A fermented Sophora japonica extract for enhancing the efficacy of colistin, its preparation method and application

Abstract

This invention discloses a fermented Sophora japonica flower extract for enhancing the efficacy of colistin, its preparation method, and its application. The preparation method of the fermented Sophora japonica flower extract includes the following steps: inoculating Aspergillus niger into a solid fermentation medium containing Sophora japonica flowers for fermentation to obtain a fermented product; separating the fermented product to obtain the fermented Sophora japonica flower extract. This fermented Sophora japonica flower extract can enhance the inhibitory effect of colistin on Salmonella (especially Salmonella pullorum), helping to reduce antibiotic use and combat the growth of drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial drug technology, specifically relating to a fermented Sophora japonica extract for enhancing the efficacy of colistin, its preparation method, and its application. Background Technology

[0002] In animal husbandry, antibiotics were once widely used to promote animal growth and prevent or treat diseases. Faced with the increasingly serious problem of antibiotic resistance, China fully implemented an "antibiotic ban" on July 1, 2020, to reduce antibiotic use. Currently, the use of antibiotics in animal husbandry is mainly limited to treating animal diseases. However, the past overuse of antibiotics in livestock production has exacerbated the problem of antibiotic resistance, which not only threatens animal health but may also transmit drug-resistant bacteria to humans through the food chain, posing a public health risk. For example, Salmonella is a bacterium prevalent in animal husbandry; it can infect not only poultry and livestock but also affect human health through contaminated meat and other animal products. Salmonella Enteritidis, an important serotype of Salmonella, is closely related to the pathogenesis of salmonellosis in humans. This bacterium is particularly associated with poultry, especially the health of chickens and the safety of chicken products. Salmonella Enteritidis can be transmitted to humans through contaminated chicken meat and eggs, causing foodborne illnesses.

[0003] Therefore, developing strategies to enhance the efficacy of existing antibiotics to reduce overall antibiotic use is crucial for ensuring food safety and animal health. Colistin, a widely used antibiotic for treating Salmonella infections, faces increasing resistance issues. Developing new methods to improve the antibacterial effects of colistin is an urgent task for the livestock industry and public health. Summary of the Invention

[0004] This invention provides a fermented Sophora japonica flower extract, which transforms the bioactive components in Sophora japonica flowers into a more effective form through fermentation with Aspergillus niger. While these active components do not possess direct bactericidal function, they can disrupt bacterial cell membranes and increase antibiotic permeability, thereby enhancing the bactericidal effect of colistin against Salmonella. By using this plant extract and colistin in combination, it is possible to reduce livestock dependence on traditional antibiotics, slow the development of antibiotic resistance, improve animal health, increase production efficiency, and reduce the risk of antibiotic-resistant bacteria spreading to humans through the food chain. This invention provides a new solution for food safety and public health, proposes a sustainable approach to addressing antibiotic resistance, helps maintain the effectiveness of existing antibiotics, and protects human and animal health.

[0005] The method for preparing fermented Sophora japonica extract provided by the present invention includes the following steps: inoculating Aspergillus niger into a solid fermentation medium containing Sophora japonica for fermentation culture to obtain fermented product; separating the fermented product to obtain the fermented Sophora japonica extract.

[0006] In the above method, the fermentation culture conditions can specifically be: cultured at 28-35℃ and 180-200 rpm for 72-108 hours.

[0007] In the above method, the composition of each liter of the solid fermentation medium containing Sophora japonica flowers is as follows: 80g-200g of crushed Sophora japonica flowers, 2.4-6g of NaNO3, 0.8-2g of K2HPO4, 4-10g of MgSO4, 0.4-1g of KCl, 0.8-2g of FeSO4, and the pH value is adjusted to 6.0.

[0008] According to one embodiment of the present invention, the composition of each liter of the solid fermentation medium containing Sophora japonica flowers is as follows: 100g of crushed Sophora japonica flowers, 3g of NaNO3, 1g of K2HPO4, 5g of MgSO4, 0.5g of KCl, 1g of FeSO4, and the pH value is adjusted to 6.0.

