Bacillus amyloliquefaciens and application thereof in soy sauce fermentation

Abstract

The invention discloses bacillus amyloliquefaciens and application of the bacillus amyloliquefaciens in soy sauce fermentation, the bacillus amyloliquefaciens is YB-18 and is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the bacillus amyloliquefaciens is CGMCC No. 22582. The bacillus amyloliquefaciens YB-18 disclosed by the invention has relatively high aminopeptidase activity in a high-salt environment, and more polypeptide substances in soy sauce can be converted into free amino acids. The bacillus amyloliquefaciens YB-18 is used for producing the soy sauce with high amino acid generation rate, the amino acid generation rate in the soy sauce reaches up to 73%, the soy sauce is mellow in taste and outstanding in delicate flavor, and a new way is provided for production of the original delicate flavor soy sauce.

Description

technical field

[0001] The invention relates to the field of food brewing technology, in particular to a bacillus amyloliquefaciens and its application in soy sauce fermentation. Background technique

[0002] Amino acid nitrogen refers to the nitrogen content in the form of amino acids. The amino acid nitrogen content directly reflects the umami taste of soy sauce. The higher the amino acid nitrogen content, the more delicious the soy sauce. At present, the amino acid production rate of non-zero additive soy sauce in the market is generally 50-60%, which has caused the lack of umami taste of non-zero additive soy sauce products. In order to solve the umami problem of soy sauce, many manufacturers adopt methods such as adding sodium glutamate or yeast extract to enhance the umami. However, the method of external addition changes the original flavor characteristics of soy sauce, and it is more feasible to increase the amino acid conversion rate of soy sauce through fermentati...

Examples

preparation example Construction

[0040] (1) Preparation of seed liquid: culturing Bacillus amyloliquefaciens YB-18 in the seed medium to obtain the seed liquid;

[0041] (2) Expansion cultivation: Bacillus amyloliquefaciens YB-18 seed liquid is according to 10 2 -10 4 The inoculum amount of each / g koji is inoculated into soy sauce koji, and the koji is made at a constant temperature to obtain soy sauce koji;

[0042] (3) The soy sauce koji obtained in step (2) is mixed with salt water, and fermented at constant temperature.

[0043] further,

[0044] The seed culture medium comprises the following components in mass fractions: 10-15% of soy sauce crude oil, 3-6% of glucose and 1-2% of edible salt. Preferably, the soy sauce crude oil is a soy sauce crude oil with a mass volume concentration of ammonia nitrogen of 1.0 g / 100 mL.

[0045] further,

[0046] Cultivate 24-30h in seed culture medium to obtain seed liquid in described step (1);

[0047] Further, in the step (2), the koji is prepared at a constan...

Embodiment 1

[0052] Screening of High Aminopeptidase Strains

[0053] Take 20g of soy sauce koji, grind it, and then add a certain amount of sterile saline to dilute in proportion to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 concentration.

[0054] Take 0.1 mL of the diluted solution and spread it evenly in the selective medium, and culture it at 37°C for 1-2 days. During the cultivation process, observe the growth of the colony, the speed and size of the transparent circle formation, and measure the circle-to-diameter ratio. According to the above method, a total of 156 colonies with obvious transparent circles were screened out, among which 35 strains were particularly prominent.

[0055] Inoculate 35 more prominent colonies into LB slant medium, culture at 37°C for 2-3 days, and number YB1-YB35 respectively.

[0056] The selective medium contains 0.02 g / L of L-leucine-4-nitroaniline, 20 g / L of glucose, 3 g / L of yeast powder, 3 g / L of peptone, and 15-20 g / L of agar powder.

Embodiment 2

[0058] Breeding of Bacillus amylolyticus YB-18 with high aminopeptidase

[0059] The obtained 35 particularly significant strains were inoculated into a liquid medium and cultured at 37°C for 3 days. The components of the liquid medium were corn steep liquor 10g / L, bran 30g / L, MgSO 4 ·7H 2 O 0.2g / L, KH 2 PO 4 4g / L, CaCl 2 0.5g / L, the pH of the liquid medium is 6.5.

