柠檬籽中类柠檬苦素反胶团萃取工艺优化及抑菌活性分析

Optimization of reversed micelle extraction and antibacterial activity of limonoids

中文关键词:  类柠檬苦素  反胶团萃取  优化  抑菌活性

英文关键词:limonoids  reversed micelle extraction  optimization  antibacterial activity

中文摘要:

      目的 探究羟丙基甲基纤维素(hydroxypropyl methyl cellulose, HPMC)辅助反胶团萃取柠檬籽中类柠檬苦素的可行性, 并对提取物质的抑菌活性进行分析。方法 以初提液中类柠檬苦素含量为响应值, 通过正交试验确定初提液制备的最优参数; 以Placktett-Burman (P-B)试验筛选影响类柠檬苦素反胶团萃取效率的主要因素, 并结合Box-Behnken (B-B)试验确定最优工艺条件; 通过测定最小抑菌浓度(minimal inhibitory concentration, MIC)、最小杀菌浓度(minimum bactericidal concentration, MBC)、碱性磷酸酶(alkaline phosphatase, AKP)活性考查类柠檬苦素对青霉菌和绿霉菌的抑菌活性。结果 初提阶段影响类柠檬苦素提取效率的最优条件为提取时间1.5 h、液料比30:1 (mL/g)、HPMC添加量0.5%、pH 6.5, 该条件下提取率为(3.34±0.02) mg/g。P-B试验结合B-B试验结果表明, 类柠檬苦素萃取的最优配方为双十八烷基二甲基氯化铵(dioctadecyl dimethyl ammonium chloride, DODMAC) 44.29 mmol/L、正己醇13.86%、溴代十四烷3.88%, 校正工艺条件下类柠檬苦素萃取率达(87.64±1.12)%。抑菌试验表明, 类柠檬苦素对绿霉菌的MIC和MBC分别为1250.00 μg/mL、2500.00 μg/mL, 对青霉菌的MIC和MBC分别为1250.00 μg/mL、5000.00 μg/mL; 通过测定AKP活力发现, 当加入抑菌活性物质后, 随着药物作用时间延长及药物纯度的提高, AKP活性逐渐增加。结论 通过HPMC辅助反胶团能有效提取柠檬籽中的类柠檬苦素, 提取条件温和、提取效率高, 该方法具有一定应用前景。采用该方法提取的类柠檬苦素对绿霉菌和青霉菌具有明显的抑制作用, 其可通过破坏细胞壁扰乱细胞的正常生理活动, 阻碍其正常生长。

英文摘要:

      Objective To explore the feasibility of extracting limonoids by hydroxypropyl methyl cellulose (HPMC) assisted reverse micelles, and analyze the antibacterial activity of the extract. Methods Taking the content of limonoids in the initial extract as the response value, the optimal parameters of the initial extract were determined by orthogonal test; the main factors affecting the extraction efficiency were screened by Placktett-Burman (P-B) test, and the optimal process conditions were determined with Box-Behnken (B-B) test. The antibacterial activity of limonoids against Penicillium digitatum and Penicillium italicum was verified by measuring the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and alkaline phosphatase (AKP) activity. Results In the initial stage, the main factors affecting extraction efficiency were the extraction time of 1.5 h, the liquid to material ratio of 30:1 (mL/g), the HPMC addition amount of 0.5%, the material liquid pH at 6.5, and at this condition the extraction rate was (3.34±0.02) mg/g. P-B test and B-B test results showed that the optimal formulation of limonoids extraction were dioctadecyl dimethyl ammonium chloride (DODMAC) 44.29 mmol/L, orthohexanol 13.86%, bromotetradnet 3.88%, and the average yield of limonoids under the corrected process conditions was (87.64±1.12)%. The bacteriostatic test showed that the MIC and MBC of limonoids acted on Penicillium digitatum were 1250.00 μg/mL and 2500.00 μg/mL, and the MIC and MBC of limonoids acted on Penicillium italicum were 1250.00 μg/mL and 5000.00 μg/mL, respectively. Furthermore, after the determination of AKP, it was found that the AKP activity gradually increased with the extension of action time and the increase of purity. Conclusion Limonoids can be effectively extracted from lemon seeds by HPMC assisted reversed micelles, and this method is promising due to its mild extraction conditions and high extraction efficiency. The inhibitory effect of limonoids on Penicillium digitatum and Penicillium italicum is manifested through its destruction on the cell wall, which will affect the normal physiological activities of the cell and hinder its normal growth.