[0009] In the above method, the Aspergillus niger strain is inoculated in the form of Aspergillus niger seed liquid, and the inoculation amount of the Aspergillus niger seed liquid is 4-10% of the volume of the solid fermentation medium.

[0010] The Aspergillus niger seed solution can specifically have a bacterial concentration of 1×10⁻⁶. 5 CFU / mL ~ 1×10 7 Seed culture of CFU / mL.

[0011] The specific method for preparing the Aspergillus niger seed solution is as follows: Take 10 mL of Aspergillus niger (spore count 1×10⁻⁶) mycelial suspension. 5 CFU / mL ~ 1×10 7 The *Aspergillus niger* strain (CFU / mL) was inoculated into 100 mL of seed culture medium and cultured at 30 °C and 180 rpm for 48 hours to obtain 100 mL of *Aspergillus niger* seed culture.

[0012] The composition of each liter of the seed culture medium is: 60g glucose, 20g peptone, 10g NaNO3, 5g MgSO4·7H2O, and 1.0g KH2PO4.

[0013] The Aspergillus niger strain suspension (spore count approximately 1 × 10⁻⁶) 7The specific preparation method of CFU / mL is as follows: Aspergillus niger is inoculated onto PDA slant medium for activation and cultured at 30℃ for 7 days to obtain activated strains; then, a strain suspension is prepared: 10 mL of sterile water is added to the strain slant under sterile conditions, and then the bacterial cells are scraped to prepare a seed solution.

[0014] The Aspergillus niger mentioned in this invention can specifically be Aspergillus niger strain number: CGMCC3.4309.

[0015] The above method involves separating the fermented product as follows: centrifuging the obtained fermented product to collect precipitate 1; washing precipitate 1 with deionized water and centrifuging to collect precipitate 2; mixing precipitate 2 with an ethanol aqueous solution, extracting with ultrasonic assistance, centrifuging, collecting the supernatant, concentrating it by low-pressure distillation, and freeze-drying the concentrate to obtain the crude extract of fermented Sophora japonica.

[0016] In the above separation method, the ethanol aqueous solution can specifically be an ethanol aqueous solution with a volume fraction of 75%-95%, and more specifically, the ethanol aqueous solution can specifically be an ethanol aqueous solution with a volume fraction of 80%.

[0017] In the above separation method, the ratio of precipitate 2 to ethanol aqueous solution is 1:5 (w / v);

[0018] In the above separation method, the ultrasonic-assisted extraction time can be 30 minutes;

[0019] In the above separation method, ethanol extraction is performed at least 3-5 times.

[0020] In the above separation method, the centrifugation conditions can be: centrifugation at 4000 rpm for 40 minutes.

[0021] Furthermore, the method also includes a step of purifying the fermented Sophora japonica crude extract, as follows:

[0022] The chromatographic conditions were as follows: a PuriFlash 4250 chromatographic system was used, with a YMC ODS-AQ column, specifications: 250×20 mm l.DS-5μm, 12nm; mobile phase A was 0.1% (v / v) formic acid aqueous solution, and mobile phase B was methanol.

[0023] Linear gradient elution was used, with the elution program set to gradually decrease mobile phase A from 100% at 0 min to 20% at 180 min within 0-180 min, while mobile phase B increased from 0% at 0 min to 80% at 180 min. The flow rate was set to 5 mL / min, the column temperature was maintained at room temperature, the detection wavelength was set to 360 nm, and the injection volume was 1 mL.

[0024] The eluent was collected with a mobile phase composition of 52%-42% mobile phase A and 48%-58% mobile phase B (collecting the eluent from the quercetin peak to its disappearance, with an elution time of 110-130 minutes). The solvent was removed by vacuum concentration, followed by freeze drying to obtain the purified product.

[0025] The fermented Sophora japonica extract prepared by the above method also falls within the scope of protection of this invention.

[0026] Another object of the present invention is to provide the application of the above-mentioned fermented Sophora japonica extract.

