槐米制备槲皮素及其与粘杆菌素联用对沙门氏菌的作用

doi:10.13304/j.nykjdb.2024.0157

摘要/Abstract

摘要：

槲皮素是一种黄酮类化合物，具有多种生物活性，大多以糖苷类化合物形式存在植物中。槐米是一种理想的制备槲皮素的原料，具有种植面积广、价格低廉等优点。探讨了黑曲霉发酵槐米制备的槲皮素与粘杆菌素联合作用对沙门氏菌（Salmonella spp.）的抑制效果。通过黑曲霉与槐米的半固体发酵及超声提取方法，从发酵产物中提取槲皮素，采用HPLC和ESI-MS/NMR技术进行纯化和结构鉴定，并利用肉汤稀释法和棋盘格法评估了槲皮素与粘杆菌素对沙门氏菌的单独及联合抑制效果。结果表明，槲皮素含量在发酵96 h达到最高，为73.7 mg·g^-1。经提取、纯化和结构鉴定的槲皮素纯度≥98%。在抗菌活性评估中，粘杆菌素对鼠伤寒沙门氏菌（S. typhimurium）、鸡白痢沙门氏菌1789（S. pullorum 1789）和鸡白痢沙门氏菌520（S. pullorum 520）的最小抑菌质量浓度（minimal inhibitory concentration，MIC）分别为2、8和4 μg·mL^-1，而槲皮素对上述菌株的MIC均超过500 μg·mL^-1。制备的槲皮素与粘杆菌素联用时，对3株沙门氏菌均表现出联合抗菌效果，尤其对鸡白痢沙门氏菌1789和520，其分数抑制浓度指数（fractional inhibitory concentration index，FICI）值均低于0.5，并且粘杆菌素的MIC值在联用后均降为1 μg·mL^-1，表明槲皮素与粘杆菌素之间存在显著的协同抑制作用。研究结果证明了通过黑曲霉发酵槐米制备的槲皮素与粘杆菌素联合对沙门氏菌具有协同抑制作用，为开发新型抗菌组合策略提供了科学参考依据。

关键词: 槲皮素, 槐米, 黑曲霉, 粘杆菌素, 沙门氏菌, 协同抑制

Abstract:

Quercetin is a flavonoid compound with various biological activities， mostly existing in the form of glycosides in plants. Sophora japonica is an ideal raw material for preparing quercetin， with advantages such as wide planting area and low price.　This study investigated the synergistic antibacterial effects of quercetin against Salmonella spp. that combined with colistin. The quercetin was prepared from the Sophora japonica buds Aspergillus niger fermentation and isolated from the semi-solid fermentation products using ultrasonic extraction methods， purified and structurally identified using HPLC， ESI-MS and NMR methods. The antibacterial activities of quercetin and colistin， both individually and in combination， against Salmonella were evaluated using dilution and checkerboard methods. The results showed that the highest product of quercetin was 73.7 mg·g^-1after fermentation for 96 h and the purity reached to 98% after purification and identification. After evaluation of antibacterial activity， the minimum inhibitory concentrations （MICs） of colistin against S. typhimurium， S. pullorum 1789 and 520 were 2， 8， and 4 μg·mL^-1， respectively， while the MICs of quercetin against the mentioned strains were all above 500 μg·mL^-1. When quercetin was used together with colistin， the synergistic antibacterial effects were observed against above Salmonella strains， particularly against S. pullorum 1789 and 520， with fractional inhibitory concentration index （FICI） values below 0.5， and the MIC value of colistin were reduced to 1 μg·mL^-1. The study demonstrated the synergistic inhibitory effect of quercetin prepared from Sophora japonica through A. niger fermentation combined with colistin against Salmonella， providing a scientific basis for developing new antibacterial combination strategies.

Key words: quercetin, Sophora japonica, Aspergillus niger, Salmonella, colistin, synergistic inhibition

中图分类号:

S567

熊小艳, 田丹丹, 弓雨欣, 乔宇, 徐小轻, 何孟欣, 石波, 彭晴. 槐米制备槲皮素及其与粘杆菌素联用对沙门氏菌的作用[J]. 中国农业科技导报, 2025, 27(6): 136-147.

