Analysis Of The Antibacterial Components Of Yunnan Litsea Cubeba And Its Mechanism Of Bacterial Inhibition

Yunnan Litsea Cubeba,deciduous trees,native to subtropical and tropical regions of Asia.Recently,research on Yunnan Litsea Cubeba has shown that its extracted essential oil has antioxidant,anti-inflammatory and anthelmintic effects.In addition,its main component is citral,which has a broad spectrum of antibacterial activity against various bacteria and fungi.However,it has many active components,except citral,but relatively few studies on this aspect.Bacillus cereus is a Gram-positive bacterium that is widely distributed in food and has become one of the top ten pathogens causing many foodborne infections.This study takes Yunnan Litsea Cubeba as the research object,and uses Bacillus cereus as the test bacteria.The active components were extracted and isolated and identified by antibacterial activity tracing.Then explore the antibacterial mechanism of Yunnan Litsea Cubeba,which can provide methods and ideas for the food pollution caused by Bacillus cereus,and also provide reference for the development and utilization of natural preservatives and antibacterial agents in Yunnan Litsea Cubeba,promoting the development of Litsea Cubeba industry.The main study contents and results are as follows:（1）By measuring the antibacterial activity of different concentrations of ethanol extracts from Yunnan Litsea Cubeba,the results showed that the anhydrous ethanol extract from Yunnan Litsea Cubeba had the best antibacterial effect on Bacillus cereus with a diameter of 13.13±0.07mm.The alcohol extract of Yunnan Litsea Cubeba was extracted with petroleum ether,chloroform,ethyl acetate,and n-butanol respectively.The antibacterial activity of each extraction phase was compared,The results showed that the chloroform extracted phase had a low inhibitory activity with an inhibition circle diameter of 12.07±0.03mm,while the ethyl acetate extracted phase had the highest inhibitory activity with an inhibition diameter of 14.65±0.02mm against Bacillus cereus.（2）After being treated with temperature,ultraviolet radiation,acid alkali,metal ions,sugar,and salt,the antibacterial properties of the ethyl acetate phase of Yunnan Litsea Cubeba have changed.The influence of temperature and UV on its antibacterial effect is relatively small,and its antibacterial activity decreases,but overall it still shows good stability.The ethyl acetate phase of Yunnan Litsea Cubeba has the best antibacterial effect at the p H value of 8,with a diameter of 14.93±0.06 mm.Different metal ions have different effects on it.Mg²⁺treatment can enhance antibacterial activity,while Fe³⁺,Fe²⁺,Al³⁺,and Ca²⁺treatments can reduce antibacterial activity.Among them,Al³⁺treatment has the worst antibacterial effect,with a diameter of 8.54±0.11 mm.Both the salt and sugar treatments enhance antibacterial activity.（3）Through static adsorption and desorption tests,the adsorption rate and resolution rate of macroporous adsorption resin HPD-450 were 55.19%and 91.99%,respectively.The resin was selected for the separation and purification of ethyl acetate phase of Yunnan Litsea Cubeba,and thin layer chromatography was conducted.Six components are collected,of which Fr.3-2 has good antibacterial activity,with a diameter of16.52±0.12mm and minimum inhibitory concentration value of 0.07mg/m L.Take this component as the research object,pass through the Sephadex LH-20 gel column,collect and then analyze its antibacterial activity.The results show that the component Fr.3-2-2has the best antibacterial effect,its minimum inhibitory concentration value is the same as the component Fr.3-2,but its antibacterial circle becomes larger,with a diameter of 18.07±0.10mm.Untargeted metabolomics analysis of Fr.3-2-2,among which there were five components with high reliability and content.Through analysis and research,it showed that gingerol and cucurbitacin B had good antibacterial effect,and determined the antibacterial effect of gingerol and cucurbitacin,and found that the antibacterial effect of gingerol was better,Its minimum inhibitory concentration is 0.03mg/m L.（4）After treating Bacillus cereus with gingerol,its growth curve,conductivity,extracellular nucleic acid and protein content,alkaline phosphatase,ATP,etc.were measured,and its morphology was observed through electron microscopy:The results indicate that compared with the control group,the growth curve of Bacillus cereus treated with gingerol could not reach the growth peak;The extracellular conductivity significantly increases,leading to electrolyte leakage;The content of extracellular nucleic acid and protein also significantly increased,indicating that it damages the permeability of the cell membrane and the integrity of the cell wall of the cell;Under electron microscopy,it was observed that the bacterial structure was incomplete,with wrinkles on the surface and leakage of contents,ultimately leading to bacterial lysis and death.