Fermentation method for bacillus subtilis to produce chlorogenic acid

A chlorogenic acid and bacterial fermentation technology, which is applied in microorganism-based methods, fermentation, biochemical equipment and methods, etc., can solve the problems of underutilization of resources, etc., and achieve easy access, simple extraction methods, and improved ability. Effect

Inactive Publication Date: 2016-05-11
SICHUAN UNIV
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  • Fermentation method for bacillus subtilis to produce chlorogenic acid
  • Fermentation method for bacillus subtilis to produce chlorogenic acid
  • Fermentation method for bacillus subtilis to produce chlorogenic acid

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Embodiment 1

[0035] Strain activation and inoculation: activate strain RP5, use PDB medium, 37°C, 200revmin -1, shake the flask for 20h. Inoculation: Take 200 grams of fresh sweet potato leaves that have been grown for 4-5 months, cut them into pieces to make a homogenate, boil for 10 minutes, then filter with a single layer of gauze, add distilled water to 1L, and obtain a crude sweet potato leaf extract. Then add (g): sucrose (40), soybean peptone (10), ammonium chloride (5), copper sulfate (0.04). Autoclaved at 115°C for 20min. Inoculate (2%) the activated bacterium liquid to the 250ml Erlenmeyer flask that 100ml culture medium is housed, and at 37 ℃, 200revmin -1 Rotating culture at 100°C for 28h.

Embodiment 2

[0037] Extraction of chlorogenic acid: after 28 hours of incubation on a shaking table. Centrifuge (5000g, 20min), take the supernatant, adjust the pH of the fermentation broth to 4, to maintain the stability of chlorogenic acid, add an equal volume of 70% ethanol and ultrasonically treat (40°C, 10min) to speed up the conversion of liquid to mass Extraction efficiency, then rest overnight. Concentrate in vacuum with a rotary evaporator to 20ml, adjust the pH to 2-3 with concentrated hydrochloric acid, then add an equal volume of ethyl acetate to extract three times, each time for 1h, take the ethyl acetate layer and concentrate in vacuum to almost dryness, add 5ml of chromatographic methanol to completely Dissolve, filter with an organic filter, remove the insoluble matter in the sample and prepare the sample solution to be tested

Embodiment 3

[0039] HPLC quantitative analysis of chlorogenic acid content in the sample solution: HPLC detection conditions, the chromatographic column is WatersC18column (75mm×4.6mm, 3.5μm), the mobile phase is acetonitrile: 0.5% phosphoric acid water (13:87, v / v), and the flow rate is 1.0 mlmin -1 , the column temperature is 35° C., the injection volume is 10 μl, and the detection wavelength is 325.2 nm. By measuring the accumulation yield of chlorogenic acid can reach 54.60mgl -1 , the ability of RP5 to ferment and produce chlorogenic acid increased by 37.75mgl -1 , is the initial fermentation of RP5 (1.58mgl -1 ) of 23.90 times and compared to the chlorogenic acid content (18.32mgl -1 ) 2.06 times. HPLC retention time and absorption wavelength see figure 1 .

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Abstract

The invention relates to a novel method for fermenting endophyte by taking sweet potato leaves as a basic material and belongs to the field of microbial fermentation. As a depside, chlorogenic acid is produced through a shikimic acid pathway generally and exists in plants such as Cortex Eucommiae, Flos Lonicerae and Flos Chrysanthemi Indici. As an important bioactivator and valuable traditional Chinese medicine, the chlorogenic acid has the antibacterial, antiviral and anti-tumor effects and the like. At present, since the chlorogenic acid is mainly extracted from natural plants but extraction technologies are complex and yield is low, seeking a novel method for producing the chlorogenic acid is particularly important. The novel method has the advantages that the endophyte capable of producing the chlorogenic acid can be obtained by fermentation of the simple and available sweet potato leaves serving as a main material, so that the chlorogenic acid synthesis capability of the endophyte is enhanced substantially, total output of the chlorogenic acid in fermentation liquor can reach 54.60mg l<-1> which is 23.90 times of initial fermentation output (1.58mg l<-1>) of RP5, output of total chlorogenic acid includes the chlorogenic acid and two kinds of isomerides thereof, and output of the two kinds of isomerides can reach 128.04mg l<-1> which is 31.74 times of initial fermentation output (1.74mg l<-1>). The novel method has considerable economic benefit.

Description

technical field [0001] The present invention relates to a new fermentation method for fermenting strains with high production of chlorogenic acid, in particular to a method that uses fresh sweet potato leaves that have been grown for 4-5 months as the basic component of the fermentation medium and the strains used are capable of producing green A strain of endophytic bacteria. Background technique [0002] Chlorogenic acid (CGA) or 3-caffeoylquinic acid (3-CQA) is a phenolic acid composed of caffeic acid and quinic acid, usually produced through the shikimic acid pathway, in plants such as Eucommia ulmoides , honeysuckle, wild chrysanthemum exists. Studies have shown that chlorogenic acid has various medicinal effects including anti-oxidation, antibacterial, anti-allergic, anti-cancer, lowering blood sugar, anti-hepatitis B virus, etc., and is widely used in traditional Chinese medicine, daily chemicals and food. Chlorogenic acid can be called a kind of plant gold, which c...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12R1/125
Inventor 张杰丁仁芳杨志荣赵建冯甦侯若彤孙群谢晨文
Owner SICHUAN UNIV
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