[0060] After the cultivation, the aminopeptidase activity of each strain was measured. The method of aminopeptidase determination was as follows: dilute the fermentation filtrate several times, take 0.4mL of the diluted solution and add it to 6mL of Tris-Hcl buffer solution with a pH value of 8.0, at 40°C Preheat the water bath for 5 minutes, then add 0.4mL 26mM L-leucine-4-nitroaniline ethanol solution (add distilled water as the control), react for 10 minutes, put it into the ice bath immediately, and measure the color at the wavelength of 405nm after 5 minutes. Under the condition of 40°C and pH value of...

technical field

[0001] The invention relates to the field of food brewing technology, in particular to a bacillus amyloliquefaciens and its application in soy sauce fermentation. Background technique

[0002] Amino acid nitrogen refers to the nitrogen content in the form of amino acids. The amino acid nitrogen content directly reflects the umami taste of soy sauce. The higher the amino acid nitrogen content, the more delicious the soy sauce. At present, the amino acid production rate of non-zero additive soy sauce in the market is generally 50-60%, which has caused the lack of umami taste of non-zero additive soy sauce products. In order to solve the umami problem of soy sauce, many manufacturers adopt methods such as adding sodium glutamate or yeast extract to enhance the umami. However, the method of external addition changes the original flavor characteristics of soy sauce, and it is more feasible to increase the amino acid conversion rate of soy sauce through fermentati...

Examples

preparation example Construction

[0040] (1) Preparation of seed liquid: culturing Bacillus amyloliquefaciens YB-18 in the seed medium to obtain the seed liquid;

[0041] (2) Expansion cultivation: Bacillus amyloliquefaciens YB-18 seed liquid is according to 10 2 -10 4 The inoculum amount of each / g koji is inoculated into soy sauce koji, and the koji is made at a constant temperature to obtain soy sauce koji;

[0042] (3) The soy sauce koji obtained in step (2) is mixed with salt water, and fermented at constant temperature.

[0043] further,

[0044] The seed culture medium comprises the following components in mass fractions: 10-15% of soy sauce crude oil, 3-6% of glucose and 1-2% of edible salt. Preferably, the soy sauce crude oil is a soy sauce crude oil with a mass volume concentration of ammonia nitrogen of 1.0 g / 100 mL.

[0045] further,

[0046] Cultivate 24-30h in seed culture medium to obtain seed liquid in described step (1);

[0047] Further, in the step (2), the koji is prepared at a constan...

Embodiment 1

[0052] Screening of High Aminopeptidase Strains

[0053] Take 20g of soy sauce koji, grind it, and then add a certain amount of sterile saline to dilute in proportion to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 concentration.

[0054] Take 0.1 mL of the diluted solution and spread it evenly in the selective medium, and culture it at 37°C for 1-2 days. During the cultivation process, observe the growth of the colony, the speed and size of the transparent circle formation, and measure the circle-to-diameter ratio. According to the above method, a total of 156 colonies with obvious transparent circles were screened out, among which 35 strains were particularly prominent.

[0055] Inoculate 35 more prominent colonies into LB slant medium, culture at 37°C for 2-3 days, and number YB1-YB35 respectively.

[0056] The selective medium contains 0.02 g / L of L-leucine-4-nitroaniline, 20 g / L of glucose, 3 g / L of yeast powder, 3 g / L of peptone, and 15-20 g / L of agar powder.

Embodiment 2

[0058] Breeding of Bacillus amylolyticus YB-18 with high aminopeptidase

[0059] The obtained 35 particularly significant strains were inoculated into a liquid medium and cultured at 37°C for 3 days. The components of the liquid medium were corn steep liquor 10g / L, bran 30g / L, MgSO 4 ·7H 2 O 0.2g / L, KH 2 PO 4 4g / L, CaCl 2 0.5g / L, the pH of the liquid medium is 6.5.

[0060] After the cultivation, the aminopeptidase activity of each strain was measured. The method of aminopeptidase determination was as follows: dilute the fermentation filtrate several times, take 0.4mL of the diluted solution and add it to 6mL of Tris-Hcl buffer solution with a pH value of 8.0, at 40°C Preheat the water bath for 5 minutes, then add 0.4mL 26mM L-leucine-4-nitroaniline ethanol solution (add distilled water as the control), react for 10 minutes, put it into the ice bath immediately, and measure the color at the wavelength of 405nm after 5 minutes. Under the condition of 40°C and pH value of...

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