CN107988109B - A kind of Flavobacterium mutant strain and its application - Google Patents

Abstract

The invention provides a flavobacterium mutant strain, the preservation number of which is CGMCC No. 14855. The strain protected by the invention is obtained by carrying out long-term fermentation and screening on the existing flavobacterium which produces fucosan sulfatase. In large-scale fermentation, the capability of the strain for producing the fucosan sulfatase by fermentation is obviously improved, and the capability of producing the fucosan sulfatase can reach 310U/ml at most.

Description

A kind of Flavobacterium mutant strain and its application

technical field

The invention belongs to the technical field of mutation screening of beneficial microorganisms, and in particular relates to a mutant strain of Flavobacterium and its application.

Background technique

Fucoidan, also known as fucoidan, is a high-molecular-weight heteropolysaccharide with sulfate groups, which exists in marine brown algae and some echinoderms. In recent years, fucoidan has been Biological activities, including anti-tumor, anti-coagulation, anti-bacterial, anti-viral, anti-oxidation, anti-inflammatory, immune regulation, anti-thrombotic, anti-complement, anti-liver disease, lowering blood lipids, anti-allergic, anti-gastric ulcer, etc., have become scientific research The "new darling" of the industry is the main focus of research and development in the field of marine drugs and functional foods.

The research found that the chemical structure of the polysaccharide is very complex, mainly composed of L-fucose and sulfuric acid groups, and also contains a small amount of galactose, mannose, uronic acid, glucose, rhamnose, xylose, hexosamine , arabinose, etc., and its chemical structure will vary significantly with the source species, harvest season, geographical location and extraction method. At the same time, the polysaccharide has a large molecular weight and high viscosity, and is prone to antigenicity and toxic side effects, which severely limit its application. The most effective way to solve the above problems is to convert high-molecular-weight polysaccharides into components with moderate molecular weight and relatively stable structure through precise degradation technology, thereby clearing the way for the application of the polysaccharides. More studies have shown that the biological activity of fucoidan sulfate is molecular-weight-dependent, that is, for a certain activity, it only exhibits the highest activity within a specific molecular weight range. From this perspective, it is also necessary to use "precision degradation technology" degrade the polysaccharide.

At present, the methods for degrading fucoidan sulfate mainly include chemical degradation, physical degradation and enzymatic degradation. The chemical degradation method is non-specific degradation, the reaction conditions are harsh, the process is difficult to control, and the product conversion rate is low and the functionality is low. Although the physical degradation method is simple and controllable, its degradation efficiency is generally low, and it is difficult to obtain products that meet a certain molecular weight range. The enzymatic hydrolysis method can specifically cut off the fucoidan chain under mild conditions, the reaction process is easy to control, and it is easy to obtain oligosaccharide products within the required molecular weight range, and the obtained products have high content and strong functionality. Enzymatic degradation of polysaccharides It is also the most ideal method recognized in academia.

Although the enzymatic method has shown great advantages in the degradation of Fucoidan, there is currently no commercial enzyme preparation sold worldwide, which greatly hinders the application of fucoidan sulfate. The main reason for this situation is that the existing enzyme-producing microorganisms generally have low enzyme-producing activities, which cannot meet the needs of large-scale production. Therefore, the screening of highly active microorganisms at this stage is an important way to solve the application problems of fucoidan sulfate.

Contents of the invention

The object of the present invention is to provide a Flavobacterium mutant strain and its application. The mutant strain can effectively increase the yield of fucoidan sulfatase in large-scale fermentation, thereby making up for the deficiencies in the prior art.

The Flavobacterium mutant strain of the present invention is the Flavobacteriaceae sp. RC2-3mut strain, which has been preserved in the Chinese Academy of Microbiology Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing on November 03, 2017. General Microorganism Center of Culture Collection Management Committee, the preservation number is CGMCC No.14855.

The bacteria screened by the present invention are used to produce fucoidan sulfatase;

The living bacteria of the Flavobacteriaceae RC2-3mut of the present invention are used to prepare bacterial fluid; the prepared bacterial fluid can be used to extract intracellular enzymes, thereby degrading fucoidan sulfate.

The bacterial strain protected by the invention is obtained by long-term fermentation screening of the existing fucoidan sulfatase-producing Flavobacterium. In large-scale fermentation, the ability of the strain to produce fucoidan sulfatase is significantly improved, and the highest enzyme production ability can reach 310U/ml.

Detailed ways

The applicant used the fermentation of Flavobacteria sp. RC2-3 strain with the preservation number CGMCC No.6932 to prepare fucoidan sulfatase, but it was found that Flavobacteriaceae sp. ) RC2-3 strain's fermentative enzyme production ability decreased obviously, and there was no potential for large-scale production. Therefore, the applicant carried out ultraviolet-induced mutations from the Flavobacteriaceae sp.RC2-3 strain, and finally obtained the target strain RC2-3mut of the present invention. The optimal growth temperature of the mutant strain was increased from 25°C to 30-35°C. In large-scale fermentation, its ability to produce fucoidan sulfatase can reach up to 310U/ml.

The present invention will be described in detail below in conjunction with the examples.

