g/mol; about 8,000 to 12,000 g/mol; about 10,000 to 1,000,000 g/mol; about 10,000 to 800,000 g/mol; about 10,000 to 700,000 g/mol; About 10,000 to 600,000 g/mol; about 10,000 to 550,000 g/mol; about 10,000 to 500,000 g/mol; about 10,000 to 450,000 g/mol; about 10,000 to 400,000 g/mol; about 10,000 to 350,000 g/mol; about 10,000 to 10,000 to 350,000 g/mol. 10,000 to 250,000 g/mol; 10,000 to 200,000 g/mol; 10,000 to 150,000 g/mol; 10,000 to 100,000 g/mol; about 10,000 to 70,000 g/mol; about 10,000 to 65,000 g/mol; about 10,000 to 60,000 g/mol; about 10,000 to 55,000 g/mol; about 10,000 to 40,000 g/mol; about 10,000 to 35,000 g/mol; about 10,000 to 30,000 g/mol; about 10,000 to 25,000 g/mol; about 10,000 to 20,000 g/mol; about 12,000 to 1,000,000 g/mol; about 12,000 to 800,000 g/mol; about 12,000 to 700,000 g/mol; about 12,000 to 650,000 g/mol; 00 12,000 to 450,000 g/mol; 12,000 to 400,000 g/mol; 12,000 to 350,000 g/mol; 12,000 to 300,000 g/mol; 0 g/mol; about 12,000 to 150,000 g/mol; about 12,000 to 100,000 g/mol; about 12,000 to 80,000 g/mol; about 12,000 to 60,000 g/mol; about 12,000 to 55,000 g/mol; about 12,000 to 50,000 g/mol; about 12,000 to 45,000 g/mol; about 12,000 to 40,000 g/mol; about 10,000 to 30,000 g/mol; about 12,000 to 25,000 g/mol; about 12,000 to 20,000 g/mol; about 15,000 to 1,000,000 g/mol; about 15,000 to 800,000 g/mol; 15,000 to 600,000 g/mol; 15,000 to 550,000 g/mol; 15,000 to 500,000 g/mol; 15,000 to 450,000 g/mol; 0 g/mol; about 15,000 to 300,000 g/mol; about 15,000 to 250,000 g/mol; about 15,000 to 200,000 g/mol; about 15,000 to 75,000 g/mol; about 15,000 to 70,000 g/mol; about 15,000 to 65,000 g/mol; about 15,000 to 60,000 g/mol; about 15,000 to 55,000 g/mol; about 15,000 to 45,000 g/mol; about 15,000 to 40,000 g/mol; about 15,000 to 35,000 g/mol; about 15,000 to 30,000 g/mol; about 15,000 to 25,000 g/mol; to 1,000,000 g/mol; about 20,000 to 800,000 g/mol; about 20,000 to 700,000 g/mol; about 20,000 to 650,000 g/mol; about 20,000 to 600,000 g/mol; 000 g/mol; about 20,000 to 450,000 g/mol; about 20,000 to 400,000 g/mol; about 20,000 to 350,000 g/mol; mol; about 20,000 to 150,000 g/mol; about 20,000 to 100,000 g/mol; about 20,000 to 80,000 g/mol; about 20,000 to 75,000 g/mol; about 20,000 to 70,000 g/mol; about 20,000 to 60,000 g/mol; about 20,000 to 55,000 g/mol; about 20,000 to 50,000 g/mol; about 20,000 to 45,000 g/mol; about 20,000 to 40,000 g/mol; to 30,000 g/mol; about 20,000 to 25,000 g/mol; about 25,000 to 1,000,000 g/mol; about 250,000 to 800,000 g/mol; 00g/mol 250,000 to 550,000 g/mol; 250,000 to 500,000 g/mol; 250,000 to 450,000 g/mol; 0 to 800,000 g/mol; 280,000 to 750,000 g/mol; 280,000 to 700,000 g/mol; 280,000 to 650,000 g/mol; 280,000 to 600,000 g/mol; 280,000 to 550,000 g/mol; to 450,000g/mol; 280,000 300,000 to 750,000 g/mol; 300,000 to 700,000 g/mol; 300,000 to 650,000 g/mol; 300,000 to 600,000 0g/mol; 300,000 to 550,000 to 500,000 g/mol; 300,000 to 500,000 g/mol; 300,000 to 450,000 g/mol; 300,000 to 400,000 g/mol; 350,000 to 800,000 g/mol; g/mol; 350,000 to 650,000 g/mol; 350,000 to 600,000g/mol; 350,000 to 550,000g/mol; 000 to 750,000g 400,000 to 700,000 g/mol; 400,000 to 650,000 g/mol; 400,000 to 600,000 g/mol; 00 to 750,000g/ mol; 450,000 to 700,000 g/mol; 450,000 to 650,000 g/mol; 450,000 to 600,000 g/mol; 00 to 750,000g/mol 500,000 to 700,000 g/mol; 500,000 to 650,000 g/mol; 500,000 to 600,000 g/mol; 0 to 700,000 g/mol; 550,000 to 650,000 g/mol; 550,000 to 600,000 g/mol; 600,000 to 800,000 g/mol; 600,000 to 750,000 g/mol; In one embodiment, when chitosan or its derivatives are used as an antimicrobial agent, the chitosan has a _Mw of 280,000 or more.

In another embodiment, the chitosan has a _Mw of 280,000 to 1,000,000 g/mol; 280,000 to 800,000 g/mol; 280,000 to 750,000 g/mol; 280,000 to 700,000 g/mol; 280,000 to 600,000 g/mol; 280,000 to 550,000 g/mol; 280,000 to 500,000 g/mol; 280,000 to 450,000 g/mol; 280,000 to 400,000 g/mol; to 800,000 g/mol; 300,000 to 750,000 g/mol; 300,000 to 700,000 g/mol; 300,000 to 650,000 g/mol; 300,000 to 600,000 g/mol; 0 g/mol; 300,000 to 400,000 g/mol; 350,000 to 800,000 g/mol; 350,000 to 750,000 g/mol; 350,000 to 700,000 g/mol; 350,000 to 650,000 g/mol; g/mol; 350,000 to 500,000 g/mol; 350,000 to 450,000 g/mol; 350,000 to 400,000 g/mol; 400,000 to 800,000 g/mol; 000 to 600,000 g 400,000 to 550,000 g/mol; 400,000 to 500,000 g/mol; 450,000 to 800,000 g/mol; 450,000 to 750,000 g/mol; 00 to 600,000 g/ mol; 450,000 to 550,000 g/mol; 450,000 to 500,000 g/mol; 500,000 to 800,000 g/mol; 00 to 600,000 g/mol 500,000 to 550,000 g/mol; 550,000 to 800,000 g/mol; 550,000 to 750,000 g/mol; 0 to 800,000 g/mol; 600,000 to 750,000 g/mol; 600,000 to 700,000 g/mol; or 600,000 to 650,000 g/mol. In one embodiment, when chitosan or its derivatives are used as an antimicrobial agent, the chitosan has a _Mw of 280,000 or more.