Xiaoyan XIONG, Dandan TIAN, Yuxin GONG, Yu QIAO, Xiaoqing XU, Mengxin HE, Bo SHI, Qing PENG. Preparation of Quercetin from Sophora japonica and Its Effect Together with Colistin on Salmonella spp.[J]. Journal of Agricultural Science and Technology, 2025, 27(6): 136-147.

图/表 9

图1 芦丁和槲皮素标准品的定量分析A：芦丁和槲皮素混合标准品的HPLC谱图；B：芦丁的标准曲线；C：槲皮素的标准曲线

Fig. 1 Quantitative analysis of rutin and quercetin standardsA： HPLC chromatogram of the mixed standards of rutin and quercetin； B： Quantitative standard curve of rutin； C： Quantitative standard curve of quercetin

图2 槐米发酵过程中芦丁和槲皮素含量的变化趋势A：槐米在发酵过程中的HPLC谱图；B：槐米在发酵过程中芦丁与槲皮素含量的变化

Fig. 2 Trends of rutin and quercetin content during the fermentation of Sophora japonicaA： HPLC spectra of Sophora japonica during fermentation； B： Changes in rutin and quercetin content of Sophora japonica during fermentation

表1 发酵槐米提取物中槲皮素分离纯化结果

Table 1 Isolation and purification result of quercetin from fermented Sophora japonica extracts

纯化步骤

Purification step

槲皮素含量

Quercetin content/（mg·g^-1）

槲皮素回收率

Quercetin recovery/%

槲皮素纯度

Quercetin purity/%

发酵槐米

Fermented Sophora japonica

73.7±4.73

100.00±0.09

7.37±0.03

槲皮素粗提物（水洗、乙醇萃取）

Quercetin crude extracts （water washing， ethanol extraction）

321.5±5.88

79.83±0.014

32.15±0.67

色谱纯化

Chromatography purification

980.5±1.21

4.14±0.023

98.00±0.034

图3 分离纯化的槲皮素HPLC谱图A：槲皮素标准品；B：分离纯化后的槲皮素样品

Fig. 3 HPLC spectrum of isolated and purified quercetinA： Standard quercetin； B： Purified quercetin

图4 从槐米发酵产物中纯化的槲皮素ESI-MS质谱图

Fig. 4 ESI-MS mass spectra of quercetin purified from the fermentation product of Sophora japonica

图5 分离纯化后槲皮素NMR谱图A：1H NMR；B：13C NMR

Fig. 5 NMR spectrum of quercetin after isolation and purification

表2 粘杆菌素和槲皮素单药使用时对沙门氏菌的MIC值

Table 2 MIC values of Colistin and Quercetin when used as single agents against Salmonella spp.

	最小抑菌浓度 Minimal inhibit concentration
菌株名称 Strain name	粘杆菌素 Colistin/（μg·mL^-1）	槲皮素 Quercetin/（μg·mL^-1）
鼠伤寒沙门氏菌 Salmonella typhimurium	2	>500
鸡白痢沙门氏菌1789 Salmonella pullorum 1789	8
鸡白痢沙门氏菌520 Salmonella pullorum 520	4

图6 槲皮素与粘杆菌素联合使用对3种沙门氏菌的抑制作用A：鼠伤寒沙门氏菌；B：鸡白痢沙门氏菌1789；C：鸡白痢沙门氏菌520

Fig. 6 Inhibition of quercetin combined with colistin on 3 strains of SalmonellaA：Salmonella typhimurium； B：Salmonella pullorum 1789；C：Salmonella pullorum 520

表3 槲皮素和粘杆菌素抑制沙门氏菌的MIC和FICI

Table 3 MIC and FICI of quercetin and colistin inhibiting Salmonella

菌株名称 Strain name	槲皮素 MIC Quercetin MIC/（μg·mL^-1）		粘杆菌素 MIC Colistin MIC/（μg·mL^-1）		FICI 值 FICI value	联用效果 Effect
菌株名称 Strain name	单用 Alone	联用 Combination	单用 Alone	联用 Combination	FICI 值 FICI value	联用效果 Effect
鼠伤寒沙门氏菌 Salmonella typhimurium	>500	250	2	1	1	相加 Additive
鸡白痢沙门氏菌1789 Salmonella pullorum 1789	>500	125	8	1	0.375	协同 Synergy
鸡白痢沙门氏菌520 Salmonella pullorum 520	>500	62.5	4	1	0.375	协同 Synergy

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