Application of Raoultella terrestris in the degradation of quercetin

Abstract

The invention discloses the application of native Raoultella in degrading quercetin. The invention separates a native Raoultella strain capable of reducing the quercetin content from the intestines of the larvae of the golden bat moth that feed on Polygonum villosa containing quercetin, and purchases the native Raoultella strain from a commercial channel. It is found that the native Raoultella can degrade quercetin and can be used to degrade quercetin, for example, to improve the growth and development of animals whose growth is inhibited by feeding on food containing quercetin, and significantly improve the growth and development of animals.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to application of Raoultella terrestris in degrading quercetin. Background Art

[0002] Quercetin is a flavonol compound with multiple biological activities. It is also known as quercetin because its alkaline aqueous solution is yellow. It is insoluble in water and tastes bitter. Quercetin is widely present in nature. Quercetin has good expectorant and antitussive effects, and has a certain antiasthmatic effect. It is used to treat chronic bronchitis. In addition, it can lower blood pressure, enhance capillary resistance, reduce capillary fragility, lower blood lipids, dilate coronary arteries, increase coronary blood flow, and has an auxiliary therapeutic effect on patients with coronary heart disease and hypertension. Many studies have shown that quercetin has an inhibitory effect on various cancer cells. However, in the list of carcinogens published by the World Health Organization's International Agency for Research on Cancer in October 2018, quercetin, an oak extract, was listed in the list of Class 3 carcinogens.

[0003] Studies have also found that some insects cannot grow and develop normally after feeding on plants or artificial feed containing quercetin. For example, the growth of the Spodoptera litura was inhibited after feeding on the roots of Polygonum villosa and artificial feed containing quercetin (Zhang Caixia, Li Yulin, Hu Fengzu. Study on the chemical composition of the whole herb of Polygonum villosa. Natural Product Research and Development, 2005, 17: 177-178; Qiu Xuehong, Han Richou. Effects of the natural feed Polygonum villosa hosted by Cordyceps sinensis on the growth and development of Plutella xylostella and Spodoptera litura. Journal of Environmental Entomology, 2016, 38: 54-60; Tu Yegou, Wu Kongming, Xue Fangsen, Guo Yuyuan. Effects of different host plants on the growth, development, reproduction and flight of Spodoptera litura. 2008, 20: 105-109).

[0004] The balance and stability of animal microecological systems is of great significance for maintaining animal health. Exploring the target microorganisms that metabolize flavonoids in animals provides an important basis for understanding the absorption and biological characteristics of quercetin in the intestines of animals after they ingest quercetin. In the human diet, quercetin is absorbed and digested in the small intestine, and then passes through the colon and degraded in the colon. This process changes the biological characteristics of quercetin itself and has a potential important impact on human health.

[0005] Raoultella terrestris belongs to the Enterobacteriaceae family, Raoultella genus, and is a Gram-negative bacillus. Summary of the invention

[0006] The invention aims to provide application of Raoultella terrestris in degrading quercetin.

[0007] The Raoultella terrigena may be any strain of the species Raoultella terrigena, such as Raoultella terrigena B49 or Raoultella terrigena ATCC 700372.

[0008] The second object of the present invention is to provide a method for degrading quercetin, which is to inoculate native Raoultella into a substance containing quercetin and utilize the native Raoultella to degrade quercetin.

[0009] Preferably, the quercetin-containing substance is animal feed, such as some insect feeds containing quercetin, such as artificial feeds of Spodoptera litura or Bat moth containing Polygonum villosa.

[0010] The second object of the present invention is to provide the use of Raoultella terrestris in the preparation of a medicament for improving the growth and development of animals whose growth is inhibited by feeding on food containing quercetin.

[0011] The animal may be a Spodoptera litura or a bat moth.

[0012] The present invention isolates a native Raoultella strain capable of reducing the quercetin content from the intestines of the larvae of the golden bat moth that feed on Polygonum villosa containing quercetin, and purchases the native Raoultella strain from a commercial channel. It is found that the native Raoultella can degrade quercetin and can be used to degrade quercetin, for example, to improve the growth and development of animals whose growth is inhibited by feeding on food containing quercetin, thereby significantly improving the growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the colonies of Raoultella terrestris (a, colonies grown on Polygonum villosa selective medium; b, LB medium) and Gram staining (c); Figure 2 This is the verification of the degradation of quercetin by native Raoultella strains. A. The bacterial cells of native Raoultella B49 and ATCC 700372 strains without quercetin added; B. The bacterial color of B49 and ATCC 700372 strains after adding quercetin; Note: 1 is the B49 strain, 2 is the ATCC 700372 strain;