In one embodiment, the molecular weight distribution of chitosan is 3,000 to 1,000,000 g/mol, for example: 5,000 to 950,000 g/mol, 10,000 to 900,000 g/mol, 50,000 to 850,000 g/mol, 75,000 to 800,000 g/mol , 90,000 to 750,000 g/mol, 100,000 to 700,000 g/mol, 150,000 to 650,000 g/mol, 200,000 to 600,000 g/mol, 300,000 to 550,000 g/mol, 400,000 to 500,000 g/mol A reasonable range of composition, for example, 5,000 to 10,000 g/mol, etc.

In one example, the number average molecular weight M of _chitosan is 10,000 to 900,000 g/mol, 10,000 to 120,000 g/mol, 15,000 to 120,000 g/mol, 20,000 to 120,000 g/mol, 25,000 to 100,000 g/mol , 35,000 to 80,000 g/mol, preferably 38,000 to 75,000, more preferably 40,000 to 70,000 or 40,000 to 65,000 g/mol, or an appropriate range formed by any of the foregoing endpoints, for example, 20,000 to 35,000 g/mol, 35,000 to 120,000 g/mol, 100,000 to 800,000 g/mol, 150,000 to 750,000 g/mol, 200,000 to 700,000 g/mol, 300,000 to 600,000 g/mol, 400,000 to 500,000 g/mol, etc.

In one example, the number average molecular weight M _n of chitosan is 300,000 to 120,000 g/mol, 300,000 to 100,000 g/mol, 300,000 to 80,000 g/mol, preferably 380,000 to 75,000 g/mol, more preferably 400,000 to 70,000 g/mol or 400,000 to 65,000 g/mol, or an appropriate range comprised of any of the foregoing endpoints, eg 200,000 to 35,000 g/mol, 350,000 to 120,000 g/mol, etc.

In one example, the number average molecular weight _M of chitosan is 120,000 to 900,000 g/mol, 150,000 to 750,000 g/mol, 200,000 to 700,000 g/mol, 300,000 to 600,000 g/mol, 300,000 to 900,000 g/mol , 400,000 to 500,000 g/mol, etc., or an appropriate range comprised of any of the foregoing endpoints.

In one example, the chitosan has a Z average molecular weight _M of 50,000 to 10,000,000 g/mol, 70,000 to 6,000,000 g/mol, 90,000 to 1,000,000 g/mol, 100,000 to 800,000 g/mol, 110,000 to 600,000 g/mol , 150,000 to 500,000 g/mol, or a suitable range comprised of any of the foregoing endpoints, such as 50,000 to 600,000 g/mol, 70,000 to 150,000 g/mol, etc.

In one example, the chitosan has a peak molecular weight _M of at least 20,000 g/mol, at least 25,000 g/mol, at least 30,000 g/mol, at least 35,000 g/mol, at least 40,000 g/mol, at least 45,000 g/mol Or at least 50,000 g/mol. In one example, the chitosan has a peak molecular weight _M of at most 100,000 g/mol, at most 90,000 g/mol, at most 80,000 g/mol, at most 70,000 g/mol, at most 60,000 g/mol, at most 55,000 g/mol or up to 50,000 g/mol. In various examples, the peak molecular weight _M of the chitosan is within a suitable range comprised of any of the aforementioned endpoints, such as 25,000 to 100,000 g/mol, 25,000 to 45,000 g/mol, 55,000 to 100,000 g/mol wait.

In one example, the chitosan has a polydispersity index (M _w /M _n ) of at most 3.1, at most 3.0, at most 2.8, at most 2.6, at most 2.4, at most 2.2, at most 2.0, at most 1.8, at most 1.6, at most 1.5, at most 1.4, at most 1.3 or at most 1.2.

In one example, the intrinsic viscosity (or intrinsic viscosity value) of chitosan is less than 1.8 dl/g, less than 1.7 dl/g, less than 1.6 dl/g, less than 1.5 dl/g, less than 1.4 dl/gg, less than 1.3 dl/g, less than 1.2 dl/g, less than 1.1 dl/g, less than 1.0 dl/g, less than 0.9 dl/g, less than 0.8 dl/g or less than 0.7 dl/g. In one example, the intrinsic viscosity (or intrinsic viscosity value) of chitosan is at least 0.2 dl/g, at least 0.3 dl/g, at least 0.4 dl/g, at least 0.5 dl/g, at least 0.6 dl/g, at least 0.7 dl/g or at least 0.8 dl/g. In various examples, the chitosan has an intrinsic viscosity (or intrinsic viscosity value) within a suitable range comprised of any of the aforementioned endpoints, such as 0.2 dl/g to 0.8 dl/g, 0.2 dl/g to 1.8 dl/g , 0.9 dl/g to 1.6 dl/g, etc.

In one embodiment, the chitosan has a degree of deacetylation of at least 75%, preferably at least 80%, more preferably at least 85%.

In one example, the chitosan PEC has a water solubility of at least 0.5 mg/100 mL of water. In some embodiments, the chitosan PEC has a water solubility of at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 22%, at least 25%, at least 30%, at least 35% or at least 40% (w/v). In a specific example, the chitosan PEC has a water solubility of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%. In one embodiment, the pH value of the chitosan PEC solution is in the range of 5.0 to 7.0; in yet another embodiment, the pH value is in the range of 6.0 to 6.5.

In one example, the metal ion can be any metal ion with a positive charge. Examples of metal ions include, but are not limited to, zinc ions, copper ions, silver ions, gold ions, tin ions, sodium ions, calcium ions, potassium ions, magnesium ions, manganese ions, lithium ions, aluminum ions, ammonium ions, and the like. In some embodiments, the metal ion is calcium ion, zinc ion, copper ion, silver ion, gold ion or tin ion or a combination thereof. Metal ions can be derived from metal salts. Examples of metal salts include, but are not limited to, metal acetates, metal bromates, metal bromides, metal carbonates, metal chlorates, metal chlorides, metal chromates, metal dichromates, metal citrates, Metal formate, metal gluconate, metal nitrate, metal perchlorate, metal phosphate, metal salicylate, metal sulfate, metal tartrate, etc. In yet another specific example, the metal ion is zinc ion, calcium ion or a combination of zinc ion and calcium ion. In yet another specific example, based on the total weight of the water-soluble chitosan polyelectrolyte complex (PEC), the content of metal ions ranges from 8% (w/w) to 40% (w/w). In some embodiments, the content of metal ions ranges from 8% (w/w) to 35% (w/w), 8% (w/w) to 30% (w/w), 8% (w/w ) to 25% (w/w), 8% (w/w) to 20% (w/w), 8% (w/w) to 15% (w/w), 10% (w/w) to 40%(w/w), 10%(w/w) to 35%(w/w), 10%(w/w) to 30%(w/w), 10%(w/w) to 25% (w/w) or 10% (w/w) to 20% (w/w).