[0027] The application provided by this invention is the use of the fermented Sophora japonica extract in the preparation of colistin antibacterial synergists.

[0028] The colistin antibacterial synergist can enhance the antibacterial and / or bactericidal activity of colistin.

[0029] Furthermore, the bacteria may be Salmonella; even further, the Salmonella may be Salmonella typhimurium or Salmonella pullorum.

[0030] Another object of the present invention is to provide an anti-Salmonella composition.

[0031] The anti-Salmonella composition provided by this invention comprises the above-mentioned fermented Sophora japonica extract and colistin.

[0032] Furthermore, the mass ratio of the fermented Sophora japonica extract to colistin is (1.9–250):1, preferably (62.5–125):1.

[0033] The colistin described in this invention is colistin sulfate, CAS number: 1264-72-8.

[0034] Furthermore, the Salmonella mentioned is Salmonella pullorum.

[0035] For inhibiting Salmonella pullorum (strain number: CVCC 1789), the combined use of 125 μg / mL fermented Sophora japonica extract and 1 μg / mL colistin can completely inhibit the growth of Salmonella pullorum 1789.

[0036] Compared to using colistin or quercetin alone, this formula reduces the amount of colistin used by 8 times and the amount of quercetin used by 4 times.

[0037] For inhibiting Salmonella pullorum (strain number: CVCC 520), the combined use of 62.5 μg / mL of fermented Sophora japonica extract and 1 μg / mL of colistin completely inhibited the growth of Salmonella pullorum 520. Compared with the use of colistin or quercetin alone, this formulation reduced the amount of colistin by 4 times and the amount of quercetin by 8 times.

[0038] Another object of the present invention is to provide an anti-Salmonella product.

[0039] The anti-Salmonella product contains the above-described anti-Salmonella composition of the present invention and a pharmaceutically acceptable carrier.

[0040] The dosage form of the above-mentioned antibacterial products may be any of the following: tablets, creams, capsules, sustained-release tablets, controlled-release tablets, oral liquids, syrups, pellets, injections, or lyophilized powder for injection.

[0041] The fermented Sophora japonica extract provided by this invention can enhance the inhibitory effect of colistin on Salmonella, which helps to reduce the use of antibiotics and combat the growth of drug resistance. Attached Figure Description

[0042] Figure 1 To analyze the contents of rutin and quercetin in Sophora japonica extract by high performance liquid chromatography (HPLC); (a) rutin standard curve, (b) quercetin standard curve, (c) HPLC chromatogram of Sophora japonica extract before solid-state fermentation, (d) HPLC chromatogram of Sophora japonica extract after solid-state fermentation.

[0043] Figure 2 The chromatograms of quercetin standard and fermented sophora japonica extract after separation and purification by high performance liquid chromatography (HPLC) are shown. (a) HPLC chromatogram of 1 mg / mL quercetin standard, (b) HPLC chromatogram of 1 mg / mL fermented sophora japonica extract sample after separation and purification.

[0044] Figure 3 To identify the principal components in fermented Sophora japonica extract by electrospray ionization mass spectrometry (ESI-MS);

[0045] Figure 4 NMR spectra of the main components of the fermented Sophora japonica extract after separation and purification; (a) 1H NMR spectrum; (b) 13C-NMR spectrum; (c) HSQC spectrum;

[0046] Figure 5 Inhibitory effect of fermented Sophora japonica extract combined with colistin on Salmonella; (a) Salmonella typhimurium (CICC 22956); (b) Salmonella pullorum 1789 (CVCC 1789); (c) Salmonella pullorum 520 (CVCC 520). Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] The strains used in the following experiments:

[0050] Aspergillus niger (strain number: CGMCC3.4309; purchased by: China General Microbiological Culture Collection Center)

[0051] Salmonella typhimurium (strain number: CICC 22956; purchased by: China Industrial Microbiological Culture Collection Center)

[0052] Salmonella pullorum (strain number: CVCC 1789; purchased by: National Veterinary Microbiology Culture Collection Center)

[0053] Salmonella pullorum (strain number: CVCC 520; purchased by: National Veterinary Microbiology Culture Collection Center)

[0054] The colistin used in the following examples is colistin sulfate, CAS number: 1264-72-8;

[0055] Sophora japonica buds: Commercially available Sophora japonica buds, from Anguo City Yuyanfang Traditional Chinese Medicine Co., Ltd.

