# US 12,111,302 B2 · Diagnostic assay for rumen undegraded protein detection

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- Number: US 12,111,302 B2
- Document ID: US12111302B2
- Status: Granted
- Kind: B2
- Application number: 17/285,460
- Filed: 2019-10-10
- Published: 2021-11-04
- Granted: 2024-10-08
- Earliest claimed: 2018-10-17
- Assignees: Novita Nutrition, LLC
- Applicants: Novita Nutrition, LLC
- Inventors: Keith E. Bruinsma; Donald L. Endres; Andrew James Manning; Kurt M. Swenson
- Web page: https://patentgrep.com/patent/US12111302B2

## CPC

- G01N 33/02: Food
- G01N 27/447: using electrophoresis
- G01N 27/44726: {using specific dyes, markers or binding molecules}
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- Y10T 436/25375: Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A diagnostic assay to determine a percentage rumen undegraded protein in a food product by comparing the percentage of modified protein content to the total crude protein content of the food product. The total crude protein content extracted from a food product sample prior to conducting gel electrophoresis to separate modified protein content from the total crude protein content. The gel band intensities of the separated protein contents analyzed to enumerate a ratio that is used to derive a correlated percentage numen undegraded protein for a given food product sample. The diagnostic assay capable of being provided onsite for quick, cost-efficient and accurate results.

## Claims

### Claim 1

A method for testing a food product for rumen undegraded protein, the method comprising:
extracting a total protein content from an assay sample of the food product to provide an extraction sample, wherein the total protein content of the extraction sample comprises a rumen undegraded protein content;
conducting gel electrophoresis on the extraction sample applied to a first gel and a second gel to separate the rumen undegraded protein content from the total protein content, thereby providing a residual total protein content;
staining the first gel after separating the rumen undegraded protein content from the total protein content, wherein staining the first gel comprises applying a stain capable of staining glycosylated proteins in a polyacrylamide gel such that the stained glycosylated proteins correspond to the separated rumen undegraded protein content;
staining the second gel after separating the rumen undegraded protein content from the total protein content, wherein staining the second gel comprises applying a stain capable of staining the total protein content in the extraction sample including both the separated rumen undegraded protein content and the residual total protein content in a polyacrylamide gel;
and conducting an intensity analysis of the stained separated rumen undegraded protein content in the first gel and the stained total protein content in the second gel to determine a percentage of rumen undegraded protein in the total protein content of the food product.

### Claim 2 (depends on 1)

The method of claim 1, wherein the food product is selected from an animal feed product and an animal feed supplement.

### Claim 3 (depends on 1)

The method of claim 1, wherein the food product is a byproduct derived from an ethanol fermentation process.

### Claim 4 (depends on 3)

The method of claim 3, wherein the food product is distillers meal or corn distillers meal.

### Claim 5 (depends on 1)

The method of claim 1, further comprising prior to the extracting, preparing the assay sample comprising grinding at least a portion of the food product to a particle size ranging from about 50 microns to about 200 microns to prepare the assay sample.

### Claim 6 (depends on 5)

The method of claim 5, wherein the portion of the food product is cooled during grinding to a temperature less than 0° C. using liquid nitrogen or liquid carbon dioxide.

### Claim 7 (depends on 1)

The method of claim 1, wherein extracting a total protein content comprises mixing an extraction buffer with the assay sample.

### Claim 8 (depends on 7)

The method of claim 7, wherein the extraction buffer comprises one or more of ethylenediamine tetra acetic acid, Tris-HCl, sodium dodecyl sulfate, and ß-mercaptoethanol.

### Claim 9 (depends on 7)

The method of claim 7, wherein extracting a total protein content further comprises heating the assay sample mixed with the extraction buffer to a temperature range of about 50° C. to about 120° C.

### Claim 10 (depends on 9)

The method of claim 9, wherein after the heating, extracting a total protein content further comprises cooling the assay sample mixed with the extraction buffer to a temperature between about 0° C. and about 21° C. and then centrifuging the assay sample mixed with the extraction buffer.

### Claim 11 (depends on 10)

The method of claim 10, wherein after the assay sample mixed with the extraction buffer is centrifuged, extracting a total protein content further comprises aspirating a first liquid phase from the assay sample and collecting the first liquid phase to provide the extraction sample.

### Claim 12 (depends on 11)

The method of claim 11, wherein extracting a total protein content is repeated at least once before conducting gel electrophoresis on the extraction sample.

### Claim 13 (depends on 11)

The method of claim 11, further comprising washing a residual assay sample defined by the assay sample after collection of the first liquid phase to provide the extraction sample, wherein washing the residual assay sample comprises mixing the residual assay sample with a washing buffer to provide a washed assay sample.

### Claim 14 (depends on 13)

The method of claim 13, wherein washing the residual assay sample further comprises centrifuging the washed assay sample, aspirating a second liquid phase from the centrifuged washed assay sample and then collecting the second liquid phase and combining it with the first liquid phase to provide the extraction sample.

### Claim 15 (depends on 14)

The method of claim 14, wherein washing the residual assay sample is repeated at least once.

### Claim 16 (depends on 14)

The method of claim 14, wherein conducting gel electrophoresis is conducted on the combined first and second liquid phases to separate a rumen undegraded protein content from the total protein content.

### Claim 17 (depends on 14)

The method of claim 14, wherein the gel electrophoresis is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a polyacrylamide percentage of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis between about 4% and 12% polyacrylamide.

### Claim 18 (depends on 1)

The method of claim 1, further comprising mixing the extraction sample with a sample buffer prior to conducting gel electrophoresis wherein the sample buffer comprises one or more of Tris-HCl, glycerol, sodium dodecyl sulfate, bromophenol blue, and ß-mercaptoethanol.

### Claim 19 (depends on 1)

The method of claim 1, wherein conducting gel electrophoresis on the extraction sample is conducted at a voltage range of about 90 volts to 130 volts until the extraction sample migrates from a starting point to an end point on the first and second gels.

### Claim 20 (depends on 1)

The method of claim 1, further compromising de-staining the first and second gels using a de-stain.

### Claim 21 (depends on 1)

The method of claim 1, wherein the intensity analysis comprises comparing a band intensity of the stained separated rumen undegraded protein content in the first gel to a band intensity of the total protein content in the second gel.