In one embodiment, the water-soluble chitosan polyelectrolyte complex (PEC) further comprises one or more amino acids complexed with chitosan and one or more metal ions. Examples of amino acids include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartate/aspartic acid , histidine, asparagine, glutamate/glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine , proline, selenocysteine, pyrrolysine, or any combination thereof, such as arginine, lysine, glutamic acid, aspartic acid; arginine, lysine, alanine, aspartic acid Partic acid; histidine, lysine, glutamic acid, aspartic acid; arginine, lysine, glutamic acid, asparagine; etc. In some embodiments, the amino acid is arginine, lysine, glutamic acid, or aspartic acid, or any combination thereof. In yet another embodiment, the amino acid is a combination of arginine, lysine, glutamic acid, and aspartic acid. Chitosan composition

The present application also relates to chitosan compositions comprising a water-soluble chitosan polyelectrolyte complex (PEC) as described herein and one or more amino acids.

In a specific example, based on the total weight of the water-soluble chitosan polymer electrolyte complex (PEC) and amino acid, the content range of the water-soluble chitosan polymer electrolyte complex is 15% (w /w) to 75% (w/w). In some specific examples, the content of the water-soluble chitosan polymer electrolyte complex ranges from 15% (w/w) to 70% (w/w), 15% (w/w) to 65% (w /w), 15%(w/w) to 60%(w/w), 15%(w/w) to 55%(w/w), 15%(w/w) to 50%(w/w ), 15%(w/w) to 45%(w/w), 15%(w/w) to 40%(w/w), 15%(w/w) to 35%(w/w), 15%(w/w) to 30%(w/w), 20%(w/w) to 70%(w/w), 20%(w/w) to 65%(w/w), 20% (w/w) to 60% (w/w), 20% (w/w) to 55% (w/w), 20% (w/w) to 50% (w/w), 20% (w /w) to 45%(w/w), 20%(w/w) to 40%(w/w), 20%(w/w) to 35%(w/w), 25%(w/w ) to 75% (w/w), 25% (w/w) to 70% (w/w), 25% (w/w) to 65% (w/w), 25% (w/w) to 60%(w/w), 25%(w/w) to 55%(w/w), 25%(w/w) to 50%(w/w), 25%(w/w) to 45% (w/w), 25%(w/w) to 40%(w/w), 25%(w/w) to 35%(w/w), 30%(w/w) to 75%(w /w), 30%(w/w) to 70%(w/w), 30%(w/w) to 65%(w/w), 30%(w/w) to 60%(w/w ), 30%(w/w) to 55%(w/w), 30%(w/w) to 50%(w/w), 30%(w/w) to 45%(w/w), 30%(w/w) to 40%(w/w), 35%(w/w) to 75%(w/w), 35%(w/w) to 70%(w/w), 35% (w/w) to 65% (w/w), 35% (w/w) to 60% (w/w), 35% (w/w) to 55% (w/w), 35% (w /w) to 50%(w/w), 35%(w/w) to 45%(w/w), 35%(w/w) to 40%(w/w), 40%(w/w ) to 75% (w/w), 40% (w/w) to 70% (w/w), 40% (w/w) to 65% (w/w), 40% (w/w) to 60% (w/w), 40% (w/w) to 55% (w/w), or 40% (w/w) to 50% (w/w).

Examples of amino acids include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartate/aspartic acid , histidine, asparagine, glutamate/glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine , proline, selenocysteine, pyrrolysine, or any combination thereof, such as arginine, lysine, glutamic acid, aspartic acid; arginine, lysine, alanine, aspartic acid Partic acid; histidine, lysine, glutamic acid, aspartic acid; arginine, lysine, glutamic acid, asparagine; etc. In some embodiments, the amino acid is arginine, lysine, glutamic acid, or aspartic acid, or any combination thereof. In yet another embodiment, the amino acid is a combination of arginine, lysine, glutamic acid, and aspartic acid.

In a specific example, based on the total weight of the water-soluble chitosan polyelectrolyte complex (PEC) and the amino acid, the content of the amino acid ranges from 15% (w/w) to 80% (w /w). In some specific examples, the content of the water-soluble chitosan polymer electrolyte complex ranges from 15% (w/w) to 75% (w/w), 15% (w/w) to 70% (w /w), 15%(w/w) to 65%(w/w), 15%(w/w) to 60%(w/w), 15%(w/w) to 55%(w/w ), 15%(w/w) to 50%(w/w), 15%(w/w) to 45%(w/w), 15%(w/w) to 40%(w/w), 15%(w/w) to 35%(w/w), 20%(w/w) to 75%(w/w), 20%(w/w) to 70%(w/w), 20% (w/w) to 65% (w/w), 20% (w/w) to 60% (w/w), 20% (w/w) to 55% (w/w), 20% (w /w) to 50%(w/w), 20%(w/w) to 45%(w/w), 20%(w/w) to 40%(w/w), 20%(w/w ) to 35% (w/w), 25% (w/w) to 75% (w/w), 25% (w/w) to 70% (w/w), 25% (w/w) to 65%(w/w), 25%(w/w) to 60%(w/w), 25%(w/w) to 55%(w/w), 25%(w/w) to 50% (w/w), 30%(w/w) to 80%(w/w), 30%(w/w) to 75%(w/w), 30%(w/w) to 70%(w /w), 30%(w/w) to 65%(w/w), 30%(w/w) to 60%(w/w), 30%(w/w) to 55%(w/w ), 30%(w/w) to 50%(w/w), 35%(w/w) to 80%(w/w), 35%(w/w) to 75%(w/w), 35%(w/w) to 70%(w/w), 35%(w/w) to 65%(w/w), 35%(w/w) to 60%(w/w), 35% (w/w) to 55% (w/w), 35% (w/w) to 50% (w/w), 40% (w/w) to 80% (w/w), 40% (w /w) to 75% (w/w), 40% (w/w) to 70% (w/w), 40% (w/w) to 65% (w/w), 40% (w/w ) to 60% (w/w), 40% (w/w) to 55% (w/w), 40% (w/w) to 50% (w/w), 45% (w/w) to 80%(w/w), 45%(w/w) to 75%(w/w), 45%(w/w) to 70%(w/w), 45%(w/w) to 65% (w/w), 45%(w/w) to 60%(w/w), 45%(w/w) to 55%(w/w), 45%(w/w) to 50%(w /w), 50%(w/w) to 80%(w/w), 50%(w/w) to 75%(w/w), 50%(w/w) to 70%(w/w ), 50% (w/w) to 65% (w/w) or 50% (w/w) to 60% (w/w).