[0014] Figure 3 It is the color of the supernatant of the bacterial solution after fermentation of quercetin by native Raoultella strain; A. native Raoultella strain B49; B. ATCC 700372 strain; Tube 1 represents negative control 1 (culture medium without strain and quercetin); Tube 2 represents negative control 2 (culture medium with strain and quercetin); Tube 3 represents the degradation treatment group (culture medium with strain and quercetin); Tube 4 represents the positive control (culture medium with strain and quercetin);

[0015] Figure 4 is the quercetin standard curve. DETAILED DESCRIPTION

[0016] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0017] Embodiment 1:

[0018] The inventors isolated an intestinal bacterium B49 from the intestine of the larvae of the golden bat moth. Bacteria B49 can grow in a 20% Polygonum villosa selective medium (9.5mM KH2PO4, 4.8mM MgSO4, 0.1mM CaCl2, 0.8mM Na2HPO4, 15-20g / L agar is added to the solid medium, and 20% Polygonum villosa root powder is added, and the solvent is water. The preparation method is to mix the components evenly and sterilize them.) The colony is as follows: Figure 1 The growth colonies of bacteria B49 in LB medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solid medium plus agar powder 15-20g / L) are shown in (a). Figure 1 As shown in (b) of Figure 1 As shown in (c) in .

[0019] The 16s rDNA was extracted and sequenced, and the nucleotide sequence was shown in SEQ ID NO.1. After Blast comparison, it was identified as Raoultella terrigena and named as Raoultella terrigena B49.

[0020] SEQ ID NO.1:

[0021]

[0022] Example 2

[0023] 1. Experimental Methods

[0024] 1.1 Experimental verification of quercetin degradation strain

[0025] Chromatographic experiments were used to verify the ability of Raoultella terrigena ATCC 700372 (purchased from Guangdong Microbial Culture Collection Center, with the collection number: GDMCC1.452) and B49 strain to degrade quercetin.

[0026] Activate the strain, pick a single colony and place it in LB liquid medium, and culture at 37°C for 16 h until OD 600 0.6, take 10ul culture solution and inoculate it in LB medium containing 0.004% (mass fraction) of quercetin for 16h, centrifuge at 5000rpm for 10min, discard the supernatant, collect the cells, wash the cells 3-4 times with 0.85% saline, then collect the cells by centrifugation, and spray 2% AlCl3 methanol solution on the cells for color development. If the strain can completely convert quercetin to produce other substances, then the cells will show the color of the cells themselves after spraying with the color developer, otherwise, it will show yellow due to the presence of quercetin.

[0027] 1.2 Determination of quercetin degradation ability of Raoultella terrestris ATCC 700372 and B49 strains

[0028] Prepare LB liquid medium, add quercetin to the medium, and make the final concentration of quercetin in the medium be 0.01, 0.02, 0.03, 0.04, 0.05 mg / ml respectively. Take 1 mL of supernatant and mix with 2.5 mL of methanol, centrifuge to remove protein, mix the prepared 2% AlCl3 methanol solution with the centrifuged extract at 1:1, let stand for 10 minutes, and measure its absorbance at 415 nm using a spectrophotometer. Use quercetin concentration as the horizontal axis and absorbance as the vertical axis to establish a standard curve.

[0029] The ability of the isolated strain to degrade quercetin was determined by measuring the residual content of quercetin in the supernatant. The strain was inoculated in liquid LB medium containing 0.004% quercetin and cultured at 37°C for 36 h until the cell concentration of the bacterial solution reached 10 8 cfu / mL. The cultured LB medium was centrifuged at 5000rpm for 20min, and the absorbance was determined according to the standard curve method. The blank was set as the corresponding culture medium without quercetin but inoculated as a negative control, and the culture medium containing quercetin but not inoculated was set as a positive control. After the determination was completed, the degradation rate (%) of the strain to quercetin was calculated according to the standard curve. Each group was repeated 3 times and the average was taken.

[0030] 2. Research Results

[0031] 2.1 Chromatographic experiments to verify the degradation of quercetin by strains

[0032] The results of bacterial chromatography experiments are shown in Figure 2 The results showed that after the bacteria of Raoultella terrestris B49 and ATCC 700372 were sprayed with the color developer, the color of the bacteria in the control group without quercetin was ( Figure 2 A) The color of the cells after adding quercetin and co-culturing with the two strains ( Figure 2 B) Similar, the two bacterial strains co-cultured with quercetin did not show the yellow color of quercetin, indicating that the two bacterial strains can degrade quercetin and there is no quercetin remaining on the bacterial surface.

