# US 12,594,308 B2 · Methods of preparing a postbiotic composition

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- Number: US 12,594,308 B2
- Document ID: US12594308B2
- Status: Granted
- Kind: B2
- Application number: 18/909,568
- Filed: 2024-10-08
- Published: 2025-04-10
- Granted: 2026-04-07
- Earliest claimed: 2023-10-09
- Assignees: Science Power Srl
- Applicants: Science Power Srl
- Inventors: Federica Nigro
- Web page: https://patentgrep.com/patent/US12594308B2

## CPC

- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- A23L 33/135: Bacteria or derivatives thereof, e.g. probiotics
- A61K 35/741: Probiotics (probiotic yeast, e.g. saccharomyces A61K36/06)
- A61K 35/744: Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K 35/745: Bifidobacteria
- A61K 35/747: Lactobacilli, e.g. L. acidophilus or L. brevis
- A61K 36/064: Saccharomycetales, e.g. baker's yeast
- A61K 47/12: Carboxylic acids; Salts or anhydrides thereof
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- C12N 1/18: Baker's yeast; Brewer's yeast
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/36: Adaptation or attenuation of cells
- 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)
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The disclosure provides methods of preparing a postbiotic composition comprising multiple (i.e., two or more) separate microbial fermentations, which may be performed in parallel or sequential steps.

## Claims

### Claim 1

A method of preparing a postbiotic composition comprising a sequential fermentation process, said sequential fermentation process comprising the steps of:
(i) inoculating a culture medium with a first fermenting microorganism;
(ii) fermenting the culture medium under conditions suitable for fermentation by the first fermenting microorganism, thereby obtaining a first fermentation matrix comprising the first fermenting microorganism;
(iii) inactivating the first fermenting microorganism in the first fermentation matrix to obtain a first fermentation product comprising the inactivated first fermenting microorganism and the first fermentation matrix;
(iv) inoculating the first fermentation product of step (iii) with a second fermenting microorganism;
(v) fermenting the first fermentation product under conditions suitable for fermentation by the second fermenting microorganism, thereby obtaining a second fermentation product comprising the second fermenting microorganism and the inactivated first fermenting microorganism;
(vi) inactivating the second fermenting microorganism in the second fermentation product to obtain a postbiotic composition comprising the inactivated first fermenting microorganism and the inactivated second fermenting microorganism, wherein at least one of the fermenting microorganisms is Lacticaseibacillus paracasei NPBO1; and
optionally, after inactivating the second fermenting microorganism of step (vi), repeating steps (iv) to (vi) one or more times with an additional fermenting microorganism.

### Claim 2 (depends on 1)

The method according to claim 1, wherein each fermenting microorganism is a different species from any other fermenting microorganism.

### Claim 3 (depends on 1)

The method according to claim 1, wherein each fermenting microorganism is selected from the group consisting of a bacterial microorganism and a yeast microorganism.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the bacterial microorganism is selected from the group consisting of a Lactobacillus species, a Lactococcus species, a Bifidobacterium species, a Lacticaseibacillus species, a Streptococcus species, an Akkermansia species, and an Escherichia species.

### Claim 5 (depends on 3)

The method according to claim 3, wherein the yeast microorganism is selected from a Saccharomyces species.

### Claim 6 (depends on 1)

The method according to claim 1, wherein each fermenting microorganism is selected from the group consisting of Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lacticaseibacillus paracasei, Bfidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Streptococcus salivarius, Akkermansia muciniphila, Escherichia coli, and Saccharomyces boulardii.

### Claim 7 (depends on 1)

The method according to claim 1, further comprising drying the inactivated fermentation product and wherein said dried inactivated fermentation product is optionally rehydrated in water prior to subsequent inoculation with a fermenting microorganism.

### Claim 8 (depends on 1)

The method according to claim 1, further comprising the step of drying the postbiotic composition.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the culture medium comprises media selected from the group consisting of MRS media, Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth, plant-derived media, functional media containing plant extracts with antioxidant, antiviral and/or antibacterial activity, culture media of natural origin, and any combination thereof.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the fermentation step is carried out at a temperature from 25° C. to 45° C.

### Claim 11 (depends on 1)

The method according to claim 1, wherein inactivation of the fermenting microorganism comprises a procedure selected from the group consisting of heat-inactivation, chemical treatment, gamma or ultraviolet irradiation, high pressure, sonication, and any combination thereof.

### Claim 12

A method of preparing a postbiotic composition comprising a sequential fermentation process, said sequential fermentation process comprising the steps of:
(i) inoculating a culture medium with a first fermenting microorganism;
(ii) fermenting the culture medium under conditions suitable for fermentation by the first fermenting microorganism, thereby obtaining a first fermentation matrix comprising the first fermenting microorganism;
(iii) inactivating the first fermenting microorganism in the first fermentation matrix to obtain a first fermentation product comprising the whole of the inactivated microbial biomass of the first fermenting microorganism and the first fermentation matrix;
(iv) inoculating the first fermentation product of step (iii) with a second fermenting microorganism;
(v) fermenting the first fermentation product under conditions suitable for fermentation by the second fermenting microorganism, thereby obtaining a second fermentation product comprising the second fermenting microorganism and the whole of the inactivated microbial biomass of the first fermenting microorganism;
(vi) inactivating the second fermenting microorganism in the second fermentation product to obtain a postbiotic composition comprising the whole of the inactivated microbial biomass of the first fermenting microorganism and the whole of the inactivated microbial biomass of the second fermenting microorganism, wherein at least one of the fermenting microorganisms is Lacticaseibacillus paracasei NPBO1; and
optionally, after inactivating the second fermenting microorganism of step (vi), repeating steps (iv) to (vi) one or more times with an additional fermenting microorganism.

### Claim 13 (depends on 1)

The method of claim 1, wherein the fermenting of the culture medium in step (ii) is carried out for a time from 2 hours to 36 hours.

### Claim 14 (depends on 13)

The method of claim 13, wherein the fermenting of the culture medium in step (ii) is carried out for a time from 6 hours to 24 hours.

### Claim 15 (depends on 1)

The method of claim 1, wherein the fermenting of the first fermentation product in step (v) is carried out for a time from 6 hours to 36 hours.