Bacillus subtilis and method for producing chymosin by using same

A technology of Bacillus subtilis and rennet, applied in the field of microbial fermentation, can solve problems such as gaps, and achieve the effects of low production cost, short production cycle and easy growth conditions

Active Publication Date: 2012-07-11
四川生力源生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with mold, the rennet activity produced by bacteria still has a certain gap.

Method used

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  • Bacillus subtilis and method for producing chymosin by using same

Examples

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Effect test

Embodiment 1

[0015] Example 1 Bacillus subtilis (Bacillus subtilis) CCTCC NO: M 2010259 screening isolation and identification 1. Bacillus subtilis T (Bacillus subtilis T) screening isolation isolated and screened. Get 6 grams of bran into a 250 milliliter sterilized Erlenmeyer flask containing 10 small glass beads, add 100 milliliters of sterile saline, vibrate and cultivate on a shaker at 150 rpm for 20 minutes, and let stand for 2 hours; The above-mentioned mixed solution of milliliter is transferred in the Erlenmeyer flask that 90 milliliters of nutrient broth medium (peptone 5g / L, glucose 5g / L, sodium chloride 5g / L) are housed, at 33 ℃, the condition of 150 rev / min Cultivate for 12 hours; take the above-mentioned culture solution, and dilute it to 10-6, 10-7, 10-8 times of dilution, and get 100 microliters of each concentration dilution and spread it on the milk medium plate (skimmed milk powder 25g / L , agar 16g / L), each concentration was repeated three times, and cultured at 33°C fo...

Embodiment 2

[0023] Example 2 Production of chymosin by liquid fermentation of Bacillus subtilis CCTCC NO:M 2010259 (1) Slant culture: Bacillus subtilis CCTCC NO:M 2010259 was inoculated on the slant medium and cultured at 33°C for 24 hours. The slant medium used was (unit: g / L): glucose 20, yeast extract 10, agar strip 20, pH natural, sterilized at 115° C. for 20 minutes.

[0024] (2) Seed culture: Take a loop of the strain grown on the slope and insert it into the seed medium with an inoculation loop, and cultivate it at 33° C. for 18 hours, with the shaker rotating at 150 rpm. The seed culture medium used is (unit: gram / liter): bran 30, sodium chloride 5, magnesium sulfate heptahydrate 5, potassium dihydrogen phosphate 2, calcium carbonate 3, pH natural; base, sterilized at 121°C for 30 minutes.

[0025] (3) Liquid fermentation culture: the fermentation medium used is (unit is gram per liter): bran 30, sodium chloride 5, magnesium sulfate heptahydrate 5, potassium dihydrogen phosphate ...

Embodiment 3

[0026] Example 3 Bacillus subtilis CCTCC NO: M 2010259 liquid fermentation production of chymosin Put the cultured seeds into a 500 ml Erlenmeyer flask, wherein the fermentation medium is 50 ml, the inoculum size is 100 microliters, and cultivated at 32°C for 96 hour, the shaker rotates at 150 rpm. The fermentation medium used was (unit: g / L): 20% glucose, 15% wheat bran, 5% sodium chloride, 5% magnesium sulfate heptahydrate, 2% potassium dihydrogen phosphate, 3% calcium carbonate, natural pH, and sterilized at 121°C for 30 minutes. Other conditions were the same as in Example 2. After the cultivation was completed, the chymosin activity of the fermented liquid was 841 SU / ml.

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Abstract

The invention relates to a strain of bacillus subtilis and a method for producing chymosin by using the same, belonging to the technical field of microbial fermentation. The invention discloses a bacterial strain producing chymosin, which is named as bacillus subtilis T and is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCCNO: M2010259. A method forapplying the bacterial strain to produce the chymosin through liquid fermentation comprises the following steps of: (1) slant culture; (2) seed culture; and (3) liquid fermentation culture: inoculating cultured seeds into a fermentation culture medium according to the inoculation quantity of 0.1-0.2%, culturing for 96-136 h at 32-36 DEG C, and controlling the rotating speed of a shaker at 150 rpm. Bacillus subtilis is common nonpathogenic bacterium and is applicable to chymosin production; the method for producing the chymosin by using the bacillus subtilis CCTCCNO: M2010259 through liquid fermentation has the advantages of high chymosin production level, short production period, low production cost, easily controlled growth conditions, convenient chymosin extraction and the like and completely has the potential of industrial production.

Description

technical field [0001] A strain of Bacillus subtilis and a method for fermenting and producing rennet with the strain, specifically a strain of Bacillus subtilis and a method for fermenting and producing rennet with the strain. The invention belongs to the technical field of microbial fermentation. Background technique [0002] Rennet is a key enzyme in cheese production. It makes raw milk coagulate into two stages: α-casein and β-casein in casein micelles will not coagulate spontaneously under the action of κ-casein, Firstly, chymosin specifically decomposes κ-casein into p-κ-casein and a macropeptide, destabilizing the casein micelles; 2+ Under the action, chemical bonds are formed between p-κ-casein and α-casein and β-casein to form a precipitate. [0003] Traditionally, rennet is extracted from the fourth stomach (abomasum) of calves. With the continuous expansion of the cheese industry, the production of rennet by slaughtering young animals can no longer meet the need...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/56C12R1/125
Inventor 丁重阳丁明亮黄博达石贵阳顾正华张梁
Owner 四川生力源生物工程有限公司
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