AUH-JLC159 and transforming method thereof for preparation of (-)-5-OH-equol

Abstract

The invention discloses an AUH-JLC159, whose preservation number is CGMCC No. 5304, and the usage thereof in preparation of (-)-5-OH-equol. The invention comprises the following steps: (1) cultivating the bacterial strain AUH-JLC159; (2) carrying out co-culture of genistein and bacterial strain AUH-JLC159 and separation and purification of the metabolite: switching the seed liquid of bacterial strain AUH-JLC159 to the BHI medium, and cultivating for 2-3 days in an anaerobic working station, thereby the substrate genistein is transformed into (-)-5-OH-equol. Purifying and separating the metabolite. The AUH-JLC159 provided by the invention can transform the substrate genistein to 5-OH-EQ with high efficiency under the anaerobic condition; thereby solving the problem of microbe biosynthesis and resource scarcity of 5-OH-EQ, and has a great promotion effects for physiology and pharmacological activities of 5-OH-EQ and new drug research and development.

Description

technical field

[0001] The invention relates to a bacterium and an application method thereof, in particular to a snagella and an application method thereof in preparing (-)-5-OH-equol by converting genistein. Background technique

[0002] Most of the isoflavones in natural soybeans exist in the form of bound glycosides, and there are only three kinds of soybean isoflavones in the form of free aglycones, namely daidzein, genistein and genistein (Glycitein), of which daidzein and genistein account for 95% to 97% of the total soybean isoflavone aglycones, and are the main components of soybean isoflavone aglycones. A large number of epidemiological and laboratory studies have shown that soybean isoflavones have obvious antioxidant, anti-inflammatory, anti-tumor effects, relieve menopausal symptoms, prevent and treat osteoporosis, and reduce the incidence of cardiovascular and cerebrovascular diseases. The physiological function of the human body has aroused widespread concern...

Examples

Embodiment 1

[0044] 1. Isolation and cultivation of strain JLC159

[0045] (1) Collection and cultivation of rooster manure samples

[0046] Use a sterilized cotton swab to pick a fresh fecal sample, put it into 1 ml of fresh BHI liquid medium, and place it in an anaerobic workstation at 37°C, as a microbial flora for screening specific functional microbial strains;

[0047] (2) Isolation and cultivation of transformed strains

[0048] ①Single colony isolation culture

[0049] Use the microbial flora in the fresh BHI liquid medium for gradient dilution, and dilute to a concentration of 10 –1 , 10 –2 、10 –3 、10 –4 、10 –5 、10 –6 、10 –7 、10 –8 , and then 100 microliters of concentration were respectively 10 –5 、10 –6 、10 –7 、10 –8 The dilution of microbial flora was uniformly coated on the pre-prepared BHI solid medium, and the BHI solid medium coated with the dilution of microbial flora was placed in an anaerobic workstation for 48 hours. Pick dozens to hundreds of single colon...

Embodiment 2

[0083] The isolation method of bacterial strain JLC159 is the same as that in Example 1.

[0084] In the anaerobic workstation, the seed liquid of the above-mentioned pre-cultivated strain JLC159 was transferred to a 250-ml Erlenmeyer flask containing 100 ml of liquid medium at a 15% inoculum size, and 40 mmol / L of genistein was added at the same time. 1.25 ml of mother liquor was cultured in an anaerobic workstation for 3 days. Use an equal volume of ethyl acetate to extract the culture in the Erlenmeyer flask twice, filter the extract and evaporate to dryness on a rotary evaporator, then add 100% methanol solution, pass through an organic membrane with a pore size of 0.45 microns, and use a preparative column to perform HPLC analysis. Separation and preparation were carried out above to obtain a conversion rate of (-)-5-OH-EQ of 83.3%.

technical field

[0001] The invention relates to a bacterium and an application method thereof, in particular to a snagella and an application method thereof in preparing (-)-5-OH-equol by converting genistein. Background technique

[0002] Most of the isoflavones in natural soybeans exist in the form of bound glycosides, and there are only three kinds of soybean isoflavones in the form of free aglycones, namely daidzein, genistein and genistein (Glycitein), of which daidzein and genistein account for 95% to 97% of the total soybean isoflavone aglycones, and are the main components of soybean isoflavone aglycones. A large number of epidemiological and laboratory studies have shown that soybean isoflavones have obvious antioxidant, anti-inflammatory, anti-tumor effects, relieve menopausal symptoms, prevent and treat osteoporosis, and reduce the incidence of cardiovascular and cerebrovascular diseases. The physiological function of the human body has aroused widespread concern...

Examples

Embodiment 1

[0044] 1. Isolation and cultivation of strain JLC159

[0045] (1) Collection and cultivation of rooster manure samples

[0046] Use a sterilized cotton swab to pick a fresh fecal sample, put it into 1 ml of fresh BHI liquid medium, and place it in an anaerobic workstation at 37°C, as a microbial flora for screening specific functional microbial strains;

[0047] (2) Isolation and cultivation of transformed strains

[0048] ①Single colony isolation culture

[0049] Use the microbial flora in the fresh BHI liquid medium for gradient dilution, and dilute to a concentration of 10 –1 , 10 –2 、10 –3 、10 –4 、10 –5 、10 –6 、10 –7 、10 –8 , and then 100 microliters of concentration were respectively 10 –5 、10 –6 、10 –7 、10 –8 The dilution of microbial flora was uniformly coated on the pre-prepared BHI solid medium, and the BHI solid medium coated with the dilution of microbial flora was placed in an anaerobic workstation for 48 hours. Pick dozens to hundreds of single colon...

Embodiment 2

[0083] The isolation method of bacterial strain JLC159 is the same as that in Example 1.

[0084] In the anaerobic workstation, the seed liquid of the above-mentioned pre-cultivated strain JLC159 was transferred to a 250-ml Erlenmeyer flask containing 100 ml of liquid medium at a 15% inoculum size, and 40 mmol / L of genistein was added at the same time. 1.25 ml of mother liquor was cultured in an anaerobic workstation for 3 days. Use an equal volume of ethyl acetate to extract the culture in the Erlenmeyer flask twice, filter the extract and evaporate to dryness on a rotary evaporator, then add 100% methanol solution, pass through an organic membrane with a pore size of 0.45 microns, and use a preparative column to perform HPLC analysis. Separation and preparation were carried out above to obtain a conversion rate of (-)-5-OH-EQ of 83.3%.

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