| As a basic peptide in glycinin,glycinin antifungal peptide(GAP)with advantages of natural preservatives has a very good inhibitory effect on bacteria and a significant antiseptic effect in milk and noodles.In this paper,the antifungal mechanism of antifungal peptides and the characteristics of GAP were summarized.The antifungal mechanism of GAP against Aspergillus niger(A.niger)and its application in sponge cake were studied.The experimental results were as follows:(1)The scanning electron microscopy,transmission electron microscopy and flow cytometry were used to detect the response of A.niger cells morphology to GAP.The results showed that 3.00 mg/mL GAP treatment decreased the numbers of normal cells of A.niger to 11.81 % with increased cell complexity.The cells were wrinkled and twisted,the spores were bound,and the hyphae were entangled.The cell wall and cell membrane were separated and damaged.The organelles began to become irregular and degenerate,and the cytoplasm began to leak.These caused the dysfunction of cell wall,cell membrane and organelle,leading to the death of cells.(2)In order to explore the response of A.niger cell membrane to GAP,the results of nucleic acid and protein leakage experiments showed that GAP could lead to cell membrane infiltration and leakage of intracellular materials.Through determination the accumulation of malondialdehyde,the accumulation of malondialdehyde on the cell membrane after GAP treatment increased,and the accumulation increased with the increase of GAP concentration,indicating that GAP could destroy the structural integrity of the cell membrane with the increasing treatment concentration.In addition,the fluorescence intensity of the cell membrane was detected by fluorescent dye,and the results found that 3.00 mg/mL GAP could reduce the fluorescence intensity on the membrane to 45.36,which destroyed the fluidity of the cell membrane and interfered its functional integrity.(3)The results of protein electrophoresis and agarose gel electrophoresis showed that after the response of the biomacromolecules in A.niger cells to GAP,the number and content of protein bands were reduced,and the concentration and width of DNA bands were reduced,indicating that the GAP treatment could interfere with the nucleic acid and protein in A.niger cells.In addition,the results of circular dichroism showed that DNA in A.niger cells responded to GAP resulted in loose double-helix structure ofDNA,weakened base accumulation effect and finally changed the secondary structure of DNA.(4)After the mitochondria treated by GAP,the content of reactive oxygen species in mitochondria was significantly increased by fluorescence spectrophotometer observation.When the treatment concentration reached 3.00 mg/mL,the content of reactive oxygen species reached 10 times than that of the control group,causing the mitochondria being damaged due to the accumulation of reactive oxygen species.It was observed by flow cytometry that the GAP resulted in the decrease of mitochondrial membrane potential and the disruption of the internal and external balance of mitochondrial membrane.In addition,the 3.00 mg/mL GAP lead to mitochondrial ATP enzyme,succinate dehydrogenase and malate dehydrogenase activity compared with the control group was reduced by 65.06 %,79.10 % and 43.35 %,respectively by spectrophotometer determination.The results showed that GAP could inhibit the mitochondrial enzyme activity,destroy the tricarboxylic acid cycle,and make A.niger die when the energy required for its growth was not satisfied.(5)By comparing the changes in the sensory characteristics,physicochemical characteristics and antimicrobial characteristics of sponge cake with the addition of GAP,the results showed that the sensory characteristics and physicochemical characteristics of sponge cake decreased to a certain extent with the increase in the addition of GAP,but the antimicrobial characteristics increased significantly.Combined with the experimental results of sensory properties and physicochemical properties,0.15 % GAP had no significant negative effect on the quality of sponge cake and achieved good antimicrobial effect.Therefore,0.15 % GAP could be used as the optimal addition amount in sponge cake. |
