| Citrus fruits are the main consumption fruit by many countries around the world and they are also commercially important fruits in Chongqing.Due to the high water content and rich nutrients,citrus fruits are susceptible to pathogen infection during postharvest storage and transportation and shelf life.Green mold,which caused by Penicillium digitatum,is the most serious fungal disease.Due to the long-term use of chemical fungicides,the public continues to pay attention to the residues of fungicides,the development of pathogen resistance,and the potential hazards of human health and environmental safety.Therefore,people are eager to find a safe and environmentally friendly antiseptic preservation method and substances to replace or reduce the use of chemical fungicides.The application of biocontrol agent instead of chemical fungicide in postharvest preservation of fruits and vegetables shows great potential.Among these antagonists,lactic acid bacteria(LAB)that do not produce toxic secondary metabolites but have an inhibitory effect against pathogens,have potential to replace chemical fungicides,and they generally regarded as safe(GRAS).In addition,as the main natural metabolite of LAB with antifungal and antibacterial activity,phenyllactic acid(PLA)has a good inhibitory effect on a wide variety of spoilage fungi and bacteria in food.In recent years,more and more attention has been paid to the identification of new LAB strains with antifungal and antibacterial activity to be used as biocontrol antagonists in food.However,the use of LAB and its metabolites phenyllactic acid to control postharvest diseases of citrus fruits has been less studied.In this experiment,pickles and cured meat were selected as the initial bacterial separation samples,and the antifungal activity of isolated LAB on P.digitatum were studied in in vitro assays,the antagonistic LAB were identified.Then 13 strains with antifungal activity screened in in vitro assays were selected to test the diseases control effect of green mold on citrus fruits.Finally,two Lactobacillus plantarum strains were selected(L.plantarum CKXP13 and L.plantarum CWXP24).The antifungal characteristics were investigated and the yield of phenyllactic acid of L.plantarum CKXP13 and L.plantarum CWXP24 cell-free supernatant were determined.Further research on the inhibitory effect and mechanism of phenyllactic acid against P.digitatum were investigated.The main research findings were summarized as follows:(1)A total of 224 strains were isolated from pickle and cured meat samples collected from Sichuan and Chongqing,and 13 strains of LAB with antagonistic ability against P.digitatum were preliminarily screened in in vitro assays and 13 strains were identified as: Lactobacillus plantarum(5 strains),Lactobacillus parafarraginis(3 strains),Lactobacillus casei(2 strains),Lactobacillus paracasei(1 strain),Lactobacillus buchneri(1 strain),Weissella paramesenteroides(1 strain).(2)Further studies were carried out on the antifungal activity of 13 antagonistic LAB strains,the results showed that the cell-free supernatant and washed cell suspension of 13 LAB strains could inhibit the growth of P.digitatum and the antifungal effect of different strains were different.Test of efficacy of LAB in controlling postharvest diseases in in vivo assay showed: the cell-free supernatant and washed cell suspension of L.plantarum CKXP13 and L.plantarum CWXP24 could effectively control green mold of citrus fruits.(3)The change of incubation time and culture initial p H value had a great influence on the antifungal activity of L.plantarum CKXP13 and L.plantarum CWXP24 cell-free supernatant.The optimal culture conditions for maintaining the antifungal activity of cell-free supernatant against P.digitatum were that the initial p H value of the medium was 6 and the incubation time was no less than 42 h.The antifungal activity of the cell-free supernatant of L.plantarum CKXP13 and L.plantarum CWXP24 decreased after the treatment of catalase and protease,which proved that the antifungal active substances produced by two L.plantarum strains included hydrogen peroxide and proteinaceous compound.p H neutralization test showed that the main antifungal active substances produced by the two L.plantarum strains included organic acids.(4)Acid production capacity of L.plantarum CKXP13 and L.plantarum CWXP24 were determined,the results showed that both strains had strong acid production capacity,the change of organic acid yield was consistent with the growth trend of the two strains.With the increase of culture time of the two strains,the organic acid products were accumulated continuously and the p H value decreased to about 4.The yield of phenyllactic acid in cell-free supernatant of two L.plantarum strains were determined by High Performance Liquid Chromatography(HPLC).The results showed that both L.plantarum CKXP13 and L.plantarum CWXP24 metabolized to produce phenyllactic acid in culture,and the yields were 42.4 mg/L and 91.9 mg/L,respectively.(5)The growth inhibition of phenyllactic acid against P.digitatum,the minimum inhibitory concentration of phenyllactic acid on P.digitatum,the lethal effect on spores and the inhibitory effect on the growth of mycelium were determined.The results showed that phenyllactic acid could significantly inhibit the growth of P.digitatum,the minimum inhibitory concentration was 2.5mg/m L,and phenyllactic acid had a strong inhibitory effect on the survival of spores and the growth of mycelium.(6)The morphological alterations of P.digitatum mycelium was observed by Scanning electron microscope(SEM).The results showed that the treatment of phenyllactic acid could lead to obvious folds and protrusions on the surface of the mycelium of P.digitatum,and the mycelium were more irregular in distribution,intertwined and twisted,with serious changes in surface morphology.In addition,phenyllactic acid destroyed the mycelium cells membrane structure of P.digitatum,resulting in the change of selective permeability,the extracellular conductivity significantly increased with the increasing concentration of phenyllactic acid.Test of efficacy of phenyllactic acid in controlling postharvest diseases in in vivo assay showed: the phenyllactic acid could effectively control green mold of citrus fruits. |
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