In yet another embodiment, the chitosan composition includes one or more organic acids. In a particular embodiment, the organic acid is a C _1-4 carboxylic acid, preferably acetic acid. In a specific example, based on the total weight of the water-soluble chitosan polyelectrolyte complex (PEC), amino acid and organic acid, if organic acid exists, the content range of the organic acid is 30% (w/ w) to 65% (w/w). In some embodiments, the organic acid exists, and the content of the organic acid ranges from 30% (w/w) to 60% (w/w), 30% (w/w) to 55% (w/w), 30%(w/w) to 50%(w/w), 30%(w/w) to 45%(w/w), 35%(w/w) to 65%(w/w), 35% (w/w) to 60% (w/w), 35% (w/w) to 55% (w/w), 35% (w/w) to 50% (w/w), 35% (w /w) to 45%(w/w), 40%(w/w) to 65%(w/w), 40%(w/w) to 60%(w/w), 40%(w/w ) to 55% (w/w) or 40% (w/w) to 50% (w/w).

The chitosan composition may be in any form, for example, powder, suspension, solution, emulsion, granule and lozenge form.

In a specific example, the chitosan composition has a viscosity of about 40 to 500 mPa.s (25°C), preferably a viscosity of 50 to 400 mPa.s (25°C), more preferably 100 to Viscosity of 150 mPa.s (25°C).

In one embodiment, the chitosan composition may contain optional additives such as stabilizers, buffers, fragrances, and the like.

In a specific example, the chitosan composition can be in the form of a concentrated stock solution, which can be diluted to obtain the chitosan composition with the aforementioned concentration. In a specific example, the concentrated stock solution of the chitosan composition may contain the chitosan polyelectrolyte complex (PEC) as described herein in an amount ranging from 1% (w/w) to 10% (w /w). In some specific examples, the concentrated stock solution of the chitosan composition comprises the chitosan polymer electrolyte complex as described herein in an amount ranging from 1% (w/w), 2% (w/w), 3%(w/w), 4%(w/w), 5%(w/w), 6%(w/w), 7%(w/w), 8%(w/w) or 9% (w/w). .

In one embodiment, the concentrated stock solution of the chitosan composition has a pH value between 3 and 5, preferably between 3.5 and 4.5, for example about 4. In a specific example, the solid content of the concentrated stock solution of the chitosan composition is 6 to 15% by weight, preferably 8 to 10% by weight. Antimicrobial efficacy and related uses of the chitosan polyelectrolyte complex (PEC) and compositions described herein

Applicants have found that the water-soluble chitosan polyelectrolyte complex (PEC) and compositions described herein can exhibit excellent performance at low concentrations, such as excellent antimicrobial efficacy. This antimicrobial effect can fight against a wide range of bacteria and viruses; and because of the low concentration, the toxicity or side effects are minimal. In a specific example, the present application provides a method for preparing an antimicrobial agent using the water-soluble chitosan polymer electrolyte complex or composition described herein, or the water-soluble chitosan polymer electrolyte complex or Use of the composition in the preparation of an antimicrobial agent.

Without being limited by theory, the chitosan involved in this application may achieve antimicrobial effect through one or more of the following mechanisms.

Chitosan cross-links with microbial membrane proteins, destroys the cell membrane or cell wall structure, and then changes the permeability to disintegrate the microorganism: the positive charge of chitosan is adsorbed to the surface of the negatively charged microbial cell through electrostatic attraction; then Combine with cell wall proteins to penetrate the cell wall; then combine with proteins and other negatively charged substances in the outer membrane through electrostatic attraction to destroy the outer membrane proteins; finally cause the release of intracellular substances (such as genetic material, ions, etc.) to cause permeability changes to kill microorganisms.

Because the chitosan involved in this application can bind to the protein of microorganisms through positive charges (such as amine groups) to denature them, or interfere with enzyme proteins in the cell wall, causing penetration, emulsification, dispersion, etc. to destroy and disintegrate microorganisms, therefore Wide applicability and less restrictions.

In addition, due to the cross-linking between chitosan and protein, the reaction is rapid, and it can act quickly even at low concentrations, and quickly and effectively exert antimicrobial effect. Low concentration reduces biological toxicity or side effects, and it is less likely to cause pollution or affect the function of organisms when applied directly to organisms or to the environment.

In a specific example, the microorganisms include, but are not limited to, microorganisms such as protozoa, Gram-negative bacteria, Gram-positive bacteria, fungi, spores, and mycoplasma. In a specific example, the microorganisms can be Pasteurella, Bacillus anthracis, Clostridium (bacillus), Salmonella (highly drug-resistant bacteria), Candida (fungus), Mycoplasma (no cell wall), Salmonella Bacillus, Staphylococcus aureus, Escherichia coli, Candida albicans or coccidia, etc. Antiviral effects and related uses of chitosan polyelectrolyte complexes (PEC) and compositions described herein

Applicants have also discovered that the chitosan polyelectrolyte complex (PEC) and compositions described herein exhibit excellent antiviral efficacy. The antiviral effect is non-specific, so it has wide applicability; and because of the low concentration, the toxicity or side effects produced are also minimal. In a specific example, the present application provides a method for preparing an antiviral agent using the water-soluble chitosan polymer electrolyte complex (PEC) or composition described herein, or the water-soluble chitosan polymer electrolyte Use of the compound (PEC) or composition in the preparation of an antiviral agent.

Without being limited by theory, the chitosan involved in this application may achieve antiviral effect through one or more of the following mechanisms:

Chitosan cross-links with viral envelope proteins, such as the viral envelope or icosahedral envelope protein, disrupting its structure and disintegrating the virus.

In a specific example, viruses include, but are not limited to, coronaviruses, enveloped viruses, unenveloped viruses, and the like. In a specific example, the virus can be Newcastle disease virus (newcastle disease virus, such as avian orthoavulavirus1, avian paramyxovirus1), infectious bursal virus (infectious bursal disease virus), avian coronavirus (avian coronavirus, which can cause infectious bronchitis ( Infectious Bronchitis, IB)), porcine respiratory coronavirus (porcine respiratory coronavirus, PRCV), porcine transmissible gastroenteritis virus (porcine transmissible gastroenteritis virus, TGEV), infectious bursal disease virus (, very virulent strain of infectious bursal disease virus , vvIBDV; such as UK661), porcine reproductive and respiratory syndrome virus (porcine respiratory and reproductive syndrome virus, PRRSV), porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), avian influenza virus (avianinfluenza virus, AIV), Swine influenza virus (SIV), foot and mouth disease virus (FMDV), swine fever virus, etc. Auxiliary effect of chitosan polyelectrolyte complex (PEC) and composition described herein on vaccines and related uses

