# US 10,765,131 B2 · Nisin production process

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- Number: US 10,765,131 B2
- Document ID: US10765131B2
- Status: Granted
- Kind: B2
- Application number: 14/760,990
- Filed: 2014-01-15
- Published: 2015-12-17
- Granted: 2020-09-08
- Earliest claimed: 2013-01-15
- Assignees: Purac Biochem B.V.
- Applicants: Purac Biochem B.V.
- Inventors: Brenda Marja Dierdorp-Andreae; Jasper Meijer; Scott Allan McElmury; Johannes Cornelius Adrianus Blonk; Lansen Court Morehouse
- Web page: https://patentgrep.com/patent/US10765131B2

## CPC

- A23B 2/7295: {Antibiotics}
- A23B 2/762: Organic compounds containing nitrogen
- A23B 2/767: Organic compounds containing sulfur
- A23B 2/783: Microorganisms; Enzymes
- A23C 9/123: using only microorganisms of the genus lactobacteriaceae; Yoghurt (A23C9/13 takes precedence)
- C07K 14/315: from Streptococcus (G), e.g. Enterococci
- C12P 7/56: Lactic acid
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- A23V 2400/231: Lactis
- A23B: PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
- A23C: DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF (preservation thereof A23B11/00)
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION

## Abstract

The present invention relates to fermentation processes. The objective of the invention was to provide methods for producing nisin containing ferments and formulating such ferments into preservatives. It was in particular an object of the present invention to provide improved processes for producing nisin containing ferments by simple and efficient (batch) culturing and allowing for simple down-stream processing either into a liquid, semi-liquid or a dry powder product, having sufficient nisin content and stability, good handling properties and satisfactory organoleptic properties. Such processes are provided, as well as the products that can be obtained with it and their use as food preservative.

## Claims

### Claim 1

A process of producing a nisin-containing lactate ferment in dry powder form, comprising the consecutive steps of:
(a) inoculating a nutrient medium comprising a solution of a fermentable substrate and a nitrogen source in an aqueous medium with nisin producing lactic acid bacteria; and
(b) incubating the inoculated nutrient medium under conditions favorable to the growth and/or metabolic activity of said lactic acid bacteria for a period sufficient to produce a fermentation broth containing at least 20 g/l of lactate equivalents and/or at least 1000 IU/ml of nisin equivalent activity, during which period the pH of the fermentation broth is controlled by addition of one or more alkalization agents comprising sodium hydroxide and calcium hydroxide, wherein the Na:Ca (w/w) ratio of the added hydroxides is between ⅙ 1/1; and
(c) spray drying the ferment obtained in step (b) to obtain a free flowing powder containing anhydrous calcium lactate in combination with sodium lactate as the bulk of the powder, by evaporating substantially all water.

### Claim 2 (depends on 1)

The process according to claim 1, wherein the Na:Ca (w/w) ratio of the added salts is between ⅙ ½.

### Claim 3 (depends on 1)

The process according to claim 1, the pH of the fermentation broth is maintained between 4.8 to 6.0.

### Claim 4 (depends on 1)

The process according to claim 1, wherein the nutrient medium comprises a dairy product.

### Claim 5 (depends on 1)

The process according to claim 1, wherein the inoculation medium comprises activated lactic acid bacteria.

### Claim 6 (depends on 1)

The process according to claim 1, further comprising (b′) acidifying the fermentation broth by adding an acid in an amount sufficient to reduce the pH of the fermentation broth to below 4.8 before step (c).

### Claim 7 (depends on 6)

The process according to claim 6, wherein the acid is lactic acid.

### Claim 8 (depends on 1)

The process according to claim 1, further comprising inactivating the lactic acid bacteria by subjecting the fermentation broth to a temperature of at least 40° C. for a period of at least 10 minutes before step (c).

### Claim 9 (depends on 1)

The process according to claim 1, wherein step (c) comprises concentrating the ferment to a concentrate having a dry solids content of at least 30 wt. %.

### Claim 10 (depends on 9)

The process according to claim 9, further comprising (d) cooling the concentrate to a temperature of below 40° C.

### Claim 11 (depends on 1)

The process according to claim 1, wherein the powder has a water content of less than 5 wt %.

### Claim 12 (depends on 1)

The process according to claim 1, wherein the nutrient medium comprises whey.

### Claim 13 (depends on 1)

The process according to claim 1, which does not comprise a step of removing non-dissolved matter from the fermented broth.

### Claim 14 (depends on 1)

A ferment produced by the process according to claim 1.

### Claim 15 (depends on 14)

The ferment according to claim 14, containing at least 0.25 mg nisin per gram of dry matter.

### Claim 16 (depends on 14)

A method of preserving an alimentary product, comprising adding the ferment of claim 14 to the product.

### Claim 17 (depends on 1)

The process according to claim 1, wherein the spray drying is after acidification of the fermentation broth and/or subjecting the fermentation broth to a temperature of at least 40° C. for a period of at least 10 minutes, to obtain a free flowing powder.

### Claim 18

A process of producing a nisin-containing lactate ferment, comprising the consecutive steps of:
(a) inoculating a nutrient medium comprising a solution of a fermentable substrate and a nitrogen source in an aqueous medium with nisin producing lactic acid bacteria; and
(b) incubating the inoculated nutrient medium under conditions favorable to the growth and/or metabolic activity of said lactic acid bacteria for a period sufficient to produce a fermentation broth containing at least 20 g/l of lactate equivalents and/or at least 1000 TU/ml of nisin equivalent activity, during which period the pH of the fermentation broth is controlled by addition of one or more alkalization agents comprising sodium hydroxide and calcium hydroxide, wherein the Na:Ca (w/w) ratio of the added hydroxides is between ⅙ 1/1,
wherein the pH of the fermentation broth is maintained between 5.0 to 6.0 in step (b); and
(c) evaporating substantially all water from the ferment to obtain a free flowing powder containing anhydrous calcium lactate in combination with sodium lactate as the bulk of the powder.