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Highly-concentrated chymosin and preparation method thereof

A highly concentrated and rennet technology, which is applied in the field of highly concentrated rennet and its preparation, can solve the problems of rapid inactivation and inability to obtain high purity, maintain stability, change the dependence of foreign imported products, and prepare The effect of simple process

Inactive Publication Date: 2016-01-27
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved in the present invention is aimed at the rapid inactivation of the existing chymosin (i.e. a metalloprotease) from the supernatant of BD3526 fermentation broth with high enzyme activity after precipitation and dialysis, so that high purity and high enzyme activity cannot be obtained. To solve the problem of rennet, a highly concentrated rennet and its preparation method are provided, the preparation method can effectively protect the activity of rennet in the process of high concentration, and finally obtain a highly concentrated curd Enzyme, this highly concentrated chymosin retains high enzyme activity

Method used

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  • Highly-concentrated chymosin and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The crude enzyme activity assay of chymosin in the Paenibacillus fermented liquid of embodiment 1

[0029] Inoculate Paenibacillus BD3526 in TYC medium (refer to Chinese patent application CN104059899A for the preparation method of TYC medium), culture at 30°C for 20h as seed liquid, and inoculate the seed liquid in bran medium at 0.8% (V / V) ; 30°C, 160r / min, cultured for 48h, after the fermentation broth was centrifuged, the supernatant of the BD3526 fermentation broth was collected (for the method, refer to the Chinese patent application with publication number CN103865842A).

[0030] Get above-mentioned fermented liquid supernatant 800mL, record rennet activity at this moment and be 2660.6SU / mL, the ammonium sulfate solid powder after grinding is slowly added in the supernatant fermented liquid, makes the ammonium sulfate saturation in the final fermented liquid supernatant be 60%, mix well.

[0031] 15000g, centrifuge at 4°C for 15min to collect the salting-out pre...

Embodiment 2

[0038] The crude enzyme activity assay of chymosin in the Paenibacillus fermented liquid of embodiment 2

[0039] Ferment and collect according to the method in Example 1 to obtain 800 mL of BD3526 fermentation broth supernatant. At this time, the measured chymosin activity was 2660.6 SU / mL, and the ground ammonium sulfate solid powder was slowly added to the fermentation broth supernatant to make The ammonium sulfate saturation of the supernatant of the final fermentation broth is 60%, and the mixture is evenly mixed.

[0040] 20000g, centrifuge at 2°C for 5min to collect the salting-out precipitate, and use 2.5mmol / L CaCl 2 Dissolve it in the solution, centrifuge at 10,000 g at 4°C for 10 minutes to remove insoluble matter, and finally obtain the supernatant solution, which is the rennet liquid produced by Paenibacillus BD3526 fermentation. The obtained coarse rennet solution was 40 mL, and the rennet activity was 19343.2 SU / mL.

[0041] In this example, the chymosin activ...

Embodiment 3

[0042] The crude enzyme activity assay of chymosin in the Paenibacillus fermented liquid of embodiment 3

[0043] Ferment and collect according to the method in Example 1 to obtain 800 mL of BD3526 fermentation broth supernatant. At this time, the measured chymosin activity was 2660.6 SU / mL, and the ground ammonium sulfate solid powder was slowly added to the fermentation broth supernatant to make The ammonium sulfate saturation of the supernatant of the final fermentation broth is 60%, and the mixture is evenly mixed.

[0044] 12000g, centrifuge at 3°C ​​for 25min to collect the salting-out precipitate, and use 75mmol / L CaCl 2 Dissolve it in the solution, centrifuge at 10,000 g at 4°C for 10 minutes to remove insoluble matter, and finally obtain the supernatant solution, which is the rennet liquid produced by Paenibacillus BD3526 fermentation. The obtained coarse rennet solution was 40 mL, and the rennet activity was 22348.3 SU / mL.

[0045] In this example, the chymosin act...

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Abstract

The invention discloses highly-concentrated chymosin and a preparation method thereof. The preparation method includes the following steps that 1, fermented supernatant fluid of paenibacillus BD3526 with the preservation number of CGMCC No.8333 is taken, precipitates obtained through an ammonium sulfate precipitation method are added into a CaCl2 solution to be dissolved, and coarse chymosin fluid is obtained; 2, the coarse chymosin fluid is charged into a dialysis bag for dialysis, wherein the CaCl2 solution serves as dialyzate; 3, after dialysis is finished, the solution in the dialysis bag is subjected to vacuum-freeze drying to obtain an enzyme preparation, namely, the highly-concentrated chymosin. The preparation method is simple, practicable, convenient to implement in practice and significant in implementation effect, effectively increases the retaining rate of activity of the chymosin and greatly improves protection on the activity of the chymosin generated by paenibacillus, the purity and enzyme activity of the obtained highly-concentrated chymosin meet industrial requirements, and it is made possible to apply the chymosin preparation prepared from paenibacillus in a fermented mode to industry.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a highly concentrated chymosin and a preparation method thereof. Background technique [0002] Microbial-derived rennet is more and more widely used in cheese industrial production due to its advantages of large yield, low cost, convenient extraction and high economic benefits. Some foreign companies have used improved strains to produce high-activity rennet, which has created huge economic benefits. However, most of the rennet used in cheese production in my country still relies on imports. At present, although a large number of high-yielding chymosin strains from different sources have been screened in China, the fermentation conditions and concentration conditions of the strains have been optimized, and related enzymatic properties have been analyzed, but the stability of rennet has been found to be poor during the concentration process. Due to short half-life, rapid ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12R1/01
CPCC12N9/52C12Y304/23004
Inventor 杭锋洪青王钦博陶源刘振民陈卫
Owner BRIGHT DAIRY & FOOD
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