Study On The Inhibition Mechanism Of Perillic Acid Against Alicyclobacillus Acidoterrestris And Its Application On Freshness Preservation

Alicyclobacillus acidoterrestris(A.acidoterrestris)is a thermo-acidophilic,gram-positive and spore-forming bacteria.The heat resistance of A.acidoterrestris grants it the ability to survive the commercial pasteurization process and makes it a key quality control target for in the juice industry.In addition,growth of A.acidoterrestris in fruit juices produces metabolites with distinctive odors,such as guaiacol,which can lead to spoilage of fruit juices and cause significant losses to the juice industry,and therefore effective control methods are needed to minimize the risk of spoilage caused by A.acidoterrestris.In this paper,we investigated the antibacterial activity of perillic acid against A.acidoterrestris,further investigated the mechanism of action of perillic acid against A.acidoterrestris at the molecular level using transcriptome sequencing technology,and analyzed the practical application of perillic acid in fruit juice preservation,so as to provide new ideas and theoretical basis for the inhibition of A.acidoterrestris and the control of spoilage of fruit juices caused by it.The main research contents of this paper include:(1)Study on the antibacterial activity of perillic acid against f A.acidoterrestrisThe minimum inhibitory concentration(MIC)of perillic acid against A.acidoterrestris was 32 μg/mL by using the doubling dilution method.The growth curves showed that perillic acid could inhibit the growth of the cells,extending the retardation period from 4 to 6 hours,delaying its entering into the logarithmic phase and thus reducing the growth rate of the bacteria.The leakage of intracellular substances,microstructure observation of the bacterium,and determination of cell membrane integrity revealed that perillic acid can disrupt the integrity and permeability of bacterial cell membrane and cell wall,causing leakage of intracellular macromolecules,such as nucleotides and proteins,and destroying the structure of the bacterium,and the greater the concentration,the more obvious the effect.The determination of intracellular reactive oxygen levels showed that perillic acid can increase intracellular reactive oxygen levels,thereby causing oxidative stress damage to cells.Crystalline violet staining experiments showed that perillic acid inhibited the formation of A.acidoterrestris biofilms in a concentrationdependent manner.The results of cytotoxicity test showed that perillic acid was less than 5% toxic to human intestinal epithelial cells(Caco-2)at a concentration of 150mg/L,which provided some basis for the safety of perillic acid.(2)Based on transcriptomics tecnology to study the inhibition mechanism of perillic acid against A.acidoterrestrisThe mechanism of inhibition of A.acidoterrestris by perillic acid was investigated based on transcriptome sequencing technology.The results showed that1022 genes were significantly expressed after perillic acid treatment,of which 656 were up-regulated and 366 were down-regulated.GO and KEGG analyses showed that these differential genes were mainly concentrated in membrane transport,cell wall,cell motility and amino acid anabolism.Most of the genes related to sugar transport protein,peptidoglycan synthesis,and amino acid synthesis were upregulated,and most of the genes related to cell motility,two-component system,and amino acid metabolism were down-regulated.These results indicated that perillic acid could destroy the cell membrane and cell wall of A.acidoterrestris,leading to increased membrane permeability,which in turn led to disruption of amino acid anabolism.At the same time,perillic acid can reduce the ability of A.acidoterrestris to defend itself against adverse external environments through colony aggregation.(3)Preparation of perillic acid antibacterial hydrogel and its effect on antibacterial preservation of apple juiceBased on the antibacterial activity of perillic acid against A.acidoterrestris,perillic acid antibacterial hydrogel was prepared and characterized with perillic acid as antibacterial active ingredient,polyvinyl alcohol(PVA)and N-succinyl chitosan(NSCS)as substrates.The results showed that the intermolecular interactions of the hydrogel form a three-dimensional network structure that encapsulates the perillic acid in the hydrogel.The antibacterial hydrogels added with different concentrations of perillic acid inhibited A.acidoterrestris in apple juice,and there was no significant difference in effect on the physicochemical indexes such as brix,color and sensory quality of apple juice,but the presence of the alkaline polysaccharide Nsuccinyl chitosan increased the pH value of the juice,resulting in a decrease in acidity.In addition,the perillic acid antibacterial hydrogel slowed down the oxidative degradation of ascorbic acid in the juice to a certain extent,thus improving its nutritional value.In summary,this study found that perillic acid has good antibacterial effect on A.acidoterrestris,and the mechanism of inhibition of perillic acid on A.acidoterrestris was investigated by combining epistatic inhibition studies with transcriptomics technology.Then the perillic acid antibacterial hydrogel was prepared and applied to apple juice to investigate its actual antibacterial effect and its effect on the physicochemical properties and sensory quality of apple juice,which provides a theoretical extension for further development of perillic acid as a natural and efficient antibacterial agent.