[0056] Example 1: Preparation of Fermented Sophora japonica Flower Extract

[0057] First, the Aspergillus niger strain was inoculated onto PDA slant medium for activation. It was cultured at 30°C for 7 days to obtain activated strains. Next, a strain suspension was prepared: under aseptic conditions, 10 mL of sterile water was added to the strain slant, and then the bacterial cells were scraped off to prepare the strain suspension.

[0058] Take 10 mL of the bacterial suspension (spore count approximately 1 × 10⁻⁶). 7 The *Aspergillus niger* strain (CFU / mL) was inoculated into 100 mL of seed culture medium. The seed culture medium consisted of: glucose 60 g / L, peptone 20 g / L, NaNO3 10 g / L, MgSO4·7H2O 5 g / L, and KH2PO4 1.0 g / L. The *Aspergillus niger* strain was cultured at 30℃ and 180 rpm for 48 hours to obtain 100 mL of *Aspergillus niger* seed culture, with a spore concentration of approximately 6 × 10⁻⁶ CFU / mL. 6 CFU / mL.

[0059] 5 mL of Aspergillus niger seed culture was inoculated into 100 mL of solid fermentation medium. The solid fermentation medium consisted of: 100 g / L crushed Sophora japonica flowers, 3 g / L NaNO3, 1 g / L K2HPO4, 5 g / L MgSO4, 0.5 g / L KCl, and 1 g / L FeSO4, with the pH adjusted to 6.0. Fermentation was carried out at 30℃ and 180 rpm for 96 hours.

[0060] After fermentation, the solid fermentation product was centrifuged to obtain 35.2 g of fermentation product. The fermentation product was washed with deionized water at a ratio of 1:5 (w / v) and centrifuged at 4000 rpm for 40 minutes to remove the supernatant. Then, the resulting 31.3 g precipitate was mixed with an 80% (v / v) ethanol aqueous solution at a ratio of 1:5 (w / v) and extracted using ultrasound-assisted extraction for 30 minutes. Afterward, the mixture was centrifuged at 4000 rpm for 40 minutes, the supernatant was collected, and the ethanol extraction process was repeated five times.

[0061] Finally, all supernatants (supernatants from five ethanol extractions) were combined and concentrated by low-pressure distillation. The concentrate was then freeze-dried to obtain 5.5 grams of fermented Sophora japonica extract powder.

[0062] Example 2: Determination of rutin and quercetin content in fermented Sophora japonica extract by HPLC

[0063] This embodiment describes a method for the quantitative analysis of rutin and quercetin in the fermented Sophora japonica extract prepared in Example 1 using high-performance liquid chromatography (HPLC). The chromatographic system used included a Waters 2695 chromatograph and a Waters 2996 UV detector, with an Amethyst C18-H column selected. The mobile phase was a mixture of acetonitrile and 0.02% aqueous phosphoric acid at a ratio of 20:80 (v / v). The chromatographic conditions were set as follows: flow rate of 1 mL / min, detection wavelength of 360 nm, column temperature maintained at 35 °C, sample loading volume of 10 μL, and total run time of 60 minutes. Under these conditions, the contents of rutin and quercetin in the fermented Sophora japonica extract were determined.

