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Engineering bacteria for producing S-equol and application

A technology of equol and engineering bacteria, which is applied in the construction and application of engineering bacteria, can solve the problems that affect the production of equol and fermentation costs, time-consuming and labor-intensive, etc., and achieve the effects of stable system, convenient use and wide application

Active Publication Date: 2014-09-10
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above patents all use a single or a small number of bacteria isolated from the intestinal tract to produce equol by anaerobic fermentation, but the study found that most of the isolated bacteria are new species of bacteria, and different medium components and fermentation Conditions will affect whether it can produce equol. In addition, the requirements for anaerobic culture are relatively high. Therefore, the method of anaerobic fermentation of original isolates to produce equol is not only time-consuming and laborious, but also may be affected by the composition of the medium and the culture conditions. Reasons that affect equol yield and fermentation cost

Method used

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  • Engineering bacteria for producing S-equol and application
  • Engineering bacteria for producing S-equol and application
  • Engineering bacteria for producing S-equol and application

Examples

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Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of equol-producing engineering bacteria derived from lactic acid bacteria

[0027] The present invention constructs an S-equol-producing engineering bacterium by using the method of gene artificial synthesis and double-enzyme cleavage connection, and the construction of the equol-producing engineering bacterium mainly includes the following steps:

[0028] 1. Artificial synthesis of equol production gene from lactic acid bacteria

[0029] L-DDRC (the nucleotide sequence is shown in SEQ ID NO: 1), L-DZNR (the nucleotide sequence is shown in SEQ ID NO: 1) and L-DZNR (the nucleotide sequence is shown in SEQ ID NO: 2), L-DHDR (nucleotide sequence shown in SEQ ID NO: 3) and L-THDR (nucleotide sequence shown in SEQ ID NO: 4) were cloned into the PUC57 vector (GenScript Biotechnology Co., Ltd.) EcoR V restriction site, and add BamH I and Not I restriction sites at the N-terminal and C-terminal of the L-DDRC gene, respectively, and add BamH I and Not I ...

Embodiment 2

[0049] Example 2: Functional verification of engineering bacteria producing S-equol from lactic acid bacteria

[0050]The present invention carries out functional verification to the ability of engineering bacterium to produce S-equol by using HPLC and liquid-mass spectrometry, and S-equol produces the functional verification of engineering bacterium and mainly comprises the following steps:

[0051] 1. Preparation of S-equol-producing engineering bacteria

[0052] Inoculate 50 μl of engineered bacteria preservation solution into 5 ml of brain-heart infusion medium (BHI) containing carbenicillin (50 μg / ml) and streptomycin (50 μg / ml) from a -80°C refrigerator, and place it in an Electrotek In an oxygen workstation (purchased from Electrotek, UK), after static cultivation at 37° C. for 24 hours, a seed solution was obtained for inoculation. Preservation solution preparation method: Centrifuge 2ml of overnight cultured bacterial solution at 8000 rpm for 3 minutes, discard the s...

Embodiment 3

[0068] Example 3 The application of lactic acid bacteria source S-equol to produce engineering bacteria

[0069] 1. Preparation of S-equol-producing engineering bacteria

[0070] Seed culture:

[0071] Inoculate 50 μl of engineered bacteria preservation solution into 5 ml of brain heart infusion liquid medium (BHI) containing carbenicillin (50 μg / ml) and streptomycin (50 μg / ml) from a -80°C refrigerator, and place in an anaerobic In the workstation, cultivate overnight at 37°C to obtain seed liquid for inoculation.

[0072] 2. Fermentation medium and fermentation conditions

[0073] (1) daidzein is added in the BHI medium as a substrate, and the final concentration of daidzein in the BHI medium is 0.05g / L, and then the seed solution obtained in step 1 is inoculated with a volume concentration of 5% inoculum (filling liquid 30ml), cultured in an anaerobic workstation at 37°C for 24h, added 5μL (accounting for 0.1% of the volume of the culture solution) 25mg / ml IPTG aqueous s...

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Abstract

The invention discloses engineering bacteria for producing S-equol. The engineering bacteria for producing S-equol are obtained by cloning L-DDRC, L-DZNR, L-DHDR and L-THDR genes from Lactococcus sp. 20-92 to escherichia coli BL21 (D3) and transforming. The engineering bacteria for producing S-equol provided by the invention are simple in fermentation condition, convenient to use, stable in system, and wide in application; the engineering bacteria for producing S-equol have the ability of transforming daidzein in BHI and LB culture media into S-equol whether under an anaerobic condition or an aerobic condition, and have the ability of transforming bean pulp in BHI and LB into S-equol. Release of daidzein of the bean pulp in the BHI is facilitated.

Description

(1) Technical field [0001] The invention relates to the construction and application of an engineering bacterium, in particular to an S-equol-producing engineering bacterium derived from lactic acid bacteria and the application of converting daidin or soybean meal into S-equol. (2) Background technology [0002] Soy isoflavones have a variety of biological functions. Since 1980, a large number of medical studies have shown that in addition to binding to estrogen receptors, soy isoflavones can effectively prevent osteoporosis, breast cancer, and endometrial cancer. Combined with androgen receptor to reduce the occurrence of prostate cancer, and has anti-oxidation, anti-hemolysis and anti-fungal activities. In animal production, a large number of studies at home and abroad have shown that soybean isoflavones can promote the growth of male animals, enhance the body's immunity, improve the quality of animal products, and improve production performance. However, the biological e...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/06C12R1/19
Inventor 尹业师王欣
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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