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Preparation method of nitrite reductase of lactobacillus casei subsp rhamnosus

A technology of subspecies rhamnose, Lactobacillus casei, applied in the directions of oxidoreductase, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem that the target product NiR cannot be obtained in large quantities, the separation and purification work is complicated, and the purification is affected. Work and application issues, to achieve good business prospects, low production costs, and less loss of enzyme activity

Inactive Publication Date: 2014-12-17
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the potential toxicity and risk of Pseudomonas for reducing nitrite in food, and the extraction of nitrite reductase from edible safe Lactobacillus casei has not been reported. The Chinese invention patent application number 201110207974.3 discloses " A production method of lactic acid bacteria nitrite reductase", which uses simple mechanical crushing and centrifugation to collect crude enzymes. Since the crude enzyme liquid contains a large amount of impurity proteins, the subsequent separation and purification work is complicated and cannot be produced in large quantities. The target product NiR is obtained. In addition, the medium component used for enzyme production in this method contains tomato juice. Due to the red color of tomatoes, red pigment is inevitably contained in the final NiR, which affects subsequent purification work and applications.

Method used

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  • Preparation method of nitrite reductase of lactobacillus casei subsp rhamnosus

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Embodiment 1

[0029] (1) The freeze-dried powder of Lactobacillus casei subsp. rhamnose was placed in MRS medium, and after static culture at 37° C. for 24 hours, a seed solution was prepared; said Lactobacillus casei subsp. rhamnose was purchased from China Industrial Microorganism Culture Collection Management Center, numbered CICC6013, named Lactobacillus casei subsp.rhamnosus6013 (abbreviated as LCR 6013).

[0030] (2) LCR 6013 seed solution was inoculated into induction medium A at a volume percentage of 5%, and induced and cultured statically at 37° C. for 24 hours to obtain an induction working starter. In terms of mass volume concentration, the formula of the induction medium A is: containing the inducer NaNO 2 10 μg / mL of MRS medium.

[0031] (3) Inoculate the induction working starter into the induction medium B at a volume percentage of 10%, and induce statically at 37° C. for 36 hours to obtain a culture solution containing NiR. In terms of mass volume concentration, the formu...

Embodiment 2

[0040] (1) Place the freeze-dried powder of Lactobacillus casei subsp. From the China Industrial Microorganism Culture Collection Management Center, the number is CICC6013, named Lactobacillus casei subsp.rhamnosus6013 (abbreviated as LCR 6013).

[0041] (2) LCR 6013 seed solution was inoculated into induction medium A at a volume percentage of 1%, and induced and cultured statically at 30° C. for 18 hours to obtain an induction working starter. In terms of mass volume concentration, the formula of the induction medium A is: containing the inducer NaNO 2 5 μg / mL of MRS medium.

[0042] (3) Inoculate the induction working starter into the induction medium B at a volume percentage of 5%, and induce statically at 30° C. for 24 hours to obtain a culture solution containing NiR. In terms of mass volume concentration, the formula of the induction medium B is: containing the inducer NaNO 2 10 μg / mL of MRS medium.

[0043] (4) The culture solution containing NiR was centrifuged, w...

Embodiment 3

[0049] (1) Place the freeze-dried powder of Lactobacillus casei subsp. China Industrial Microorganism Culture Collection Management Center, numbered CICC6013, named Lactobacillus casei subsp.rhamnosus6013 (abbreviated as LCR 6013).

[0050] (2) LCR 6013 seed solution was inoculated into induction medium A at a volume percentage of 3%, and induced and cultured statically at 40° C. for 20 hours to obtain an induction working starter. In terms of mass volume concentration, the formula of the induction medium A is: containing the inducer NaNO 2 20 μg / mL of MRS medium.

[0051] (3) Inoculate the induction working starter into the induction medium B at a volume percentage of 15%, and induce statically at 40° C. for 24 hours to obtain a culture solution containing NiR. In terms of mass volume concentration, the formula of the induction medium B is: containing the inducer NaNO 2 25 μg / mL of MRS medium.

[0052] (4) The culture solution containing NiR was centrifuged, washed twice ...

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Abstract

The invention discloses a preparation method of nitrite reductase of lactobacillus casei subsp rhamnosus. The method comprises the following steps: firstly, carrying inductive culture of nitrite reductase on lactobacillus casei subsp rhamnosus 6013; adding an enzyme protective agent to extract coarse nitrite reductase when the wall is broken; after carrying out vacuum-freezing and drying, finally preparing pure nitrite reductase sequentially through anion exchange chromatography and / or sephadex chromatography. The activity of NiR extracted from lactobacillus casei subsp rhamnosus is high and the condition for degrading nitrite reductase by NiR is simple. According to the disclosed condition, the enzyme activity loss of NiR is less, and the obtained NiR is high in purity and can be used for solving problem of nitrite in three fields such as vegetable fermentation, meat products and aquatic water.

Description

technical field [0001] The invention relates to the field of biological products, and relates to a preparation method of nitrite reductase in Lactobacillus casei subsp. rhamnosus6013 (abbreviated as LCR 6013). Background technique [0002] Nitrite is a potential carcinogen, which is easy to accumulate in the process of vegetable fermentation, bringing potential food safety problems to the product, and excessive intake of nitrite can induce methemoglobinemia, so strict control of nitrite in food The salt content is very important. Many studies have shown that nitrite is the precursor of nitrosamines, which are strong carcinogens that can induce various cancers in the digestive system, such as gastric cancer, intestinal cancer and liver cancer. In view of a series of problems such as the possibility of excessive nitrite pollution in food, the potential food safety problems of nitrite in meat products, and the potential food safety problems caused by excessive nitrite in aquac...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12R1/245
CPCC12N9/0044C12Y107/01004
Inventor 刘冬梅陈浩周劲松张馨月
Owner SOUTH CHINA UNIV OF TECH
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