Study On The Antibacterial Activity And Mechanism Of Ethanol Extract Of Yunnan Litsea Cubeba

As a special food of Yunnan province,Yunnan Litsea Cubeba contains a variety of chemical components such as volatile oil,steroids,alkaloids and flavonoids.The essential oil has been confirmed to have good antioxidant,antibacterial and other biological activities.There is no relevant report on the antibacterial effect of EELC（Ethanol Extract of Yunnan Litsea Cubeba）.In this study,five different ethanol concentrations（0%,55%,75%,95%,100%）were used to extract the fruits of Yunnan Litsea Cubeba.The Diameter of inhibition zone（DIZ）and Minimal inhibitory concentration（MIC）of the extracts against Escherichia coli and Staphylococcus aureus investigated,and to screen the best antibacterial activity EELC against S.aureus and E.coil.The effects of temperature,heat treatment time,p H and ultraviolet light on the bacteriostatic stability of EELC were analyzed.The growth kinetics curve,extracellular conductivity content,nucleic acid and protein content,intracellular ATP content,extracellular alkaline phosphatase（AKP）activity,scanning electron microscope and transmission electron microscope of EELC on S.aureus and E.coil were used to reveal the bacteriostatic mechanism of EELC.Finally,EELC was used to keep grass carp meat fresh,which can provide a theoretical basis for the further development of Yunnan Litsea Cubeba.The main conclusions are as follows:（1）The bacteriostatic effect of ethanol extract from Yunnan Litsea Cubeba extracted with 5 different ethanol concentrations（0%,55%,75%,95%,100%）was found:55%EELC had the best inhibitory effect on S.aureus.The DIZ and MIC were18.25±0.26mm and 19μg/m L,respectively.75%EELC had the best inhibitory effect on E.coli,with the DIZ and MIC of 17.81±0.71 mm and 819μg/m L,respectively.On this basis,subsequent experiments were performed.（2）The study of EELC stability by temperature,heat treatment time,p H and UV light showed that:Both 55%EELC and 75%EELC showed good antibacterial stability at different temperatures,among which 55%EELC had the best inhibitory effect at 40℃,75%EELC had the best antibacterial activity at 100℃,and the antibacterial activity remained stable when treated for 5 h under the best inhibitory effect,while 55%and 75%EELC showed a trend of decreasing at first and then increasing when p H was 4～10,and the antibacterial activity was more stable under alkaline condition.There was no significant effect on the inhibitory effect of10～60min irradiated by UV light.（3）After S.aureus and E.coil were treated with 55%and 75%EELC respectively,the OD₆₀₀value of the growth curve after 0 h was significantly lower than that of the control group（0MIC）.The extracellular conductivity of S.aureus and E.coil cultured for 2 h was significantly higher than that of the control group.And the protein content and OD₂₆₀value of the two tested bacteria were higher than that of the control group;the AKP activity of S.aureus and E.coil was significantly higher than that of the control group at 3,6,9,and 12 hours（P<0.01）;and after being treated with 55%and 75%EELC at 1 hour,the ATP content in the cells was significantly lower than that in the control group（P<0.01）.The observation of scanning electron microscope and transmission electron microscope showed that the S.aureus cells of the control group were arranged like grape shape,the cell surface was smooth and the cell content was abundant,and the E.coil gathered in slender rod shape,and the cell was intact.After treatment,the cell membrane was obviously damaged and the contents of the cells were leaked,and voids appeared in the cells.After treatment,the cell membrane of the bacteria was obviously damaged and the contents of the cells leaked,and cavities appeared in the cells.Therefore,55%and 75%EELC can affect the synthesis of macromolecules such as nucleic acid by acting on S.aureus and E.coil metabolism,growth activity and nutrient transport system,interfere with their normal energy metabolism,change cell membrane permeability and damage The cell wall promotes the massive leakage of cytoplasm and alkaline phosphatase,accelerates the death and invasion of bacteria from the outside to the inside,and finally causes the bacteria to stop their life activities.（4）55%and 75%EELC on the preservation of fresh grass carp meat pieces showed that the water loss rate of the fish pieces treated with 55%and 75%EELC was significantly lower than that of the control group（P<0.05）;The p H values??of the treatment group and the treatment group showed a trend of gradually increasing,then decreasing,and then rising again.On the 9th day,the p H values??of the treatment group were 6.43 and 6.54,which were significantly lower than 9.03 of the control group（P<0.05）;and the fish pieces The b^*value and whiteness from 1 to 9days were significantly lower than those of the control group（P<0.05）,and the L^*value and a^*value were significantly higher than those of the control group（P<0.05）;the TVB-N values??of the fish pieces in the treatment group were significantly It was lower than that of the control group（P<0.05）;with the prolongation of storage time,the total number of colonies and the number of coliforms in all groups showed an upward trend,but the total number of colonies and the number of coliforms in the treatment group were significantly lower than those in the control group（P<0.05）.