[0064] Before fermentation, the original rutin content of Sophora japonica buds was determined to be 121.6 mg / g, and the quercetin content was 12.0 mg / g. After 96 hours of fermentation, the quercetin content significantly increased to 73.7 mg / g. However, the rutin content was not detected in HPLC analysis. Based on the relative molecular weights of rutin and quercetin (608.521 g / mol and 302.24 g / mol, respectively), the calculation methods were as follows: Decrease in moles of rutin = Initial rutin content (mg / g) / Relative molecular weight of rutin (g / mol); Increase in moles of quercetin = [Quercetin content after fermentation (mg / g) - Quercetin content before fermentation (mg / g)] / Relative molecular weight of quercetin (g / mol). The calculations show that the decrease in moles of rutin is equal to the increase in moles of quercetin, confirming that rutin was 100% converted to quercetin during fermentation. This result indicates that the fermentation process of Aspergillus niger can effectively convert rutin into quercetin.

[0065] Example 3: Isolation and purification of fermented Sophora japonica flower extract

[0066] A sample of Sophora japonica buds after 96 hours of fermentation (the 96-hour fermentation product in Example 1) was washed with deionized water, the volume of which was five times the weight of the centrifuged buds. After thorough mixing, the mixture was centrifuged at 4000 rpm for 40 minutes. This process was repeated five times to ensure the removal of water-soluble impurities from the fermentation product. The supernatant was discarded after each centrifugation. Subsequently, the precipitate was subjected to three ultrasonic extractions using anhydrous ethanol at a ratio of 1:5 (w / v), each lasting 30 minutes. After each extraction, the mixture was centrifuged at 4000 rpm for 40 minutes. The extracts were combined and concentrated under reduced pressure, and finally, the crude extract was obtained by freeze-drying.

[0067] The crude extract was dissolved in methanol to prepare a 5 mg / mL solution for the preparative liquid chromatography (HPLC) separation and purification step. The chromatographic conditions were as follows: a PuriFlash 4250 HPLC system was used, with a YMC ODS-AQ 250×20 mm L.DS-5 μm, 12 nm column. Mobile phase A was 0.1% formic acid in water (v:v), and mobile phase B was methanol. Linear gradient elution was employed, with the elution program set to gradually decrease mobile phase A from 100% at 0 min to 20% at 180 min, while simultaneously increasing mobile phase B from 0% at 0 min to 80% at 180 min. The flow rate was set to 5 mL / min, the column temperature was maintained at room temperature, the detection wavelength was set to 360 nm, and the injection volume was 1 mL. The collected mobile phase composition is: mobile phase A is 52%-42%, and mobile phase B is 48%-58% of the eluent (collected from the time of quercetin elution to the time of quercetin peak disappearance, within 110-130 minutes, during which time quercetin is eluted). The solvent is removed by vacuum concentration, followed by freeze drying to obtain the purified product.

[0068] To determine the content and purity of quercetin, 10.0 mg (accurate to 0.0001 g) each of quercetin standard and purified Sophora japonica extract were accurately weighed and dissolved in DMSO. The solutions were then transferred to 10.0 mL volumetric flasks and diluted to a final concentration of 1.0 mg / mL. The quercetin content in the sample was calculated using a standard curve of quercetin. The purity calculation formula is as follows:

[0069]

[0070] in:

[0071] m1: Mass of quercetin in the purified extract as determined by HPLC.

[0072] m0: Mass of the separated and purified extract weighed.

[0073] 98%: Purity of standard quercetin

[0074] HPLC analysis and purity calculations showed that the purity of quercetin in the purified extract was 98%.

[0075] Example 4: Identification of the main components in fermented Sophora japonica extract using electrospray ionization mass spectrometry (ESI-MS)

[0076] In this embodiment, we used electrospray ionization mass spectrometry (ESI-MS) to identify the main components of the fermented Sophora japonica extract obtained after preparative liquid chromatography purification. Mass spectrometry analysis was performed in positive ion mode under the following operating conditions: spray voltage set to 3.5 kV, capillary voltage to 13 V, and capillary temperature maintained at 325 °C. Nitrogen was used as both the spray gas and auxiliary gas, with a spray gas flow rate of 10 L / min and an auxiliary gas flow rate of 0.15 L / min. Collision-induced dissociation analysis was performed using a collision gas; the fragmentation voltage was 1.00 V in automatic mode and 0.80 V in manual mode. The mass spectrometry scan range was set between m / z 100 and 1000. The sample was injected directly using a flow injection pump at a flow rate of 0.5 mL / h.

