Antibacterial Activity And Mechanism Of Limonene Against Listeria Monocytogenes And Its Application

Food borne pathogenic microorganism is one of the main factors affecting global food safety and human health.Limonene is a kind of monoterpene with good antibacterial activity,which is widely used in food preservation,food additives and other fields.However,the antibacterial activity and mechanism of limonene against Listeria monocytogenes(L.monocytogenes)are rarely studied.In this paper,the antibacterial activity of limonene against L.monocytogenes,the effect on cell wall and membrane,the effect on cell respiratory metabolism and respiratory chain were studied,and the antibacterial mechanism of limonene against L.monocytogenes was revealed.At the same time,limonene was applied in chilled meat,which would benefit the development and application of limonene as a safe,natural and non-toxic new natural preservative in the future.It provides a reliable theoretical basis for the establishment of the method.In this paper,limonene was applied to L.monocytogenes.The effects of limonene on the morphology and structure of L.monocytogenes were evaluated by determining the scanning electron microscopy,AKP activity,PI fluorescence staining,conductivity,leakage of nucleic acid and protein.The effect of limonene on energy metabolism of L.monocytogenes was investigated by measuring ATP and ATPase activity.The respiratory function of L.monocytogenes was evaluated by measuring the changes of membrane potential and respiratory metabolic activity,the respiratory inhibition pathway of limonene was determined by measuring the respiratory inhibition test with oxygen electrode,and the key enzymes of respiratory inhibition pathway were determined to explore the effect of limonene on the respiratory metabolism of L.monocytogenes.The possible inhibitory site of limonene on the respiratory chain of L.monocytogenes was explored.Secondly,limonene was applied to chilled meat,and its effect was explored by determining volatile basic nitrogen,thiobarbituric acid value,total bacterial count,nuclear magnetic resonance imaging and sensory evaluation.The results are as follows.(1)Limonene had significant inhibitory activity on L.monocytogenes,and the minimum inhibitory concentration was 20 m L/L.the growth curves results showed that limonene could effectively inhibit the growth and reproduction of L.monocytogenes.(2)Limonene can penetrate the damaged cells,combine with intracellular nucleic acids and emit red fluorescence under fluorescence microscope,which indicated that the cell morphology was seriously damaged,cell wall and cell membrane were damaged,and cell wall and cell membrane permeability were increased.(3)The activities of Na⁺K⁺-ATPase and Ca²⁺-ATPase in the control group and the experimental group showed a downward trend.With the addition of limonene(1 MIC),the activities of Na⁺K⁺-ATPase and Ca²⁺-ATPase and the content of ATP decreased significantly.The disorder of ATPase activity indicated that limonene could inhibit the synthesis of ATP by inhibiting energy metabolism.(4)The decrease of respiratory metabolic activity and the increase of membrane potential showed that respiratory metabolism of L.monocytogenes was inhibited by limonene.Respiratory inhibition test showed that limonene inhibited respiratory metabolism mainly by inhibiting TCA pathway of L.monocytogenes,followed by glycolysis(EMP)pathway.In addition,after limonene treatment,PK activity increased first and then decreased,SDH activity decreased first and then increased and then decreased,and MDH activity decreased significantly.In general,the activities of SDH,MDH and PK in treatment group were significantly lower than those in the experimental group,which indicated that the activities of key enzymes in respiratory metabolism pathway were inhibited.Finally,the decrease of respiratory chain complex activity and the results of differential expression of respiratory chain proteins confirmed that limonene affected respiration and energy metabolism by inhibiting the function of respiratory chain complex,and the inhibitory site of limonene on the respiratory chain of L.monocytogenes may exist in the transfer process of NADH to coenzyme Q.(5)After limonene(1 MIC)treatment,the TVB-N and TBA values of the meat samples were significantly lower than those of the control group at 2?10 d,indicating that limonene may delay the protein decomposition and have certain antioxidant effect on meat.The increase of the total number of colonies,the results of magnetic resonance imaging and sensory evaluation showed that limonene could delay the spoilage of chilled meat and inhibit the water diversion loss of chilled meat to a certain extent.