It has also been found that when the chitosan polyelectrolyte complex (PEC) or composition described herein is used together with a vaccine, particularly in poultry animals, it provides significant benefits in reducing the side effects of the vaccine and enhancing the efficacy of the vaccine. unexpected effect. Using the chitosan polyelectrolyte complex or composition described herein in combination with a vaccine can keep the efficacy of the vaccine unchanged or higher; however, it can reduce the side effects caused by the vaccine. Thus, the use of a chitosan polyelectrolyte complex (PEC) or composition as described herein, eg, as a vaccine adjuvant, in combination with a vaccine can increase the viability of an individual. Chitosan (composition) can be safely applied to avian embryos, subadults or adults. In one embodiment, the present application provides a method of using the water-soluble chitosan polyelectrolyte complex (PEC) or composition described herein in the preparation of an adjuvant, adjuvant or reinforcing agent. In a specific example, the present application provides the use of the water-soluble chitosan polyelectrolyte complex (PEC) or the composition described herein in the preparation of an adjuvant, auxiliary agent or reinforcing agent.

In one example, a chitosan polyelectrolyte complex (PEC) or composition as described herein is used as a food additive for vaccinated poultry. In a specific example, the application provides a method for preparing a food additive using the water-soluble chitosan polymer electrolyte complex (PEC) or composition described herein, or the chitosan polymer electrolyte complex ( PEC) or composition in the purposes of preparing food additives. In one example, a chitosan polyelectrolyte complex (PEC) or composition as described herein is co-administered to poultry with a vaccine as an adjuvant or booster. In one example, co-administration comprises administering the vaccine simultaneously with, before or after administration of the chitosan (composition). "Administration" of chitosan may include, but is not limited to, intravenous injection, nasal administration, oral administration, feeding, and the like.

The following examples illustrate the implementation of the application and explain the technical characteristics of the application, and are not intended to limit the scope of protection of the application. Any changes or equivalence arrangements that can be easily accomplished by those familiar with the technology fall within the scope of this application, and the scope of protection of the rights of this application should be based on the scope of the attached patent application. example

Analysis of the compositions disclosed herein

The components and their contents in the compositions disclosed herein can be analyzed by various chemical analysis methods, such as gas chromatography-mass spectrometry (GC-MS), energy dispersive X-ray analysis (EDS), Fourier transform infrared spectroscopy method (FTIR) and X-ray powder diffraction (XRPD), etc.

Agents used in the following examples

Yelicon Q1: The main ingredients are 2% chitosan (molecular weight from 300,000 to as high as 400,000) and 0.6% acetic acid.

Yelikang Q2 to Q6 solution (the chitosan PEC composition of the present application): the main components include the following: Element Weight ratio (w/w) Yelicon Q2 Yelicon Q3 Yelicon Q4 Yelicon Q5 Yelicon Q6 Acetic acid (~0.6% w/v) solution 83.0 83.0 83.2 83.2 82.5 Chitosan Yelicon Q2: MW: 10,000 to 50,000 Da Yelicon Q3: MW: 100,000 to 300,000 Da Yelicon Q4: MW: 250,000 to 350,00 Da Yelicon Q5: MW: 300,000 to 500,000 Da Yelicon Q6: Molecular Weight: 600,000 to 800,000 Da 10.0 8.5 6.5 4.5 3.5 amino acid 5.0 4.0 4.5 5.8 9.0 Zinc Sulfate and/or Calcium Sulfate 2.0 4.5 5.8 6.5 5.0

Jindun No.1: commercially available (supplied by Yile Company) nano-silver water-based agent, the concentration is 10,000 ppm.

Weike, gram 1000: commercially available (supplied by Yile Company) nanometer silver powder.

Yalezin: The main ingredients are 1.2% acetic acid and 3% chitosan (molecular weight from 300,000 to as high as 400,000).

Klebsiella Probiotics 1000: Nano silver powder containing Bacillus subtilis and light calcium carbonate.

example 1

In vitro safety test and toxicity test for mammalian cells

The chitosan composition of the present application (Yerlikon Q1 solution), Jindun No. 1 (diluted 10 times in this example), gram 1000 (the concentration of nano silver powder after deployment in this example is about 0.1%) and Wei Ke (In this example, the concentration of nanosilver after deployment is about 0.1%) The safety of VERO cells (African green monkey kidney cells), MARC cells (African green monkey embryonic kidney cells) and MDCK cells (mammalian cell lines) were respectively tested. Assay, assessing the cytotoxic concentration (CC50). The results are shown in FIG. 1A , FIG. 2A and FIG. 3A , the chitosan reagent of the present application shows higher safety than the known fungicides.

Further evaluate the antiviral ability of various agents under different challenge viruses to different cells.

VERO cell /PEDV

Firstly, VERO cells were used as the experimental object, and PEDV (Porcine Epidemic Diarrhea Virus, Porcine Epidemic Diarrhea Virus) was used as the challenge virus, which was diluted 5000 times for later use. After the drug is diluted or combined in different ratios, it is mixed with the virus at a volume of 1:1 to attack the cells, and the drug protection is tested. The cytopathic pathology (CPE) and cell viability are observed the next day after administration. The evaluation method is to use the Alamar Blue cell growth rate detection test as an indicator, take MOCK as the control group without drug administration and virus, and set the recovery rate as 100%; PC is the challenge control group without drug administration. Figure 1B is the test results, it can be known that the chitosan composition of the present application diluted 1000 to 10000 times (i.e. Yelicon Q, containing 2 to 20 to ppm of chitosan after dilution) obtained the cell survival rate can reach About 40% to 42%; Weike (after dilution) obtained a cell survival rate of about 28% when diluted 1,000 times, and the cell survival rate decreased when diluted to 10,000 times; and Weike (after dilution) and Yelikon Q (after dilution) The cell survival rate obtained when the 1:1 ratio mixture is diluted 10,000 times is about 25%.

In addition, VERO cells are also used as experimental subjects to test the antiviral ability of drugs. The 10-fold diluted PEDV virus liquid and the 100-fold diluted drug were mixed at a volume of 1:1 to prepare a drug/virus mixture, which was then administered to the cells to test the antiviral ability of the drug, and the cytopathic pathology (CPE) was observed the next day after administration. and cell viability; PC is the control group without drug administration; the antiviral potency is used as the evaluation index. Figure 1C shows the test results. It can be seen that the 100-fold diluted chitosan composition of the present application (i.e. Yelicon Q), the 100-fold diluted gram 1000/Yerlikon Q mixture, and the 100-fold diluted Vacron can all effectively inhibit PEDV The cytopathic effect caused by the virus; the gram 1000 diluted 100 times has the ability to resist PEDV; the 100-fold diluted Golden Shield No. 1 cannot effectively inhibit the cytopathic effect.

