Study On Antibacterial Effect And Mechanism Of Inosine On Alicyclobacillus Acidoterrestris

Alicyclobacillus acidoterrestris is the main microorganism that leads to the spoilage of fruit juice,which has a significant impact on the fruit and food processing industry.By studying the antibacterial activity and mechanism of inosine against A.acidoterrestris,it is conducive to creating a new,efficient and safe hazard control method,and also lays a theoretical foundation for the application of inosine as a food preservative in fruit processing industry.In this paper,the antibacterial activity of inosine was determined,and its antibacterial mechanism was further explored.By constructing the protein interaction network of A.acidoterrestris after inosine treatment,the functional module process and regulation mechanism of inosine were analyzed from the protein level,and the antibacterial effect of inosine was further verified.The main research results of this paper are as follows:1.The quality problems of acidic juices such as nutritional loss,flavor deterioration and precipitation caused by the pollution of acidophilic and thermophilic Alicyclobacillus acidoterristris have become the bottleneck restricting the processing and export trade of fruits and vegetables.In this paper,the antibacterial activity of inosine on growth of A.acidoterristris and its influence on the quality of orange juice were studied by determination of optical density,measurement of biofilm formation,scanning electron microscopy(SEM)detection,plate colony count,physicochemical properties and sensory quality identification of fruit juice.The results showed that inosine with different concentrations could effectively inhibit the growth and reproduction of the vegetative cells of A.acidoterristris under acidic conditions,and had no significant effect on the physicochemical properties such as acidity,sugar content,color,taste and sensory quality of fresh orange juice.The addition of inosine as low as 5 m M could reduce the biofilm formation ability of A.acidoterristris,damage the cell wall and cell membrane,and lead to cell lysis and death.When the initial contaminated bacteria content is up to 10~5CFU/m L,the total number of A.acidoterristris can still be reduced by one order of magnitude,which control the number of vegetative bacteria so that it can not produce guaiacol that reaches the olfactory threshold.It is expected to be a new and efficient preservative for inhibiting the pollution of A.acidoterristris in acidic juice production.2.To further explore the antibacterial mechanism of inosine against A.acidoterrestris.Under the condition of inosine treatment for 120 min,the adsorption rate of xylene by the strain increased from 37%to 55%,indicating that inosine could improve the hydrophobicity of A.acidoterrestris.When the inosine concentration was 5 m M,the intracellular protein content decreased significantly,indicating that inosine caused the damage of bacterial cell membrane and the loss of content.The results of flow cytometry showed that the main peak shifted significantly after the cells were treated with 5 m M inosine for 6 h.Corresponding to the results of fluorescence inverted microscope,a large number of cells were stained red,indicating that the cell membrane was seriously damaged.Inosine at low concentrations can effectively destroy the growth of A.acidoterrestris and even lead to death.3.To further explore the antibacterial mechanism of inosine against A.acidoterrestris.Under the condition of inosine treatment for 120 min,the adsorption rate of xylene increased from 37%to 55%,indicating that inosine can improve the hydrophobicity of A.acidoterrestris,and the improvement of hydrophobicity indicates that the surface of cell membrane is damaged.When the concentration of inosine was 5 m M,the content of intracellular protein decreased significantly,indicating that inosine caused the damage of bacterial cell membrane and the loss of content logistics;The results showed that the main peak shifted significantly after 5 m M inosine treatment,indicating that the cell membrane was seriously damaged;Corresponding to the results of fluorescence inverted microscope,a large number of bacteria were stained red,indicating that the cell membrane was seriously damaged.The addition of inosine also led to the increase of cell membrane permeability of A.acidoterrestris.Erythromycin could penetrate into the cell,and OD₆₀₀decreased from 0.953 to 0.86.The results showed that low concentration of inosine could effectively destroy the normal growth and metabolism of A.acidoterrestris.This experiment verified the antibacterial activity of inosine against A.acidoterrestris,and the application of inosine in freshly squeezed clarified fruit and vegetable juice had no significant effect on its physical and chemical quality and sensory quality.The antibacterial mechanism was also studied,and it was found that inosine could destroy the cell membrane and cell wall of A.acidoterrestris,induce the loss of protein and content,and may be involved in the process of DNA cleavage,purine nucleotide metabolism,and purine nucleoside biosynthesis,which inhibited the growth and reproduction of bacteria.