[0077] Mass spectrometry analysis revealed two significant molecular ion peaks at m / z = 325.02 and m / z = 303.04, respectively. These peaks represent the binding of a sodium ion (Na+, M = 23) and a hydrogen ion (H+, M = 1) to a quercetin molecule (molecular weight M = 302). Based on these results, we can confirm that the main component of the fermented Sophora japonica extract is consistent with the theoretically expected molecular weight of quercetin, thus verifying that the main component of the fermented Sophora japonica extract is quercetin.

[0078] Example 5: Identification of the main components in fermented Sophora japonica extract using nuclear magnetic resonance spectroscopy (NMR)

[0079] In this embodiment, we used nuclear magnetic resonance (NMR) spectroscopy to identify the main components of the fermented Sophora japonica extract obtained after preparative liquid chromatography purification. The instrument used for analysis was a Bruker Avance-500 NMR spectrometer manufactured by Bruker Corporation, Germany. We analyzed the proton NMR spectrum of the extract sample. 1 H), carbon spectrum ( 13 C) and carbon-hydrogen single quantum correlation (HSQC) were measured in detail. In the experiment, DMSO was used as the solvent, and tetramethylsilane (TMS) was added as an internal standard to ensure the accuracy of the analytical results.

[0080] exist 1 In the 1H-NMR spectrum, the chemical shifts of aromatic hydrogen atoms ranged from 6.17 to 7.68 ppm, exhibiting the characteristic signals of aromatic hydrogen atoms. The chemical shifts of the phenolic hydroxyl group (-OH) ranged from 9.34 to 12.48 ppm; these data are found in [reference needed]. Figure 4 a. In 13 In the C-NMR spectrum, the chemical shift of the carbonyl carbon appears at 176 ppm, while the chemical shifts of the aromatic carbon atoms range from 93.9 to 164.3 ppm. Specific data are shown below. Figure 4 b. Using HSQC (carbon-hydrogen single quantum correlation) spectroscopy, we obtained the corresponding carbon signals δC 98.58, 93.9, 115.1, 116.1, and 120.3 ppm, which can be attributed to the five methylene carbon atoms C6, C8, C2′, C5′, and C6′, respectively. See details... Figure 4 c.

[0081] In summary 1 H, 13 Based on C and HSQC NMR spectral data, we identified quercetin as the main component in the fermented Sophora japonica extract.

[0082] Example 6: Determination of the minimum inhibitory concentration (MIC) of fermented Sophora japonica extract and colistin against Salmonella.

[0083] To determine the antibacterial activity of fermented Sophora japonica extract and colistin against Salmonella, this example uses the broth dilution method to determine their minimum inhibitory concentrations (MICs) against three Salmonella strains. The experimental strains included Salmonella Typhimurium (CICC 22956), Salmonella Pullorum 1789 (CVCC 1789), and Salmonella Pullorum 520 (CVCC 520). Single colonies were inoculated into Mueller-Hinton Broth (MHB) medium and cultured at 37°C and 180 rpm / min on a shaker until the OD600 nm reached 0.5, at which point the colony count was approximately 1 × 10⁻⁶. 6 CFU / mL. The fermented Sophora japonica extract was dissolved in DMSO to prepare a stock solution of 100 mg / mL, with each well containing no more than 1% of the total volume, and the highest concentration added was 500 μg / mL. Colistin was dissolved in deionized water to prepare a stock solution of 1024 μg / mL, with the highest concentration added being 256 μg / mL. The above fermented Sophora japonica extract stock solution and colistin stock solution were serially diluted twelve times to different concentrations, and then 1×10⁻⁶ CFU / mL was added. 6 CFU / mL bacterial suspensions were inoculated for MIC testing. Mixtures containing twelve different concentrations of antibacterial agent and bacterial suspension were incubated at 37°C for 18 hours, and the absorbance of each sample was measured at 600 nm using a microplate reader. A concentration with an absorbance change of less than 0.05 was considered the minimum inhibitory concentration (MIC) for effectively inhibiting bacterial growth. Each experiment was repeated three times to ensure the reliability of the results.