MARC-145 cell /PRRSV

MARC-145 cells were used as the experimental object, and PRRSV (Porcine Respiratory and Reproductive Syndrome Virus, Porcine Reproductive and Respiratory Syndrome Virus) was used as the challenge virus, diluted 100 times and then used. The diluted PRRSV virus liquid and the 100-fold diluted drug were mixed at a volume of 1:1 to obtain a drug/virus mixture; another 10-fold dilution of the drug/virus mixture was obtained to obtain a drug/virus mixture containing a 1000-fold diluted drug . The drugs were administered to the cells to test the antiviral ability of the drug, and the cytopathic pathology (CPE) and cell viability were observed the next day after administration. The evaluation method is to use the Alamar Blue cell growth rate detection test as an indicator, take MOCK as the control group without drug administration and virus, and set the recovery rate as 100%; PC is the challenge control group without drug administration. Figure 2B shows the test results. It can be seen that all drugs diluted 100 to 1000 times have antiviral ability; however, the cytotoxicity of drugs other than the chitosan composition of the present application (namely Yelikon Q) still needs to be considered.

MDCK cell /SIV

MDCK cells were used as the experimental object, and SIV (Swine Influenza Virus, swine influenza virus) was used as the challenge virus, which was diluted 10 times before use. The diluted SIV virus solution and the 100-fold diluted drug were mixed at a volume of 1:1 to prepare a drug/virus mixture. The drugs were administered to the cells to test the antiviral ability of the drug, and the cytopathic pathology (CPE) and cell viability were observed the next day after administration. The evaluation method is to test the virus potency as an indicator, and PC is the control group without drug administration. Figure 3B shows the test results. It can be seen that the 100-fold diluted Yelicon Q, Gram 1000+ Yelicon Q and Weike have better antiviral ability; the 100-fold diluted Jindun No. Gram 1000 is not effective against viruses. However, the cytotoxicity of drugs other than the chitosan composition of the present application (ie Yelicon Q) must be considered.

example 2

Virus culture and poisoning in vitro test for poultry

For various coronaviruses, enveloped viruses and non-enveloped viruses, the chitosan composition of the present application (Yerlikon Q1 solution) was used to evaluate the killing effect.

After the virus was treated with different concentrations of the chitosan composition of the present application (Jelizin, groups 1 to 6), alcohols (alcohol, group 7) and silver nanoparticles (Weike, group 8) , the virus was cultured, and the antiviral effect of each group was evaluated by virus potency. The results are shown in Table 1 below: Table 1 Chicken coronavirus (avian coronavirus) treatment virus amount: 1 x 10 ⁸ group control 1 2 3 4 5 6 7 8 Drug concentration none 1 ppm 5 ppm 10 ppm 20 ppm 40 ppm 80 ppm 75% alcohol 250 times dilution Virus price 8.17 7.28 4.41 1.23 0.14 0 0 4.63 1.52 Porcine respiratory coronavirus (PRCV) treatment virus volume: 1 x 10 ⁸ group control 1 2 3 4 5 6 7 8 Drug concentration none 1 ppm 5 ppm 10 ppm 20 ppm 40 ppm 80 ppm 75% alcohol 250 times dilution Virus price 8.67 4.97 1.19 0.36 0 0 0 5.09 1.83 Porcine transmissible gastroenteritis virus (TGEV) treatment virus volume: 1 x 10 ⁷ group control 1 2 3 4 5 6 7 8 Drug concentration none 1 ppm 5 ppm 10 ppm 20 ppm 40 ppm 80 ppm 75% alcohol 250 times dilution Virus price 7.89 5.27 1.84 0.21 0 0 0 5.67 2.01 Poultry Newcastle disease virus (avian paramyxovirus 1; APMV-1) (with envelope) treatment virus amount: 1 x 10 ⁷ group control 1 2 3 4 5 6 7 8 Drug concentration none 5 ppm 10 ppm 20 ppm 40 ppm 60 ppm 80 ppm 75% alcohol 250 times dilution Virus price 7.21 4.37 0 0 0 0 0 3.19 1.01 Infectious bursitis bursitis (vvIBDV) (non-enveloped) Treated virus volume: 1 x 10 ⁷ group control 1 2 3 4 5 6 7 8 Drug concentration none 5 ppm 10 ppm 20 ppm 40 ppm 60 ppm 80 ppm 75% alcohol 250 times dilution Virus price 7.23 5.79 2.14 0 0 0 0 4.57 1.34

It can be seen from the contents of Table 1 that when the chitosan composition of the present application contains chitosan at a concentration of 1 to 5 ppm, it can exhibit the ability to suppress various coronaviruses similar to those treated with alcohol or nano silver , and when it contains chitosan at a concentration of 10 to 20 ppm, it can effectively suppress various coronaviruses and is superior to alcohol or nano-silver treatment.

example 3

Microbial culture and toxicity test in vitro

For various microorganisms, including Clostridium (Bacillus), Salmonella (highly drug-resistant bacteria), Candida (fungus), Mycoplasma (no cell wall), Staphylococcus aureus, Escherichia coli and Candida albicans, etc., The chitosan composition of the present application (Yerlikon Q1 solution) was used to evaluate the killing effect.

Treat Clostridium, After Salmonella, Candida and Mycoplasma were cultured, these microorganisms were cultured, and the antiviral effect of each group was evaluated by virus titer. The results are shown in Table 2 below: Table 2 Clostridium (Bacillus) Treatment bacteria amount: 1 x 10 ⁸ group control 1 2 3 4 5 6 7 8 Drug concentration none 5 ppm 10 ppm 20 ppm 40 ppm 60 ppm 80 ppm 75% alcohol 250 times dilution Colony number (log10) 8.13 6.84 4.31 2.04 0.92 0 0 7.92 2.13 Salmonella (highly drug-resistant bacteria) The amount of bacteria treated: 1 x 10 ⁸ group control 1 2 3 4 5 6 7 8 Drug concentration none 5 ppm 10 ppm 20 ppm 40 ppm 60 ppm 80 ppm 75% alcohol 250 times dilution Colony number (log10) 8.13 3.28 0 0 0 0 0 3.79 0.11 Candida (fungus) Treatment bacteria amount: 1 x 10 ⁸ group control 1 2 3 4 5 6 7 8 Drug concentration none 5 ppm 10 ppm 20 ppm 40 ppm 60 ppm 80 ppm 75% alcohol 250 times dilution OD value 2.47 2.16 1.58 0.67 0 0 0 1.97 0.87 Mycoplasma (without cell wall) The amount of treated bacteria: 1 x 10 ⁸ group control 1 2 3 4 5 6 7 8 Drug concentration none 5 ppm 10 ppm 20 ppm 40 ppm 60 ppm 80 ppm 75% alcohol 250 times dilution OD value 2.56 1.84 0.79 0 0 0 0 2.06 0.64