Embodiment 1. Mutagenesis screening of bacterial strains

Flavobacterium RC2-3 with the preservation number CGMCC No.6932 was inoculated in liquid medium (Fucoidan 0.2%, peptone 1%, ammonium nitrate 5mg/mL, prepared with membrane seawater, pH natural), at 25°C, Cultivate under the condition of 180rpm for 12h to obtain the seed culture liquid, inoculate the seed culture liquid into fresh liquid culture medium according to the inoculation amount of 5%, and cultivate at 25°C and 150rpm for 48h to obtain the Flavobacterium bacterial liquid.

The bacteria were collected by centrifugation, washed twice with sterile normal saline, added with sterile normal saline to make the number of bacteria reach the order of 10 ⁶ , and vibrated to form a cell suspension for ultraviolet mutagenesis. The irradiation time of ultraviolet mutagenesis is 10-45s respectively, and the time gradient interval is 5s. The selective plate was coated with the bacterial suspension that had not been treated with ultraviolet radiation, and cultured in the dark for 24 hours, and was used as the control group to calculate the lethal rate.

Wherein the composition of selective medium plate is as follows: Fucoidan 0.2%, peptone 0.2%, ammonium nitrate 5mg/mL, agar 2%, prepare with membrane seawater;

The results showed that with the prolongation of the ultraviolet irradiation time, the lethality rate of the bacteria gradually increased, and when the time was 40s, the lethality rate was 90%. When the time reaches 45s, the lethal rate is close to 100%, so the present invention selects an irradiation time of 40s for ultraviolet mutagenesis.

Treat the bacterial suspension with the irradiation time with a lethal rate of 90% as the mutagenic dose and spread it on the selection medium plate to obtain the purified strain of the mutant bacteria;

The above slant strains were inoculated into 50mL liquid culture medium (0.2% of Fucoidan, 1% of peptone, 5mg/mL of ammonium nitrate, prepared with membrane-passed seawater, with natural pH), cultured at 35°C and 180rpm on a shaker for 12h as a seed solution, Then the seed liquid was inoculated into the fermentation medium (0.5% of Fucoidan, 1% of beef extract, prepared with membrane seawater, natural pH) by 10% inoculum, and fermented in a 5L fermenter (charging 3.5L) at 35 Cultivate at 150 rpm for 72 hours, and measure the content of Fucoidan by the methylene blue method. The mutant strains were screened according to the level of Fucoidan content, and finally the RC2-3mut strain of the present invention was obtained.

Wherein the methylene blue method measures the method of Fucoidan content as follows:

Take 9 mL of distilled water, add 50 μL of the sample to be tested, mix well, add 1 mL of 0.41 mmol/L methylene blue solution, mix well, and measure the absorbance at 559 nm. Fucoidan standard (Sigma) was used as a standard curve for calculation.

Embodiment 2: Detection of fermented enzyme production activity of RC2-3mut strain

The RC2-3mut strain was inoculated in a liquid medium (0.2% of Fucoidan, 1% of peptone, 5 mg/mL of ammonium nitrate, prepared with membrane seawater, and the pH was natural), and cultured at 35°C for 12 hours on a shaker at 180 rpm as a seed solution, and then The seed solution was inoculated in the fermentation medium (0.5% of Fucoidan, 1% of beef extract, prepared with membrane-passed seawater, natural pH) according to 10% inoculation amount, and fermented in a 5L fermenter (loading 3.5L), at 35°C, Cultivate for 72h under the condition of 150rpm. Sample 1 mL every 24 hours under sterile conditions, inactivate the enzyme at 80°C for 15 minutes, and centrifuge at 10,000 rpm for 10 minutes to get the supernatant. With high performance gel size exclusion chromatography (chromatographic conditions are as follows: Aglient1100 high performance liquid chromatography; TSK-gel G3000PW × 1 (30cm × 7.5mm) chromatographic column; 40 ℃ column temperature; 0.2MNaCl is mobile phase; 0.5mL/min flow rate ; differential detector) to detect changes in the molecular weight of polysaccharides. The results showed that after culturing for 0h, 24h, 48h, and 72h, the content of Fucoidan with a retention time of 13.6min was 100%, 63%, 20%, and 15%, respectively.

The enzyme activity detection method is: take intracellular enzyme and mix equal volumes of 0.2% Fucoidan (pH 8.0, dissolved in 20mM Tris-HCl), shake and culture at 25°C and 120rpm for 2h, and measure reducing sugar content by Somogyi-Nelson method . One unit of enzyme activity is defined as the amount of enzyme required to produce 1 nanomol of reducing sugar per hour at 25°C and pH 8.0. It was determined that the intracellular enzyme activity of the strain RC2-3mut after 72 hours of culture reached 310U/mL. It proves that the bacterial strain screened by the present invention has better enzyme production performance in large-scale fermentation.

The above experimental results show that the enzyme-producing bacteria screened by the present invention have better fermentation and enzyme-producing ability, and can effectively degrade high-molecular-weight fucoidan sulfate.

Claims (4)

1. A flavobacterium strain (Flavobacterium sp.) RC2-3mut is characterized in that the preservation number of the flavobacterium is CGMCC No. 14855.

2. Use of flavobacterium according to claim 1 for the production of fucoidan sulfatase.

3. A method for producing fucosan sulfatase, wherein the method comprises fermenting the flavobacterium of claim 1 to produce fucosan sulfatase.

4. A method for producing fucosan oligosaccharide, wherein the method is produced by fermentation using the Flavobacterium of claim 1.