[0084] Based on the data in Table 1, we found that the fermented Sophora japonica extract did not show inhibitory activity against any of the three Salmonella strains at the highest added concentration of 500 μg / mL. Therefore, we considered the MIC of the fermented Sophora japonica extract to be >500 μg / mL. In contrast, the MIC values ​​of colistin against Salmonella typhimurium (CICC 22956), Salmonella pullorum 1789 (CVCC 1789), and Salmonella pullorum 520 (CVCC 520) were 2 μg / mL, 8 μg / mL, and 4 μg / mL, respectively. These results indicate that Salmonella pullorum exhibits a certain degree of resistance to colistin.

[0085] Table 1. MIC values ​​of colistin and fermented sophora flower extract when used alone.

[0086]

[0087] Example 7: Synergistic effect index (FICI) of fermented Sophora japonica extract combined with colistin against Salmonella.

[0088] To evaluate the synergistic inhibitory effect of fermented Sophora japonica extract combined with colistin on Salmonella, this example uses a checkerboard method to test three Salmonella strains. The experimental strains included Salmonella Typhimurium (CICC22956), Salmonella Pullorum 1789 (CVCC 1789), and Salmonella Pullorum 520 (CVCC 520). First, the fermented Sophora japonica extract and colistin were serially diluted to obtain solution sequences with concentration gradients of 1.9-250 μg / mL and 0.1-16 μg / mL, respectively. Then, the above solutions were mixed with 1×10⁻⁶... 6 Salmonella strains at a concentration of CFU / mL were mixed to evaluate the synergistic inhibitory effect of the two drugs. Negative controls (wells containing only MHB medium) and positive controls (wells containing the culture medium of the test strain) were set up to ensure the accuracy of the experimental results. After incubation at 37°C for 18 hours, the absorbance of each well was measured at 600 nm using a microplate reader to determine the growth status of the strain.

[0089] The synergistic effect was evaluated by calculating the fractional inhibitory concentration index (FICI), as shown in the following formula:

[0090] FICI = FIC 粘杆菌素 +FIC 发酵槐米提取物

[0091]

[0092] Based on the FICI value, the criteria for evaluating synergistic effects are as follows:

[0093] FICI ≤ 0.5: There is a synergistic effect between the drugs. The smaller the FICI value, the more obvious the synergistic effect;

[0094] 0.5 < FICI ≤ 1: There is an additive effect between the drugs;

[0095] 1 < FICI ≤ 2: There is no synergistic effect between the drugs;

[0096] FICI > 2: There is an antagonistic effect between the drugs.

[0097] When fermented Sophora japonica extract and colistin were used in combination, we calculated the fractional inhibitory concentration index (FICI) of three different Salmonella strains. The results showed that the FICI value for Salmonella typhimurium was 1, indicating an additive effect between the drugs, that is, the inhibitory effect of the combination of these two drugs on this strain was not better than that of using them alone; the FICI value for Salmonella pullorum 1789 was 0.375, and the FICI value for Salmonella pullorum 520 was 0.375, indicating a synergistic effect between the drugs. The combination of these two drugs could significantly improve the inhibitory effect on this strain. See Table 2 for details. These data indicated different degrees of combined inhibitory effects, especially significant for the combination of the two Salmonella pullorum strains, meeting the criteria for synergistic inhibition.

[0098] As Figure 5 shown in (a), when inhibiting Salmonella typhimurium in combination, 250 μg / mL of fermented Sophora japonica extract and 1 μg / mL of colistin could completely inhibit the growth of Salmonella typhimurium. Compared with using colistin or quercetin alone, this formulation reduced the dosage of colistin by 2 times and the dosage of quercetin by 2 times.

