# US 11,306,128 B2 · Methods of purifying Mucin

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- Number: US 11,306,128 B2
- Document ID: US11306128B2
- Status: Granted
- Kind: B2
- Application number: 16/723,551
- Filed: 2019-12-20
- Published: 2020-06-25
- Granted: 2022-04-19
- Earliest claimed: 2018-12-21
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Jess Dreher Reed; Sergio Madrigal-Carballo; Michael Allen Polewski; Christian Gerald Krueger; Emilia Alfaro-Viquez
- Web page: https://patentgrep.com/patent/US11306128B2

## CPC

- C07K 1/006: {of peptides containing derivatised side chain amino acids}
- C07K 14/4727: {Mucins, e.g. human intestinal mucin}
- C07K 1/145: {by extraction or solubilisation}
- C07K 1/30: by precipitation
- C07K 1/36: by a combination of two or more processes of different types
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Methods of purifying mucin, purified mucin, and products comprising the purified mucin. The methods include combining a mucin-containing substance with water and one or more purification agents to form a purification mixture, incubating the purification mixture for a time sufficient to form a mucin precipitate in a liquid phase, and separating the mucin precipitate from the liquid phase. The purification agents include one or more of a surfactant, a chelating agent, and a protic solvent. The mucin purified from the methods can be used alone or in combination with a biopolymer such as a tannin and chitosan and can be used to generate materials in the form of a gel, a foam, a film, or a powder.

## Claims

### Claim 1

A method of purifying mucin from a mucin-containing substance comprising:
combining the mucin-containing substance with water, a surfactant, a chelating agent, and a protic solvent to form a purification mixture:
incubating the purification mixture for a time sufficient to form a mucin precipitate in a liquid phase; and
separating the mucin precipitate from the liquid phase, wherein the mucin precipitate comprises the mucin.

### Claim 2

A method of purifying mucin from a mucin-containing substance comprising:
combining the mucin-containing substance with water and more than one purification agent selected from the group consisting of a surfactant, a chelating agent, and a protic solvent to form a purification mixture, wherein the surfactant comprises at least one of an anionic surfactant, a nonionic surfactant, and a zwitterionic surfactant;
incubating the purification mixture for a time sufficient to form a mucin precipitate in a liquid phase; and
separating the mucin precipitate from the liquid phase, wherein the mucin precipitate comprises the mucin,
wherein the combining comprises combining the mucin-containing substance with the water, the surfactant, and the chelating agent.

### Claim 3 (depends on 2)

The method of claim 2, wherein the incubating is conducted at a temperature of about 0-90° C.

### Claim 4 (depends on 2)

The method of claim 2, wherein:
the surfactant comprises an anionic surfactant;
the chelating agent comprises at least one of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate dehydrate, nitriloacetic acid, and diethylenetriamine pentaacetic acid;
the protic solvent comprises an alcohol; and
the mucin-containing substance comprises mucus.

### Claim 5 (depends on 2)

The method of claim 2, wherein the combining comprises combining the mucin-containing substance with a purification solution comprising the water and the more than one purification agent selected from the group consisting of the surfactant, the chelating agent, and the protic solvent.

### Claim 6 (depends on 5)

The method of claim 5, wherein the purification solution comprises the water, the surfactant, the chelating agent, and the protic solvent.

### Claim 7 (depends on 5)

The method of claim 5, wherein the purification solution has a pH from about 6 to about 8 and comprises:
the surfactant in an amount of about 0.3-10% w/v;
the chelating agent in an amount of about 0.3-6% w/v; and
the water in an amount greater than about 90% v/v.

### Claim 8 (depends on 5)

The method of claim 5, wherein the combining comprises combining the mucin-containing substance with the purification solution in a volume ratio of from about 50:1 to about 1:50 (vol. mucin-containing substance:vol. purification solution).

### Claim 9 (depends on 2)

The method of claim 2, wherein the separating comprises:
centrifuging the mucin precipitate in the liquid phase to form a mucin precipitate pellet comprising the mucin precipitate and a liquid phase supernatant; and
removing the liquid phase supernatant from the mucin precipitate pellet.

### Claim 10 (depends on 9)

The method of claim 9, wherein the mucin precipitate pellet comprises a first fraction and a second fraction, the first fraction is denser than the second fraction, the second fraction comprises the mucin precipitate, and the method further comprises separating the first fraction from the second fraction.

### Claim 11 (depends on 2)

The method of claim 2, further comprising, after the separating, washing the mucin precipitate with an acidic wash solution having a pH of about 2-6.

### Claim 12 (depends on 2)

The method of claim 2, further comprising, after the separating, washing the mucin precipitate with an organic solvent.

### Claim 13 (depends on 2)

The method of claim 2, further comprising, after the separating, drying the mucin precipitate.

### Claim 14 (depends on 13)

The method of claim 13, wherein the dying comprises freeze-drying the mucin precipitate.

### Claim 15 (depends on 2)

A purified mucin prepared according to claim 2.

### Claim 16 (depends on 15)

A material comprising the purified mucin of claim 15.

### Claim 17 (depends on 16)

The material of claim 16, wherein the material is in the form of a gel, a foam, a film, or a powder.

### Claim 18 (depends on 16)

The material of claim 16, wherein the material further comprises a biopolymer.

### Claim 19 (depends on 16)

The material of claim 16, wherein the material further comprises a biopolymer selected from the group consisting of a tannin and chitosan.

### Claim 20 (depends on 2)

The method of claim 2, wherein the mucin-containing substance comprises mucus from at least one of a respiratory system and a gastrointestinal system of an animal.

### Claim 21 (depends on 2)

The method of claim 2, wherein the mucin-containing substance comprises mucus from at least one of lungs and intestines of an animal.

### Claim 22 (depends on 2)

The method of claim 2, wherein the mucin comprises mucin 2.

### Claim 23 (depends on 2)

The method of claim 2, wherein the surfactant comprises an anionic surfactant.