# US 10,870,877 B2 · Methods, apparatuses and systems for analyzing microorganism strains from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon

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- Number: US 10,870,877 B2
- Document ID: US10870877B2
- Status: Granted
- Kind: B2
- Application number: 15/948,965
- Filed: 2018-04-09
- Published: 2018-08-09
- Granted: 2020-12-22
- Earliest claimed: 2015-06-25
- Assignees: Native Microbials, Inc.
- Applicants: Native Microbials, Inc.
- Inventors: Mallory Embree
- Web page: https://patentgrep.com/patent/US10870877B2

## CPC

- C12Q 1/06: Quantitative determination
- A23K 10/16: Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions (A23K10/12 takes precedence)
- A23K 10/18: of live microorganisms
- A23K 20/10: Organic substances
- A23K 50/10: for ruminants
- A23K 50/30: for swines
- A23K 50/70: for birds
- A23K 50/75: for poultry
- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- C12Q 1/689: for bacteria
- G01N 33/569: for microorganisms, e.g. protozoa, bacteria, viruses
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 2600/158: Expression markers
- C12Q 2600/178: miRNA, siRNA or ncRNA
- G01N 2333/33: from Clostridium (G)
- G01N 2333/37: from fungi
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A23K: FODDER
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

Methods, apparatuses, and systems for screening, analyzing and selecting microorganisms from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, synthesizing microbial ensembles based thereon, and forming and administering endomicrobial feed supplements are disclosed. Methods for identifying and determining the absolute cell count of microorganism types and strains, along with identifying the network relationships between active microorganisms and environmental parameters, are also disclosed.

## Claims

### Claim 1

A method, comprising: improving rumen function in a ruminant by administering an endomicrobial feed supplement thereto, the endomicrobial feed supplement including a synthetic microbial ensemble, including:
selecting at least three active microorganism strains, the selected at least three active microorganism strains included in a set of active microorganism strains identified based on results from an analysis of a plurality of rumen samples collected from a sample population of ruminants, the results including a categorization of active microorganisms strains according at least one of predicted function and chemistry, the analysis including:
detecting the presence of one or more microorganism types and determining an absolute number of each of the one or more microorganism types for each rumen sample;
measuring unique markers and quantity thereof for each rumen sample, each unique marker being a marker of a microorganism strain of one microorganism type of the one or more microorganism types;
determining an absolute cell count of each microorganism strain in each rumen sample from the absolute number of each of the one or more microorganism types and relative quantity of unique markers;
determining an activity level for each microorganism strain in each rumen sample;
selecting, based on activity level, a set of active microorganism strains and determining their respective absolute cell counts for each rumen sample;
identifying a performance indicator associated with an animal and related to each rumen sample;
measuring at least one metadata associated with the performance indicator related to each rumen sample; identifying network relationships (1) between active microorganism strains and (2) between active microorganism strains and the at least one metadata associated with the performance indicator; and
categorizing active microorganism strains based on the network relationships between the active microorganism strains and between the active microorganism strains and the at least one metadata according to at least one predicted function and chemistry;
preparing at least one active microorganism strain of the at least three active microorganism strains for inclusion in an endomicrobial feed supplement;
forming the endomicrobial feed supplement by mixing the at least one active microorganism strain of the at least three active microorganism strains with at least one medium; and
packaging the formed endomicrobial feed supplement for subsequent administration as a top dress, in a partial mixed ration, and/or in a total mixed ration to the animal to elicit a change in the performance indicator associated with the animal.

### Claim 2 (depends on 1)

The method of claim 1, further comprising identifying a subset of network relationships that exceed a specific threshold, the categorizing the active microorganism strains being based on the subset of network relationships.

### Claim 3 (depends on 1)

The method of claim 1, further comprising assigning each active microorganism strain to one of at least two groups according to the at least one predicted function and chemistry thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the analysis includes generating matrices populated with linkages denoting metadata and microorganism strain associations.

### Claim 5 (depends on 1)

The method of claim 1, wherein the analysis includes determining co-occurrence of at least one active microorganism strain and another active microorganism strain and/or the at least one metadata.

### Claim 6 (depends on 5)

The method of claim 5, wherein the determining co-occurrence is based on graph theory.

### Claim 7 (depends on 1)

The method of claim 1, wherein the absolute number of cells of each microorganism type of the plurality of microorganism types for each sample of the plurality of samples is determined via flow cytometry.

### Claim 8 (depends on 1)

The method of claim 1, wherein the unique first markers comprise a unique genomic DNA marker.

### Claim 9 (depends on 1)

The method of claim 1, wherein the unique markers comprise a unique genomic RNA marker.

### Claim 10 (depends on 1)

The method of claim 1 comprising preparing the at least three active microorganism strains for inclusion in an endomicrobial feed supplement.

### Claim 11 (depends on 10)

The method of claim 10 further comprising forming the endomicrobial feed supplement by mixing the at least three microorganism strains with the at least one medium.