Study On The Synergistic Inhibitory Mechanism Of Eugenol And Citral Against Penicillium Roqueforti And Aspergillus Niger

The mildew and spoilage of bread will not only cause huge economic losses,some fungal toxins caused by fungi will also pose a serious threat to human health.How to control the mildew and spoilage of bread and prolong its shelf life effectively has become the primary problem hindering the development of bread industry in China.Compared with chemical synthetic antistaling agents,natural antistaling agents have become a research hotspot in food fresh-keeping industry at home and abroad because of their no environmental pollution,pesticide residues and threats to health.However,natural preservatives have some problems such as high cost,low efficiency and unclear target.As the main natural plant preservative,essential oils（EOs）has the property of low aroma threshold,short action time,easy oxidation and decomposition and that its minimum inhibitory concentration（MIC）in food matrix is higher than that in vitro,which limits its application in food preservation.Therefore,in order to solve these problems effectively,this paper investigated the synergistic mechanism of eugenol and citral(S_EC)on Penicillium roqueforti and Aspergillus niger from the perspective of synergetic antibacterial,in order to reduce the dosage and cost of EOs in food preservation and improve its antibacterial effect.On this basis,the active antibacterial packaging of EOs containing porous starch microcapsules was prepared.The addition amounts of microcapsules in bread preservation was determined according to the release kinetics model at different temperatures.The main research contents of this paper include four parts:1.Screening of combinations with synergistic antibacterial effect and preliminary evaluation of its application in bread preservationIn this study,10 kinds of natural preservatives that have been reported commonly used in baking products preservation were selected to study the antibacterial activities of P.roqueforti and A.niger which are common spoilage fungi in baked products and also the main microbial species causing bread spoilage.According to MIC,10 kinds of natural preservatives were compounded by checkerboard method.The results showed that only one group,eugenol and citral(S_EC),had synergistic antibacterial effect on the two kinds of fungi.The MIC value of the S_EC was reduced by 75%⁷6%compared with the MIC value of single active compounds.This combination can effectively reduce the action concentration of single active compounds.Through molecular docking,it was found that eugenol and citral could bind to the active sites of β-1,3-glucan synthase and chitin synthase in the cell wall of P.roqueforti and A.niger through different amino acid residues,thus inhibiting the normal synthesis of cell wall.When S_EC was used in 1MIC,the inhibition rate of spore germination of two kinds of fungi was more than 87%,and had obvious inhibitory effect on mycelium growth and mycelium morphology.At the same time,the shelf-life experiment showed that S_EC could prolong the shelf-life of bread better than eugenol or citral alone,and had no significant effect on the sensory quality of bread.2.The mechanism of synergistic effect of S_EC on cell membrane of P.roqueforti and A.niger was revealed at the molecular levelThe combination of S_EC caused serious damage to the integrity of cell membrane of P.roqueforti and A.niger.S_EC can combine with tryptophan（TRP）,tyrosine（Tyr）and phenylalanine（PHE）in membrane protein,and destroy the structure of cell membrane.The binding of S_EC to them mainly by hydrogen bond,supplemented by van der Waals force.GC-MS and electrophoresis analysis showed that after S_EC treatment,the proportion of unsaturated fatty acids in the cell membrane of the two fungi increased,the membrane fluidity increased,and the membrane protein was degraded.At the same time,S_EC combination induced ROS accumulation by regulating the expression of NOX gene,and citral played a major role in the synergistic effect of S_EC on cell membrane oxidative damage.The lipophilicity of eugenol and citral contributes to their dissolution and penetration in the cell membrane and reduces the synthesis of ergosterol,while eugenol contributes more to the inhibition of ergosterol synthesis than citral.Because the integrity of cell membrane was destroyed,cell contents leaked,DNA degradation and conductivity increased.Eugenol plays a leading role in the process of S_EC damage to cell membrane integrity and permeability.The changes of cell morphology and ultrastructure further confirmed this conclusion.3.Effects of S_EC on mitochondrial damage and energy metabolism of P.roqueforti and A.nigerThe S_EC can lead to severe deformation or collapse of mitochondrial morphology of the two fungi,resulting in a significant increase of membrane potential,and finally a phenomenon of membrane potential super.The contribution of eugenol to the synergistic effect of S_EC on mitochondrial morphology and membrane potential was greater than that of citral.Studies on the activities of key enzymes in the tricarboxylic acid cycle（TCA）showed that S_EC inhibited the activities of succinate dehydrogenase（SDH）,isocitrate dehydrogenase（IDH）,pyruvate dehydrogenase（PDH）,malate dehydrogenase（MDH）,citrate synthetase（CS）and α-ketoglutarate dehydrogenase（α-KGDH）,and the enzyme activity decreased by 71.45%⁹8.38%.In addition,S_EC decreased ATP synthesis and ATPase activity,disrupted the normal TCA cycle and destroyed the normal function of mitochondria.Further energy metabolism experiments showed that the contents of β-galactosidase,alkaline phosphatase,total adenylate content and energy charge were decreased in varying degrees by S_EC treatment.The decrease rates of the activities of the two enzymes ranged from 12% to19%,and the energy charge decreased from 6.9% to 13%.In addition,S_EC inhibited the gene expression of SDH,IDH and FH in P.roqueforti,whereas it induced the expression of these genes in A.niger.The mutations of these gene expression disrupted the normal TCA cycle and energy metabolism seriously,and eventually lead to apoptosis.4.A new method to prolong the shelf life of bread: active antibacterial small package containing S_EC(P_SMA)A kind of S_EC-containing porous starch microcapsule antibacterial small package(P_SMA)was developed,based on S_EC and porous starch.In vitro test showed that P_SMA had good inhibitory effect on the two fungi.P_SMA belongs to first-order kinetic release at 25 ℃,and between diffusion and first-order release at 35 ℃ and 45 ℃.When stored at 25 ℃,P_SMA could prolong the shelf life of bread to 13 d in PP and HDPE packaging.In these two kinds of packaging,the shelf life of bread with the antibacterial small package containing eugenol and citral was 11 days and 9 days respectively.P_SMA can inhibit the growth of mold and yeast in bread more effectively than the small package with antibacterial activity of eugenol and citral alone.When the amount of microcapsules in the antibacterial package was 1 g、1.5 g and 2 g,the synergistic minimum inhibitory concentration（0.190 mg/m L）was reached or exceeded on the 13 th,7th and 5th day of storage,respectively Through the shelf-life experiment,it was found that the antibacterial activity of S_EC in the gas phase was higher than that in the liquid state.Finally,sensory evaluation showed that the use of P_SMA in PP and HDPE packaging had no significant effect on the sense of bread. The research results of this project provide a scientific theoretical basis for the development of high-efficiency natural antibacterial agents,and also provide reference for extending the shelf life of baking products.