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A kind of preparation method of Bacillus licheniformis rennet

A technology of Bacillus licheniformis and chymosin, applied in the biological field, can solve the problems of low rennet activity, large loss of enzyme activity, and long fermentation time, and achieve the effects of shortening the fermentation cycle, high activity, and simplifying the preparation process

Active Publication Date: 2022-08-02
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, the activity of chymosin produced by Bacillus licheniformis screened by natural strains is not high, and the fermentation time is long, the enzyme activity is lost during the purification process, and the properties of chymosin cannot better meet the requirements of cheese production in the later stage

Method used

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  • A kind of preparation method of Bacillus licheniformis rennet
  • A kind of preparation method of Bacillus licheniformis rennet
  • A kind of preparation method of Bacillus licheniformis rennet

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] figure 2 It is a flow chart of a preparation method of Bacillus licheniformis chymosin in the embodiment of the present invention.

[0029] like figure 2 As shown, the present embodiment provides a preparation method of Bacillus licheniformis chymosin, comprising the following steps:

[0030] Step 1, spread and streak Bacillus licheniformis BL312 on the modified medium for 14-16 hours, then inoculate it in the modified medium for culturing Bacillus licheniformis to produce chymosin at 37°C, 150-170r / min shaking culture Seed solution was obtained in 12-14 hours.

[0031] The modified medium was prepared by mixing 15g casein peptone, 5g yeast extract, 50g glucose, 0.2g L-cystine, 1gNaCl, 2gNa 2 HPO 4 ·12H 2 O, 2g NaHCO 3 and 2% agar after mixing, adding deionized water to make up to 1000mL, and sterilizing at 115°C for 20min.

[0032] Step 2, the seed liquid is inoculated into the bran medium with 5% inoculum, and the fermentation liquid is obtained after shaking...

Embodiment 2

[0065] In the present embodiment, the beef extract is used as a culture medium to obtain Bacillus licheniformis rennet, comprising the following steps:

[0066] Step 1, spread Bacillus licheniformis BL312 on the beef extract medium, streak culture for 22 to 24 hours, and then inoculate it in the beef extract medium for culturing Bacillus licheniformis to produce chymosin at a temperature of 37°C and a rotating speed of Under the condition of 150~170r / min, shake and cultivate for 18~20 hours to obtain the seed liquid.

[0067] Step 2: Inoculate the seed liquid in 30 g / L bran medium with 5% inoculum at 37°C, shake and culture at 150 r / min for 80-85 hours to obtain a fermentation broth, and freeze and centrifuge the obtained fermentation broth to obtain the supernatant.

[0068] In step 3, the supernatant was mixed with ammonium sulfate, precipitated at 4°C for 2 hours, and then the precipitate was collected by high-speed freezing and centrifugation. The precipitate was dissolved...

Embodiment 3

[0077] The operation steps and conditions of this example are the same as those of Example 1, except that the seed liquid in step 2 is inoculated with 5% inoculation amount on 20g / L, 30g / L, 40g / L, 50g / L, 60g / L bran respectively In the medium with skin content, the percentage is the volume percentage.

[0078] Table 6 Effects of different bran contents on rennet activity

[0079]

[0080]

[0081] As shown in Table 6, when Bacillus licheniformis BL312 adopts the improved culture and fermentation method, the bran content is in the range of 20g / L-30g / L. The higher the bran content, the earlier the production of rennet and the higher the activity. , but beyond this range, the rennet activity will be lower and lower, and the bran content in the bran medium is 30g / L to obtain the maximum rennet activity.

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Abstract

The invention provides a method for preparing Bacillus licheniformis rennet, which is prepared by using Bacillus licheniformis BL312, and the preparation method includes the following steps: Step 1, inoculating Bacillus licheniformis BL312 on a modified medium after coating and streaking culture, and culturing to obtain Seed liquid; Step 2, inoculate the seed liquid on the bran medium, culture to obtain a fermentation liquid, freeze and centrifuge the fermentation liquid and take the supernatant; Step 3, settle the mixed liquid of the supernatant and ammonium sulfate, and then freeze and centrifuge the liquid , and collect the precipitate, dissolve the precipitate in Tris-HCl buffer, lyophilize after dialysis to obtain crude enzyme; Step 4, dissolve the crude enzyme in Tris-HCl buffer, permeate through the filter membrane, and then pass through DEAE-Sepharose Bacillus licheniformis chymosin was obtained after separation and purification by Fast Flow ion exchange column, dialysis and lyophilization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of Bacillus licheniformis chymosin. Background technique [0002] As a key enzyme in cheese processing and production, rennet has a crucial impact on the quality of cheese. With the increasing market demand year by year, the method of extracting rennet by slaughtering young animals alone can no longer meet the market demand, and a large number of slaughtering young animals is limited by the growth cycle and economic factors. Cheese is hard to accept for vegans, and a variety of factors have prompted a constant search for new sources of rennet. In recent years, some microbial rennets derived from fungi, yeasts and bacteria have shown their commercial value in cheese making and processing. Nevertheless, the search for efficient and high-quality rennet substitutes is still a major research topic today. . Due to the late start of rennet research in my ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/54C12R1/10
CPCC12N9/54C12Y304/21039
Inventor 艾连中张尧夏永军王光强熊智强张汇
Owner UNIV OF SHANGHAI FOR SCI & TECH