# US 12,004,543 B2 · Salt-reduced fermentation method for high-salt dilute-state fermented soy sauce

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- Number: US 12,004,543 B2
- Document ID: US12004543B2
- Status: Granted
- Kind: B2
- Application number: 17/540,528
- Filed: 2021-12-02
- Published: 2022-03-24
- Granted: 2024-06-11
- Earliest claimed: 2021-10-20
- Assignees: Jiangnan University
- Applicants: Jiangnan University
- Inventors: Fang Fang; Guangyao Hu; Guocheng Du; Jian Chen
- Web page: https://patentgrep.com/patent/US12004543B2

## CPC

- A23L 27/50: Soya sauce
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/205: Bacterial isolates
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- C12R 2001/01: Bacteria or Actinomycetales {; using bacteria or Actinomycetales}
- C12R 2001/07: Bacillus
- Y02A 40/90: in food processing or handling, e.g. food conservation
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present disclosure discloses a salt-reduced fermentation method for high-salt dilute-state fermented soy sauce, and belongs to the technical field of fermentation engineering. The present disclosure separates and screens Weissella paramesenteroides JL-5 and Bacillus amyloliquefaciens JDF-2 which inhibit the growth of spoilage bacteria from low-salt soy sauce mash, and by changing the microbial fermentation process of high-salt dilute-state soy sauce, the strains obtained by screening are used for the fermentation of low-salt soy sauce mash. Without changing the flavor and quality of soy sauce, low-salt fermentation of the soy sauce is enabled to proceed normally, and the biogenic amine content is less than 100 mg L−1, and the number of spoilage bacteria in the soy sauce are reduced. The prepared low-salt soy sauce has an amino acid nitrogen content of greater than 1.2 g·100 mL−1, contents of various spoilage bacteria of less than 1.0×103 CFU·g−1, a NaCl concentration of no more than 10 g·100 mL−1, and a sodium content of less than or equal to 420 mg·10 mL−1. Without the addition of preservatives, the quality stability is the same as that of soy sauce without salt reduction.

## Claims

### Claim 1

A method for producing high-salt dilute-state soy sauce, comprising cooking, koji making, and fermentation;
the fermentation comprising: mixing finished koji with brine at a volume ratio of 1:(2-3), so that the final concentration of salt in the system is less than or equal to 12 g·100 mL−1, adding Weissella paramesenteroides (W. paramesenteroides) JL-5 and Bacillus amyloliquefaciens (B. amyloliquefaciens) JDF-2, or a composition containing the W. paramesenteroides JL-5 and the B. amyloliquefaciens JDF-2, and performing fermentation at 25-30° C. for at least 30 days;
wherein the W. paramesenteroides JL-5 is maintained in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2021707; and
wherein the B. amyloliquefaciens JDF-2 is maintained in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2021737.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the concentration of the W. paramesenteroides JL-5 is greater than or equal to 1.0×107 CFU·g−1, and the concentration of the B. amyloliquefaciens JDF-2 is greater than or equal to 1.0×106 CFU·g−1.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the finished koji is prepared by a method comprising: steaming defatted soybeans at 121° C. (0.1 MPa) for 20 min., uniformly mixing cooled soybean with wheat flour at a mass ratio of (4-6):1, adding a soy sauce koji starter accounting for 1-1.5% of the total mass of the mixed raw materials, performing uniform mixing, and performing culturing at 28° C.-30° C., wherein the koji making process takes 40 hours-48 hours.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the composition is a starter containing the W. paramesenteroides JL-5 and the B. amyloliquefaciens JDF-2, wheat koji, bran koji or other types of koji for fermentation.

### Claim 5 (depends on 1)

The method according to claim 1, comprising the following steps:
(1) koji making process: steaming defatted soybeans at 121° C. (0.1 MPa) for 20 min., uniformly mixing the cooled soybean with wheat flour at a mass ratio of (4-6):1, adding a soy sauce koji starter accounting for 1-1.5% of the total mass of the mixed raw materials, performing uniform mixing, and performing culturing at 28° C.-30° C., wherein the koji making process takes 40 hours-48 hour; and
(2) fermentation process: mixing the finished koji prepared in step (1) with brine containing NaCl at a volume ratio of 1:(2-3), so that the final concentration of salt in the system is less than or equal to 12 g·100 mL−1, adding the W. paramesenteroides JL-5 and the B. amyloliquefaciens JDF-2, or adding the starter, wheat koji, bran koji or other types of koji for fermentation, and performing fermentation at 25° C.-30° C. for 40 days; during fermentation, on day 3 to day 5, adding Zygosaccharomyces rouxii ZQ01, and performing stirring during the fermentation.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the concentration of the W. paramesenteroides JL-5 in a soy sauce mash containing salt with a concentration of 12 g·100 mL−1 is at least 1.0×107 CFU·g−1.

### Claim 7 (depends on 5)

The method according to claim 5, wherein the number of the W. paramesenteroides JL-5 added and the number of the B. amyloliquefaciens JDF-2 added are in a same order of magnitude.

### Claim 8 (depends on 5)

The method according to claim 5, wherein the method makes the produced soy sauce have at least one of the following features (a)-(e):
(a) the number of spoilage bacteria in the produced soy sauce is reduced compared to a control soy sauce not made by the method;
(b) the salt content in the produced soy sauce is less than or equal to 12 g·100 mL−1;
(c) the biogenic amine content in the produced soy sauce is reduced compared to a control soy sauce not made by the method;
(d) the stability during the shelf life of the produced soy sauce is improved compared to a control soy sauce not made by the method; and
(e) the formation of volatiles in the produced soy sauce is promoted.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the spoilage bacteria comprise at least one of: Bacillus subtilis, Bacillus halodurans, Bacillus megaterium, Staphylococcus saprophytics, Kurthia zopfii, Kurthia gibsonii, and Lactobacillus pobuzihii.

### Claim 10

A starter containing Weissella paramesenteroides JL-5 and/or Bacillus amyloliquefaciens JDF-2, wheat koji, bran koji or other types of koji for fermentation, wherein the W. paramesenteroides JL-5 is maintained in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2021707; and
the B. amyloliquefaciens JDF-2 is maintained in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2021737.