For Staphylococcus aureus (ATCC 6538), Escherichia coli (8099) and Candida albicans (ATCC 10231), the 1000-fold dilution of Jelicin+cab was used to test the bactericidal activity according to the GB 15979-2002 standard. The neutralizing agent is D/E neutralizing broth, the action temperature is 21~22°C, the action is 30 minutes, repeated 3 times, and the incubation temperature is 36°C. The test results are shown in Table 3 below: Table 3 bacteria Staphylococcus aureus Escherichia coli Candida albicans The average bactericidal rate after 30 minutes of action (3 groups) >99.65% 99.86% >99.93%

From the results in Table 2 and Table 3, it can be seen that the minimum effective inhibitory concentration for each bacterial species is about 10 ppm to 60 ppm.

example 4

In vivo environmental test of microbial culture and inhibition

In vivo environmental test for Salmonella which is highly harmful to poultry. Apply highly resistant Salmonella to the surface of the eggshell, and then spray with different concentrations of the chitosan composition of the present application (Yerlikon Q1 solution), and then apply the treated eggshell surface to the petri dish Cultivate and evaluate the antibacterial effect, the results are shown in Table 4 below: Table 4 salmonella group control 1 2 3 4 Drug concentration 0 ppm 5 ppm 10 ppm 20 ppm 100 ppm Bacteria count (log 10) 6.0 4.7 3.7 0 0

The results showed that the growth of bacteria was completely inhibited at a chitosan concentration of 20 ppm, and the number of bacteria could be effectively reduced at a chitosan concentration of 5 ppm and 10 ppm.

example 5

In vivo safety test

In order to evaluate the safety of the chitosan composition (Yerlikon Q1 solution) of the present application, the hatching rate of hatching eggs after administration is used as an index. The control group was not administered the chitosan composition, and the experimental group was administered the chitosan composition at a concentration of 20 ppm in the incubator by atomization. The results show that the hatching efficiency of the two groups is similar (the hatching rate of the control group is 90%, and the hatching rate of the experimental group is 94%), indicating that the chitosan composition of the present application will not have a negative impact on the hatching efficiency of the eggs.

example 6

in vivo test

In order to evaluate the effect of the chitosan composition (Yerlikang Q1 solution) of the present application on the treatment of microorganisms or viral infections, 30 day-old test chickens (130 in total, 10 in each group, including completely unchallenged chickens) In vivo experiments were carried out with the control group administered with the drug (13 groups in total). The test chickens were treated with poultry Newcastle disease virus (avian paramyxovirus 1; APMV-1) (with mantle), infectious bursitis (UK661 is a very virulent strain; vvIBDV) (without mantle), mycoplasma ( No cell wall), Clostridium (bacillus), Salmonella (highly drug-resistant bacteria) and Candida (fungus) were challenged, and the chitosan composition of the present application was administered on the fourth day after the challenge For six consecutive days, clinical symptoms, survival rate and virus inspection were recorded. Test conditions and mortality rates are shown in Table 5: Table 5 pathogen APMV-1 wxya Mycoplasma Drug concentration 0 10 ppm 0 10 ppm 0 20 ppm mortality rate 100% 30% 90% 20% 80% 10% pathogen Clostridium Salmonella Candida Drug concentration 0 60 ppm 0 10 ppm 40 ppm mortality rate 100% 30% 100% 10% 90% 20%

From the results in Table 5, it can be seen that the chitosan composition of the present application has a significant therapeutic effect on experimental chickens infected with microorganisms or viruses.

The curative effect of the chitosan composition (Yerlikon Q1 solution) of the present application was also evaluated against common parasitic infections. Eimeria tenella was used as the challenge coccidia strain to challenge 30-day-old laying hens (42 chickens, 6 chickens in each group). Orally infect 50,000 coccidia/feather with cecal coccidia oocysts, administer the chitosan composition of the present application for six consecutive days on the fourth day after challenge, record the mortality rate and check the number of coccidia in the feces (OPG, number of coccidia/gram of feces). The test conditions and results are shown in Table 6 below: Table 6 coccidia infection group control 1 2 3 4 5 6 Drug concentration none 5 ppm 10 ppm 20 ppm 40 ppm 60 ppm 80 ppm mortality rate 83% 83% 67% 50% 50% 17% 0% OPG 840000 760000 480000 280000 130000 48000 0

The test results show that the chitosan composition of the present application can effectively treat coccidia infection and reduce mortality.

example 7

in vivo test

In order to evaluate the effectiveness of the chitosan of the present application (Yerlikon Q1 solution) or chitosan composition (Yerlikon Q2 solution) in combination with the vaccine, specific pathogen-free (spf) chickens (total 50 rats, 10 rats in each group, totally 5 groups including the control group) were carried out in vivo test. Test chickens were challenged with infectious bronchitis virus (IBV) type TW-I. The positive control group and the experimental group were intranasally administered live attenuated vaccines (TW-I type and TW-II type) on day 0. All five groups were then challenged (intranasally) with virus level 10 ⁶ (EID ₅₀ ) on day 14 (two weeks). Cloacal swab tests were performed on day 19 (5 days post-challenge) and all individuals were sacrificed on day 21 (7 days post-challenge) to examine the kidneys. Test conditions and results are shown in Table 7: Table 7: IBV infection group negative control positive control 1 2 3 Reagent none vaccine Vaccine+ 1: 250g Dysentery Probiotics Vaccine+ 1: 250 Yelicon Q2 Vaccine + 1: 250g Dysentery + 1: 250 Yelicon Q2 Diseases in the kidneys 100% individual 60% individual 20% individual 30% individual 10% individual Lesion degree severely blanched and swollen slight swelling No obvious lesions No obvious lesions No obvious lesions Virus content in cloaca ~10 ⁷ ~10 ⁷ ~10 ⁶ ~10 ⁶ ~10 ⁷ virus in kidney ~10 ⁶ ~10 ⁴ ~10 ³ ~0 ~10 ³

Therefore, it is clear that chitosan (composition) when used in combination with a vaccine does not affect the efficacy of the vaccine, while it can reduce negative effects such as damage in the kidneys and potentially improve the viability of the individual.

example 8

spf In Vivo Experiments on Embryos

Also in order to evaluate the effectiveness of the chitosan (Yerlikon Q1) or chitosan composition (Yerlikon Q4 and Q5 solutions) of the present application when used in combination with vaccines, specific pathogen-free (spf) vaccines at the age of 0 days Carry out in vivo tests, see Table 8 below for detailed notes: Table 8 group Inoculation conditions number Test purposes 1 Yelicon Q1 (1:100) + AIV 8 3 for virus titer, 5 for survival 2 AIV 8 3 for virus titer, 5 for survival 3 Yelicon Q4 (1:100) + NDV 5 all for survival 4 NDV 5 all for survival 5 Yelicon Q5 (1:100) + IBV 5 all for survival 6 IBV 5 all for survival 7 PBS control 3 all for survival AIV : Avian Influenza Virus NDV : Newcastle Disease Virus IBV : Infectious Bronchitis Virus

Specifically, spf embryo eggs were incubated at room temperature for 6 hours, followed by inoculation according to the conditions noted in Table 8. On day 3, samples were taken from the viral titer panel (AIV) for assessment of viral titer. On days 4-7, the survival rates of all surviving groups were recorded daily. The results of virus titer and survival are shown in Figure 4A to Figure 4C. The results show that the claimed chitosan can interfere or destroy various common virus infection/reproduction in avian embryos/sub-adults, and can also prevent the death of avian embryos/sub-adults infected (possibly) by various common viruses.

