Bacillus cereus mutant strain for high production of phospholipase C as well as fermentation method and application of bacillus cereus mutant strain

A Bacillus cereus, fermentation method technology, applied in the fields of microbial technology, microbial engineering and pharmaceutical engineering, can solve problems such as no practical application value, and achieve the effects of strong stress resistance, long survival time and easy preservation

Inactive Publication Date: 2015-10-07
陈明锴 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the PLC produced by many Gram-negative

Method used

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  • Bacillus cereus mutant strain for high production of phospholipase C as well as fermentation method and application of bacillus cereus mutant strain
  • Bacillus cereus mutant strain for high production of phospholipase C as well as fermentation method and application of bacillus cereus mutant strain
  • Bacillus cereus mutant strain for high production of phospholipase C as well as fermentation method and application of bacillus cereus mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A Bacillus cereus mutant strain with high production of phospholipase C is obtained by screening in the following manner:

[0035] Preparation of mutagenic bacteria solution: After activating the original preserved Bacillus cereus Shenzhen strain 754-1 twice, transfer it to 50ml LB culture solution / 500ml Erlenmeyer flask, and culture at 28±2°C at 200rpm for 8-12 hour, inoculate in 100ml culture solution / 500ml Erlenmeyer flask according to the inoculation amount of 8-12%, totally three bottles. Shake at 200rpm and culture for three days at 28±2°C, smear, microscopic examination, all the spores fall off. The three bottles of fermentation were combined, mixed evenly, counted with a hemocytometer, and set aside.

[0036] Bacterial fluid mutagenesis: after the counted Bacillus cereus Shenzhen strain 754-1 was diluted, plasma mutagenesis was performed.

[0037] Screening of mutant strains was performed by the egg yolk agar filter paper method, and then screened by the egg y...

Embodiment 2

[0045] The application of a high-yield phospholipase C bacillus cereus mutant strain in the preparation of phospholipase C (phospholipase, abbreviated PLC) preparation comprises the following steps:

[0046] 1) Preparation of seed solution

[0047] Put the Bacillus cereus 754-1 mutant strain obtained by screening into 50ml culture solution / 500ml Erlenmeyer flask filled with LB medium, shake at 200rpm at 28±2°C and culture for 12 hours, smear, microscopic examination, uniform coloring , growing uniformly, counted on a hemocytometer to 10 per ml 8 spare.

[0048] 2) Fermentation tank fermentation

[0049] Inoculate the seed solution prepared in step 1) in a 50L semi-automatic fermenter with an inoculum size of 10%, fill it with 30L LB-III culture solution, cultivate it at 28±2°C, 200rpm, and ventilate 1:1 (V:V) for 18 hours. put the can.

[0050] The formula of the LB-III culture solution is: 0.7% peptone, 0.3% beef extract, 0.2% yeast extract, 0.1% glucose, 0.1% urea, all i...

Embodiment 3

[0057] The preparation of PLC enzyme preparation comprises the following steps:

[0058] The three batches of fermented and purified PLC in Example 2 were adjusted to a thick liquid with 10% dry matter.

[0059] The thick liquid is formed by adding 5% skimmed milk and 5% maltodextrin.

[0060] Put the viscous material on trays with a thickness of about 1cm per tray, pre-freeze at -40°C for 4 hours under 0.01Mpa vacuum, then sublimate to 0°C, maintain for 10 hours, and finally raise the temperature to 25°C at a low speed, and dry for 6 hours. The PLC enzyme powder is obtained respectively, and the powder is white to milky white, loose, easy to powder, has no peculiar smell, and contains 3-8% of water. Tested by NPPC method, the results are shown in Table 4. From Table 4, the yield of freeze-drying is 89.23-93.44%; and the total yield of this test is 78.52-86.65%.

[0061] Table 4 NPPC method detects three batches of freeze-drying, PLC activity

[0062]

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Abstract

The invention discloses a bacillus cereus mutant strain for high production of phospholipase C (PLC) as well as a fermentation method and an application of the bacillus cereus mutant strain. A naturally screened bacillus cereus Shenzhen strain 754-1 is subjected to mutation screening, so that a bacillus cereus III-2-6 is obtained and has a preservation number of CCTCC NO:M2015277. The bacillus cereus mutant strain is high in PLC enzyme production activity by comparing with an original starting strain, is easy to produce by fermenting in an improved LB culture medium, and is short in growth period which is 16-22 hours, and by virtue of NPPC method detection, the PLC enzyme activity unit is more than that of 24.5u/ml fermentation liquor, so that the bacillus cereus mutant strain is suitable for production of the phospholipase C on large scale.

Description

technical field [0001] The present invention relates to the fields of microbial technology, microbial engineering and pharmaceutical engineering, in particular to a mutant strain of Bacillus cereus with a high yield of phospholipase C, a fermentation method of the mutant strain, and the production of phospholipid by the mutant strain Enzyme C application. technical background [0002] 1. Phospholipase C physiological activity and market [0003] Biological cell membranes are composed of more than 50% phospholipids and their lipids, and phospholipids are composed of glycerophospholipids and sphingomyelins. Phospholipids not only play a very important role in cell metabolism, signal transduction and maintenance of cell physiological activity, but also play a key role in platelet adhesion and aggregation. The adhesion and aggregation of platelets can cause the formation of human thrombus, which can prevent the bleeding of body tissues, which is conducive to hemostasis, and pr...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/16C12R1/085
CPCC12N1/20C12N9/16C12Y301/04003C12N1/205C12R2001/085
Inventor 陈明锴陈涛田华
Owner 陈明锴
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