[0099] As Figure 5 shown in (b), when inhibiting Salmonella pullorum 1789 in combination, 125 μg / mL of fermented Sophora japonica extract and 1 μg / mL of colistin could completely inhibit the growth of Salmonella pullorum 1789. Compared with using colistin or quercetin alone, this formulation reduced the dosage of colistin by 8 times and the dosage of quercetin by 4 times.

[0100] As Figure 5 shown in (c), when inhibiting Salmonella pullorum 520 in combination, 62.5 μg / mL of fermented Sophora japonica extract and 1 μg / mL of colistin could completely inhibit the growth of Salmonella pullorum 520. Compared with using colistin or quercetin alone, this formulation reduced the dosage of colistin by 4 times and the dosage of quercetin by 8 times.

[0101] Table 2 FICI values of antibacterial activity of the combination of fermented Sophora japonica extract and colistin

[0102]

Claims

1. A method for preparing a fermented Sophora japonica extract, comprising the following steps: inoculating Aspergillus niger with... Aspergillus niger The mixture was inoculated into a solid fermentation medium containing Sophora japonica flowers for fermentation culture to obtain a fermented product; the fermented product was then separated to obtain the fermented Sophora japonica flower extract. The method for separating the fermented product is as follows: the obtained fermented product is centrifuged to collect precipitate 1; precipitate 1 is washed with deionized water and centrifuged to collect precipitate 2; precipitate 2 is mixed with an ethanol aqueous solution, extracted with ultrasonic assistance, centrifuged, the supernatant is collected, and concentrated by low-pressure distillation, and the concentrate is freeze-dried to obtain fermented Sophora japonica crude extract; The method further includes a purification step of the fermented Sophora japonica crude extract, as follows: The chromatographic conditions were as follows: a PuriFlash 4250 chromatographic system was used, with a YMC ODS-AQ column, 250×20 mm, DS-5 μm, 12 nm, mobile phase A being 0.1% (v / v) formic acid aqueous solution, and mobile phase B being methanol; Linear gradient elution was used, with the elution program set to gradually decrease mobile phase A from 100% at 0 min to 20% at 180 min within 0-180 min, while mobile phase B increased from 0% at 0 min to 80% at 180 min. The flow rate was set to 5 mL / min, the column temperature was maintained at room temperature, the detection wavelength was set to 360 nm, and the injection volume was 1 mL. The eluent was collected with a mobile phase composition of 52%-42% mobile phase A and 48%-58% mobile phase B. The solvent was removed by vacuum concentration and then freeze-dried to obtain the purified product. The ethanol-water solution is an ethanol-water solution with a volume fraction of 80%.

2. The preparation method according to claim 1, characterized in that: The fermentation conditions are: 28-35℃ and 180-200 rpm for 72-108 hours; Alternatively, the composition of each liter of the solid fermentation medium containing Sophora japonica flowers is as follows: 80g-200g of crushed Sophora japonica flowers, 2.4-6g of NaNO3, 0.8-2g of K2HPO4, 4-10g of MgSO4, 0.4-1g of KCl, 0.8-2g of FeSO4, and the pH value is adjusted to 6.

0.

3. The preparation method according to claim 1 or 2, characterized in that: The Aspergillus niger strain Aspergillus niger The culture medium is inoculated with Aspergillus niger seed liquid, and the inoculation amount of Aspergillus niger seed liquid is 4-10% of the volume of the solid fermentation medium. The Aspergillus niger seed liquid has a bacterial concentration of 1×10⁻⁶. 5 CFU / mL ~ 1×10 7 Seed culture of CFU / mL.

4. The preparation method according to claim 1, characterized in that: The ratio of precipitate 2 to the ethanol aqueous solution is 1:5 w / v; The ultrasonic-assisted extraction time is 30 minutes; In the separation method, ethanol extraction is performed 3-5 times.

5. The use of the fermented Sophora japonica extract prepared by any one of claims 1-4 in the preparation of colistin antibacterial synergists; The bacteria mentioned is Salmonella pullorum. Salmonella pullorum Or Salmonella typhimurium Salmonella typhimurium .

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