香芹酚-酪蛋白纳米颗粒制备及其对枇杷果实炭疽病的抑制作用

Preparation of Carvacrol-Loaded Casein Nanoparticles and Its Inhibitory Activity against Colletotrichum acutatum on Loquat Fruit(Eriobotrya japonica)

通过制备香芹酚(carvacrol,CL)-酪蛋白(casein,CS)的纳米颗粒(nanoparticles,NPs),分析并比较CL-CS纳米颗粒(CL-CS-NPs)与游离CL、酪蛋白+CL混合物在固体培养基上的留存差异性,以及三者对离体培养炭疽菌(Colletotrichum acutatum)菌丝生长的抑制作用和接种在枇杷果实上炭疽菌诱发枇杷病斑的抑制作用,考察CL-CS-NPs对枇杷果实炭疽病的抑制效果。结果表明,12 mg/mL CL和20 mg/mL酪蛋白溶液经超声处理可制备稳定的CL-CS-NPs(152.6 nm),CL-CS-NPs组的缓释时间较游离CL组和酪蛋白+CL混合物组分别延长4 d和2 d,表明纳米化CL缓释效果明显,应用CL-CS-NPs可以延长CL对病菌的作用时间;枇杷炭疽菌离体培养实验结果表明,CL-CS-NPs完全抑菌时间达到5 d,酪蛋白+CL混合物组与游离CL组均为3 d;枇杷果实接种炭疽菌实验结果表明,接种5 d后,喷涂CL-CS-NPs分散液的枇杷果实基本没有出现腐烂,接种7 d后CL-CS-NPs的平均病斑直径仅为1.5 mm、发病率仅为10%;而喷涂酪蛋白+CL混合物的枇杷果实接种5 d后病斑直径虽相对较小,但发病率高达80%,接种7 d后的病斑平均直径达到4.8 mm,腐烂比较严重。综上,将CL-CS-NPs分散液直接喷涂在枇杷果实上,能够取得良好的抑制由炭疽菌引起的枇杷果实腐烂的效果。

Objective:To prepare casein(CS)nanoparticles(NPs)loaded with carvacrol(CL)for improved antifungal activity of CL.Methods:Carvacrol-loaded casein nanoparticles(CL-CS-NPs)were prepared through ultrasonic treatment.The difference in the CL retention rate of CL-CS-NPs,when incubated on solid culture medium,versus free CL and a physical mixture of casein and CL was detected by high performance liquid chromatography(HPLC).The in vitro growth inhibitory effects of free CL,casein+CL and CL-CS-NPs on Colletotrichum acutatum mycelium were evaluated as well as the inhibitory effects on Colletotrichum acutatum inoculated into loquat fruit(Eriobotrya japonica(Thunb.)Lindl.cv.Baisha).Results:Stable CL-CS-NPs(152.6 nm in diameter)could be prepared by adding CL(12 mg/mL)to casein solution(20 mg/mL)followed by ultrasonic treatment.The sustained release time of CL-CS-NPs was 4 days and 2 days longer than that of free CL group and casein+CL mixture group,respectively,indicating that nano-CL had an obvious sustained release effect,and CL-CS-NPs showed potential to prolong the time of action of CL on pathogens.The in vitro culture experiment showed that CL-CS-NPs completely inhibited Colletotrichum acutatum for 5 days,while casein+CL mixture and free CL were effective for 3 days.Loquats sprayed with CL-CS-NPs dispersion did not rot basically at 5 days after inoculation,and the average lesion diameter and disease incidence were only 1.5 mm and 10%at 7 days.Moreover,those treated with casein+CL mixture had relatively small lesions but a high disease incidence(80%)at 5 days,and seriously rotted with an average lesion diameter of up to 4.8 mm at 7 days.Conclusion:Spraying the CL-CS-NPs dispersion onto loquat fruit can have a significant inhibitory effect on anthracnose rot caused by Colletotrichum acutatum.