Mixed bacterial flora for degrading fumonisin B1 and crude enzyme preparation thereof

A technology of mixed flora and fumonisins, applied in the direction of enzymes, enzymes, bacteria, etc., to achieve the effect of less loss of nutrients, simple fermentation method, and mild treatment conditions

Inactive Publication Date: 2019-01-18
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Mixed bacterial flora for degrading fumonisin B1 and crude enzyme preparation thereof
  • Mixed bacterial flora for degrading fumonisin B1 and crude enzyme preparation thereof
  • Mixed bacterial flora for degrading fumonisin B1 and crude enzyme preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Isolation and screening of mixed flora degrading fumonisin B1

[0030] Prepare MM medium:

[0031] Weigh 2.5g K 2 HPO 4 , 2.5g KH 2 PO 4 , 1.0g (NH 4 ) 2 HPO 4 , 0.2g MgSO 4 ·7H 2 O, 0.01g FeSO 4 , 0.004g MnSO 4 ·7H 2 O is placed in a volumetric flask, add water to make up to 1L, and adjust the pH to 7. Autoclave at 121°C for 30 minutes.

[0032] Weigh 10 g of the bacteria residue sample and dissolve it in 100 mL of sterilized distilled water, shake at 37°C for 12 hours. Dilute 1 mL of shaking solution to 10 mL, inoculate 100 μL of the diluted solution into 2 mL of MM medium (sterilized) containing 50 mg / L fumonisin B1, and culture at 28°C for 7 days with shaking at 200 rpm. The content of fumonisin B1 in the culture medium was determined by LC-MS / MS. The results show that: in the 7d culture period, the degradation ability of the culture medium to fumonisin B1 reaches more than 99%. Further, 100 μL of the culture solution was added to 2 mL of ...

Embodiment 2

[0034] Example 2: Analysis of the community structure of the mixed flora

[0035] Take 200 μL of the mixed flora described in Example 1 and inoculate it into MM medium containing 50 mg / L fumonisin B1, shake and culture at 200 rpm for 24 h at 28 ° C, centrifuge at 15000 rpm for 10 min, and store the bacteria at -20 °C conditions.

[0036] According to the QIAamp Fast DNA Stool Mini Ki kit (Qiagen, Germany), the total DNA in the bacteria was extracted, and the bacterial DNA was successfully identified by electrophoresis. Using the method of high-throughput sequencing, primers 926F (5'-AAACTYAAAKGAATTGACGG-3') and 518R (5'-ATTACCGCGGCTGCTGG-3') were used to amplify the V4-V5 region fragment of bacterial 16S rDNA (primers were provided by Shanghai Sangon Bioengineering Co., Ltd. Ltd Synthetics). After PCR products were detected by agarose gel electrophoresis, they were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for Illumina paired-end sequencing analysis. , the sequence...

Embodiment 3

[0039] Embodiment 3: the degradation efficiency of mixed flora to fumonisins B1

[0040] Investigate the degradation ability of mixed flora to fumonisin B1 under different medium and culture conditions.

[0041] (1) Degradation efficiency of FB1 by mixed flora under different medium conditions

[0042] Configure different media, as follows: ① Inorganic salt medium (MM): weigh 2.5g K 2 HPO 4 , 2.5gKH 2 PO 4 , 1.0g (NH 4 ) 2 HPO 4 , 0.2g MgSO 4 ·7H 2 O, 0.01g FeSO 4 , 0.004g MnSO 4 ·7H 2 O is placed in a volumetric flask, add water to make up to 1L, and adjust the pH to 7. Autoclave at 121°C for 30 minutes; ②LB medium: Weigh 5g of yeast extract, 10g of peptone, and 10g of NaCl into a volumetric flask, add water to 1L, adjust the pH to 7, and autoclave at 121°C ③ Nutrient broth medium (NB): Weigh 10.0g peptone, 3.0g beef powder, 5.0g NaCl, dissolve in 1L distilled water, and autoclave at 121°C for 30min; ④Contain 1% glucose MM medium (MM+PT): Dissolve 10 g of glucos...

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Abstract

The invention discloses a mixed bacterial flora for degrading fumonisin B1 and a crude enzyme preparation thereof. The mixed bacterial flora for degrading fumonisin B1 comprises the following components: Pseudomonas (Pseudomonas), Chaetomonas (Comamonas) and Sphingobacterium (sphingobacterium), wherein the total effective viable number of the mixed microflora accounts for more than 90% of the total number of the mixed microflora. The mixed bacterial flora degrading fumonisin B1 of the invention is amplified and cultured in MM culture medium containing sucrose, the bacterial body obtained by centrifugation is broken by ultrasonic wave, and the supernatant is lyophilized to obtain crude enzyme preparation. The mixed bacterial flora degrading fumonisin B1 of the invention is amplified and cultured in MM culture medium containing sucrose. The fumonisin B1 degradation rate of the mixed microflora and the crude enzyme preparation thereof obtained by the invention is above 90% in 24 hours. The mixed microbial flora and its crude enzyme preparation have the characteristics of simple fermentation and low production cost, and have broad application prospects for detoxification of fumonisin B1 in feedstuffs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mixed bacterial group for degrading fumonisin B1 and a crude enzyme preparation thereof. Background technique [0002] Fumonisins (FB) are a class of toxic secondary metabolites produced by Fusarium moniliforme and Verticillium. At present, there are 11 known FB species, among which fumonisin B1 (FB1) has the widest pollution range, the strongest toxicity, and the greatest health hazard to humans and animals. FB1 can extensively contaminate feed and its raw materials. Surveys in recent years have shown that the pollution rate of FB1 in feed in my country generally exceeds 90%, causing harm to animal husbandry that cannot be ignored. FB1 not only interferes with the normal physiological functions of plants, but also has various specific toxic effects on livestock and poultry, can cause leukomalacia in horses and rabbits, pulmonary edema in pigs, and has nephrotoxicity ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/00C12R1/38C12R1/01
CPCC12N1/20C12N9/00
Inventor 赵志勇王建华周昌艳张艳梅陈珊珊赵晓燕杨宪立
Owner SHANGHAI ACAD OF AGRI SCI
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