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Bacillus subtilis capable of stably producing chymosin with high yield by mutation and application

A technology of Bacillus subtilis and chymosin, applied in the direction of application, bacteria, hydrolytic enzymes, etc., can solve the problems of being susceptible to shear damage, gaps, and affecting enzyme production, etc., to shorten the production cycle, reduce fermentation costs, and stabilize strains sex good effect

Inactive Publication Date: 2010-03-10
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mold growth produces a large amount of mycelium, which is susceptible to shear damage and affects enzyme production
But so far, compared with mold, there is still a certain gap in the rennet activity produced by it.
Moreover, the chymosin produced by Bacillus subtilis has the defect of high proteolytic activity, which affects the flavor and taste of cheese

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042]Example 1, Bacillus subtilis YB-3 capable of high-yielding chymosin invented by mutation breeding

[0043] According to the mutagenesis breeding method provided by the present invention, the bacillus subtilis capable of high-yielding chymosin is bred by mutagenesis, and its steps are as follows:

[0044] (1) Cultivation of the starting strain

[0045] The strain Bacillus subtilis QL-2 screened out by our laboratory was used as the starting strain;

[0046] The starting strain Bacillus subtilis QL-2 was inoculated on the sterilized modified NA liquid medium, and rejuvenated at 30°C for two generations; then the stable growing strain was inoculated in the sterilized liquid casein medium and cultured for two generations, so that It reaches the normal growth cycle, that is, the starting strain is obtained, and it is ready for use:

[0047] The casein medium consists of (g / L): casein 10, beef extract 3, NaH 2 PO 4 12H 2 O5, NaCl5, pH=7.0. Sterilize at 121°C for 20 minut...

Embodiment 3

[0065] Embodiment 3, the fermentative production enzyme of Bacillus subtilis YB-3 that can produce chymosin

[0066] Inoculate Bacillus subtilis YB-3 on sterilized modified NA liquid medium for rejuvenation and culture for 3 generations at 30°C; then inoculate the stably grown strain in sterilized liquid casein medium for 3 generations to make it reach normal growth cycle, spare.

[0067] The casein medium consists of (g / L): casein 10, beef extract 3, NaH 2 PO 4 12H 2 O5, NaCl5, pH = 7.0. Sterilize at 121°C for 20 minutes.

[0068] seed culture

[0069] The Bacillus subtilis YB-3 cultured to the logarithmic growth phase was inoculated into the sterilized seed medium, and cultured at 30° C. with shaking at 200 rpm for 36 hours, and set aside.

[0070] Described seed culture medium is made up of (g / L): whey powder 20, bran 30, NaH 2 PO 4 12H 2 O5, NaCl5, CaCl 2 1, pH=7.0. Make a solution except whey powder and sterilize at 121°C for 20 minutes; whey powder is made int...

Embodiment 4

[0081] Embodiment 4, the application of fermented rennet crude enzyme liquid

[0082] Adjust the pH of the crude enzyme solution to 6, incubate at 40°C for 15 minutes, and add it at a ratio of 1:10 until it contains 0.01mol / LCaCl 2 In the skim emulsion, heat preservation at 40°C for 20min. After the skim emulsion is coagulated into blocks, the enzyme is inactivated at 70°C, and the coagulated milk is used to cut into cheese; or it is evaporated to dryness at high temperature to prepare casein, and the curd effect is greatly improved compared with the original strain.

[0083] The milk degreasing steps are as follows: boil the fresh milk on a low heat, leave the fire source, and after it cools down, pick off a layer of cream skin formed on the surface with a glass rod, then boil, cool, and peel the skin, repeat this process several times. times until there is no more crust.

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PUM

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Abstract

The invention discloses a bacillus subtilis capable of stably producing chymosins with high yield by mutation and an application. The original strain used is a bacillus subtilis which is obtained by the screening of a laboratory and used for producing products such as cheeses and the like by fermentation, and is preserved in China Center for Type Culture Collection (CCTCC) in February 2008, with the preservation name of bacillus subtilis QL-2 (Bacillus subtilis QL2) and the preservation No. of M208023. The mutation breeding comprises the following steps of: taking the bacillus subtilis QL-2 which is preserved in CCTCC with the culture preservation No. of M208023 as the original strain, and then culturing the bacillus subtilis QL-2 on a rejuvenation culture medium and a fermentation culture medium in sequence so as to obtain the original strain suspension; using a vortex device to make the strain suspension into single spore suspension; and then conducting ultraviolet mutation and culture for a plurality of times to the single spore suspension; and finally obtaining the bacillus subtilis YB-3 (Bacillus subtilis YB-3 , CCTCC NO: M209075) capable of stably producing chymosins with high yield. The bacillus subtilis capable of stably producing chymosins with high yield can be used for preparing chymosin crude enzymes by liquid fermentation, which can be used for preparing cheeses and caseins, and shortens the clotting time of milk.

Description

technical field [0001] The invention relates to a high-yield and stable chymosin-producing Bacillus subtilis obtained by mutagenesis and its application. Specifically, it involves a strain of Bacillus subtilis (Bacillus subtilis) YB-3 capable of high-yielding chymosin, which was preserved in the China Center for Type Culture Collection on April 17, 2009, address: China.Wuhan.Wuhan University, Zip code: 430072, Tel: (027)68754052, Fax: (027)68754833, E-mail: cctcc@whu.edu.cn, its preservation number is CCTCC NO: M209075, and its mutagenesis selection method, which belongs to food biological technology field. Background technique [0002] Rennet (EC4.4.1.4) belongs to acid protease, also known as aspartic acid protease, is an indispensable preparation for cheese production, and its output value accounts for 15.5% of the total output value of the entire enzyme preparation. At present, the main source of the enzyme is the gastric mucosa of unweaned calves, some plant tissues a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/64A23C19/032C12R1/125
Inventor 李红玉李洋刘河涛
Owner LANZHOU UNIVERSITY
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