[0033] The supernatants of quercetin degradation by Raoultella terrestris B49 and ATCC 700372 were compared, and the results showed that the content of residual quercetin in the bacterial solution was positively correlated with the color of the bacterial solution ( Figure 3 The color of the supernatant of the two strains (tube 3) is darker than that of the LB medium (tube 1) and the negative control (tube 2), but lighter than that of the positive control (tube 4), indicating that the B49 and ATCC700372 strains can degrade quercetin, but a part of the quercetin in the supernatant of tube 3 is not degraded by the strains.

[0034] 2.2 Determination of the ability of strains to degrade quercetin

[0035] The quercetin standard curve established by fermentation is as follows Figure 4 , the regression equation is y = 7.794x-0.0026. R2 = 0.9975. The content of residual quercetin in the supernatant of the fermentation quercetin solution of B49 and ATCC 700372 strains was determined, reflecting the degradation ability of B49 and ATCC 700372 strains on quercetin (Table 1).

[0036] Table 1 Degradation ability of B49 and ATCC 700372 strains on quercetin

[0037]

[0038] Example 3

[0039] 1. Experimental Methods

[0040] 1.1 Growth determination of Spodoptera litura fed with artificial diets containing different quercetin contents

[0041] A single colony of Raoultella terrestris B49 was inoculated into LB and cultured at 37°C and 200 rpm until the OD value was 0.8. 50ul of quercetin of different concentrations was added to 940ul of LB medium to make the final concentrations of quercetin 0.25, 1.25 and 2.5 mg / ml, respectively. 50ul of DMSO was added to the solvent control. 10ul of B49 bacterial solution with an OD value of 0.8 and 10ul of LB culture solution were added to the treatment group and the positive control group, respectively. The culture was placed under the corresponding culture conditions for 36h to make the bacterial solution concentration reach 10 8 cfu / ml. Take 4 ml of the above-mentioned culture solution with different treatments and mix them with 96 g of artificial feed of Spodoptera litura, and feed them to the 4th instar larvae of Spodoptera litura that have just molted. The quercetin content in the artificial feed of the three positive controls that have not been degraded by B49 is 0.01, 0.05 and 0.1 mg / g respectively. Change the food every other day and observe the growth, development and death of the larvae.

[0042] 2. Research Results

[0043] 2.1 Growth of Spodoptera litura fed with artificial diets containing different quercetin contents

[0044] The growth of Spodoptera litura fed with artificial diets containing different amounts of quercetin is shown in Table 2. There was no significant difference in the larval period, larval survival rate, prepupal stage, pupal stage, eclosion rate, average pupal weight, etc. between the 4th instar larvae of Spodoptera litura fed with artificial diets containing 0.01, 0.05 and 0.1 mg / mL quercetin and the control group with only DMSO solvent. -1 The growth of larvae fed artificial diets with quercetin was retarded, the larval life, prepupal and pupal stages were significantly prolonged, the larval survival rate and eclosion rate were significantly reduced, and the average pupal weight was significantly reduced. However, when the same concentration of quercetin was fermented in vitro by Raoultella solani for 36 hours and then added to the artificial diet, the growth and development of the fourth-instar larvae of Spodoptera litura was improved. The fourth-instar larvae fed on 0.01 mg / mL fermented by Raoultella solani -1 The survival rate, eclosion rate, prepupal stage, pupal stage and average pupal weight of larvae after quercetin treatment were not significantly different from those of the two control groups, but were significantly higher than those of the fermentation group without native Raoultella addition. The larval development period was also significantly shortened compared with the fermentation group without native Raoultella addition. -1 The quercetin treatment group significantly shortened the pupal stage of Spodoptera litura and significantly increased the eclosion rate; in vitro fermentation of Raoultella terrestris with 0.1 mg / mL -1 In the quercetin treatment group, the pupal period of the 4th instar larvae of Spodoptera litura was significantly shortened, and the emergence rate was significantly increased; the prepupal and pupal periods of Spodoptera litura were significantly shortened, and the emergence rate and average pupal weight were significantly increased.

[0045] Table 2. Development of the 4th instar larvae of Spodoptera litura fed with artificial diets containing different levels of quercetin

[0046]

Claims

1. Raoultella terrestris Raoultella terrigena ) Application of ATCC 700372 in the degradation of quercetin.

2. A method for degrading quercetin, characterized in that: The method comprises inoculating Raoultella ATCC 700372 into a substance containing quercetin, and utilizing Raoultella ATCC 700372 to degrade quercetin.

3. The method according to claim 2, characterized in that The quercetin-containing substance is animal feed.

4. The method according to claim 3, characterized in that The animal feed is artificial feed for Spodoptera litura or Bat moth containing Polygonum villosa.

Images