Preparation Of IONPs @pDA-Nisin Composites For Capaturing And Killing Alicyclobacillus Acidoterrestris And Analysis Of The Antibacterial Molecular Mechanism

Alicyclobacillus acidoterrestris,a acidothermophilic spore-forming bacteria,could survive from the pasteurization processes and germinate at room temperature.It is able to produce guaiacol inducing a medicinal and smoky taste causing the spoilage of beverages.The off-odur smelling in juices can be detected when the concentration of guaiacol is only 2 ppb which is produced by 10⁴-10⁵ CFU/m L of A.acidoterrestris cells.This spoilage caused by A.acidoterrestris has been found in many kinds of fruit juices and beverages,but it most commonly accured in apple juice.Nisin,as the natural food bio-preservative approved by almost 50 countries.However,the application of nisin in liquid productis is still limited due to the loss of antibacterial activity by various factors,such as interaction with components in food systems and enzymatic degradation.Furthermore,the prolonged exposure of micro-organisms results in the resistance of nisin,which is an occurred danger in food safety and human health.The main purpose of the present study was to propose a novel method to improve the activity of nisin by modificatiing the IONPs@p DA,which not only retain the effective antimicrobial activity of nisin,but also avoid producing the drug-resistant bacteria through magnetic separation.Newly bacteriocidal composites were synthesized and characterized by TEM,VSM,XRD,FT-IR,and XPS.The recovery rate of IONPs@p DA-nisin composites was measured to evaluate the residue and recoverability.What’s more,study on the antibacterial mechanism at cell level and gene expression levels was carried out.The acute toxicity study were carried out to determine the potential toxicity of the antibacterial nanoparticles.Finally,the evaluation of the antimicrobial activities of multifunctional composites on A.acidoterrestris cells and spores in three apple juices was performed.The affection on the sensory quality of three kinds of apple juices were investigated after the separation of multifunctional nanoparticles.The main research contents and results are as follows:（1）Nisin-loaded IONPs@p DA composites with heat stable and p H tolerance was synthesized as a simple and no drug resistance antibacterial material to Alicyclobacillus acidoterrestris.The optimal preparation conditions of IONPs@p DA-nisin composites was as below:the optimal ratio（IONPs@p DA:nisin）was 1:1,the optimal treatment time was30 min,and the optimal p H value was 6.86.In addition,the composites have a crystal structure and a diameter of 25.61±2.9 nm with a saturation magnetization（MS）of 43.7995 emu g^-1.According to the investigation of FT-IR and XPS,nisin was successfully conjugated onto the IONPs@p DA nanoparticles.The recovery rate values of the IONPs@p DA at different concentrations were all 99.99%in 30 min.The polydopamine coated magnetic nanoparticles（IONPs@p DA）were synthesized as an effective adsorbent for nisin.Research on the adsorption theory was conducive to development of the nisin-IONPs@p DA composites.The experimental kinetic data was fitted with the equations of adsorption kinetics and adsorption isotherms.The adsorption data was well fitted with the Lagergren pseudo-second-order model and the Freundlich model,indicating the occurrence of chemical adsorption and the multilayer adsorption.The adsorption process was endothermic reaction and the content of nisin increased with the rise of temperature.（2）The membrane damaged was obtained by the live/dead staining,SEM and TEM on A.acidoterrestris which pretreated by the proposed composites.In addition,the composites exhibited highly effective antibacterial activity against target strains in only 5 min.（3）Based on the results of RNA sequencing（RNA-seq）analysis,a total of 334 DEGs were obtained,which mainly related to transcriptional regulator,energy metabolism,membrane transporters,membrane and cell wall synthesis and cell motility.Therefore,the changes in transcriptional regulator caused by aggregating of the composites and target strain led to a series variation in homeostasis,which mostly happened in the decreased energy metabolism,pore-formation in membrane and depressed synthesis of cell wall.Meanwhile,the cell motility and spore germination were both depressed and finally intracellular substances flowed out.（4）To evaluate the toxicity of the composites,the acute toxicity test was taken.The results were demonstrated the biosecurity and nontoxicity of the IONPs@p DA-nisin.（5）IONPs modificated with polydopamine were covalently immobilized with nisin to inhibit the growth of Alicyclobacillus acidoterrestris cells and spores in apple juices（p H 3.57,14゜Brix）.The minimum bactericidal concentration（MBC）of IONPs@p DA-nisin composites against Alicyclobacillus acidoterrestris cells and spores in three kinds of apple juice（reconstituted apple juice,fresh apple juice,and commercial appl juice）were 1.25mg/m L and 2.5 mg/m L,respectively.The number of cells and spores decreased to 10 CFU when treated with the composites at the concentration of 20 mg/m L for 10 min.The content of polypheonls,organic acid,and sugar were measured,and results indicate that where was no influence of the composites on the apple juices.The content of volatile compounds almost the same compared to the control samples.In summary,newly synthesized bacteriocidal composites exhibited characteristic of highly efficient sterilization,bio-safety,and nontoxicity.The composites showed the damage of cell integrity by the changes in membrane.DEGs were mostly encoding transcriptional regulator,energy metabolism,membrane transporters,membrane and cell wall synthesis and cell motility.Notably,four transcriptions,including two transcriptional regulators,cell motility,sporulation,and ribosomal proteins,were firstly found due to the composites challenge.The composites exhibited highly effective antibacterial activity against A.acidoterrestris with only5 min in apple juice.Furthermore,the sensory and qualities of apple juices were not affected by the composites.Thus,the results of the present work demonstrated the potential used of IONPs@p DA-nisin composites to solve the practical spoilage problem by A.acidoterrestris cells and spores.What’s more,the study offers a novel method to control the microorganism without inducing the drug resistance and provide insights in controlling the microorganism.