Example 9

In vivo assays for monitoring long-term antibody titers/potencies

To assess the long-term effect of chitosan (composition) on the maintenance or enhancement of antibody titer/potency when used as a food additive, an 8-week monitoring program was carried out under the conditions indicated in Table 9 below: Table 9 group feeding conditions number annotation 1 control 6 Vaccinated, no other food additives 2 Klebsiella Probiotics 1000 6 Vaccinations and indicated food additives 3 Yelicon Q6 6 Vaccinations and indicated food additives 4 Chloritis Probiotics 1000+ Yelicon Q6 6 Vaccinations and indicated food additives

Specifically, 0-day-old spf chickens were vaccinated with BIOFORS (for NDV-IBV) on day 14 (i.e., the end of week 2), and were vaccinated on days 28, 42, and 56 (i.e., at the end of week 2). 4 weeks, 6 weeks and 8 weeks) blood samples were taken for HI potency test of NDV or IBV. The HI titer assay for NDV or IBV is performed by contacting erythrocytes, NDV or IBV virus with antibody to assess long-term antibody titer/potency.

The results showed that the antibody titer to NDB in Group 1 (control) decreased slightly from week 4 to week 8 (HI force value (log ₂ ) decreased from 10 to 9). Although the antibody titers of groups 2 to 4 were slightly lower at the 4th week, they were still able to protect effectively. (HI force value (log ₂ ) of 9 or higher) compared to or even higher than that of . Similar results were shown in tests of IBV antibody titers. Obviously, the chitosan (composition) of the present application can be used as a food additive for maintaining or enhancing the long-term antibody titer/potency of vaccinated birds.

Figures 1 to 3 are the cytotoxicity tests of the chitosan composition of the present application (Yerlikon Q1) and control reagents on VERO cells (African green monkey kidney cells), MARC cells (African green monkey embryonic kidney cells) and MDCK cells result. 4A to 4C show the results of virus titer and survival of chitosan (composition) of the present application (Yerlikon Q1, Q4, Q5) and control reagents.

Claims (24)

A water-soluble chitosan polyelectrolyte complex (PEC), which comprises chitosan complexed with one or more metal ions, wherein the chitosan has one or more of the following characteristics: (a) weight average molecular weight (M _w ) of 8,000 to 1,000,000 Daltons (Dalton, Da); and; (b) a number average molecular weight (M _n ) of 10,000 to 900,000 Daltons. The complex according to claim 1, wherein the chitosan has a solubility of at least 1 g solute/100 mL water. As the compound of claim 1, it has an intrinsic viscosity (or limit viscosity value) less than 1.8 dl/g. The complex according to claim 1, wherein the chitosan has a deacetylation degree of at least 75%. The complex as claimed in item 1, wherein the metal ion is zinc ion, copper ion, silver ion, gold ion, tin ion, sodium ion, calcium ion, potassium ion, magnesium ion, manganese ion, lithium ion, aluminum ion, ammonium ion ions or combinations thereof. The compound of claim 1, wherein the metal ion is calcium ion, zinc ion, copper ion, silver ion, gold ion or tin ion or a combination thereof. The composite of claim 1, wherein the content of metal ions ranges from 8% (w/w) to 40% (w/w) based on the total weight of the composite. The complex according to claim 1, further comprising one or more amino acids complexed with the chitosan and one or more metal ions. As the compound of claim item 1, wherein the amino acid is glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid ( aspartate/aspartic acid), histidine, asparagine, glutamate/glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine , cysteine, proline, selenocysteine, pyrrolysine, or any combination thereof. The complex according to claim 1, wherein the amino acid is arginine, lysine, glutamic acid or aspartic acid or any combination thereof. A chitosan composition comprising the water-soluble chitosan polymer electrolyte complex (PEC) according to any one of claims 1 to 10 and one or more amino acids. As the chitosan composition of claim 11, wherein based on the total weight of the water-soluble chitosan polymer electrolyte complex (PEC) and amino acid, the content range of the chitosan is 15% (w /w) to 75% (w/w). As the chitosan composition of claim item 11, wherein the amino acid is glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, tianmen Aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline acid, selenocysteine, pyrrolysine, or any combination thereof. The chitosan composition according to claim 11, wherein the amino acid is arginine, lysine, glutamic acid or aspartic acid or any combination thereof. As the chitosan composition of claim 11, wherein based on the total weight of the chitosan polyelectrolyte complex (PEC) and amino acid, the content range of the amino acid is 15% (w/w ) to 80% (w/w). The chitosan composition according to claim 11, further comprising one or more organic acids. A water-soluble chitosan polyelectrolyte complex (PEC) according to any one of claims 1 to 10 or a chitosan composition according to any one of claims 11 to 15 is used to prepare an antiviral agent or Use of antimicrobial agents. A water-soluble chitosan or its derivatives for the preparation of antiviral agents or antimicrobial agents, wherein the chitosan has the following characteristics: (a) weight average molecular weight ( _Mw ) of 280,000 to 1,000,000 Dales and; (b) a number average molecular weight (M _n ) of 300,000 to 900,000 Daltons. The use according to claim 17 or 18, wherein the antiviral agent or antimicrobial agent is in the form of solution, suspension, powder, emulsion, granule or buccal tablet. The use of claim 17 or 18, wherein the antiviral agent is used for treating or preventing infection or disease of coronavirus, enveloped virus, non-enveloped virus or any combination thereof. Such as the use of claim 17 or 18, wherein the antiviral agent is used for treating or preventing infection or disease caused by coronavirus, Newcastle disease virus, infectious bursal disease virus or avian influenza virus . Such as the purposes of claim 17 or 18, wherein the antimicrobial agent is used for the treatment or prevention of protozoa, Gram-negative bacteria, Gram-positive bacteria, fungi, spores, mycoplasma or any combination of infection or disease. Such as the use of claim 22, wherein the protozoan is a coccidia. Such as the use of claim 22, wherein the Gram-positive bacteria is Staphylococcus aureus; the Gram-negative bacteria are Escherichia coli or Salmonella; or the fungus is Candida albicans.