Study Of Tolerance Responses Of Meat-Borne Pseudomonas Fluorescens Biofilms To Acidic Electrolyzed Water

Pseudomonas fluorescens is recognized as one of the most important spoilage bacteria due to the ability to form biofilms on food processing equipment surfaces,which can cause severe cross-contamination.Though there were various ways of spoilage bacteria contaminating food,biofilm have been considered as one of the primary cause of cross-contamination.The detached biofilms which were triggered by environmental changes are also hazardous because they can behave similarly to adhered biofilms and survive disinfection challenges due to neutralization or the partial consumption of the disinfectant by extracellular polymeric substances(EPS).It is critical to enhance the control of detached and adhered biofilms.Acidic electrolyzed water(AEW)has been seen as an effective disinfectant with low available chlorine concentration(20-80 mg/L),high oxidation-reductive potential(>+1000 mV)and low pH(<3.0),which is safe and not any toxicity to the human being.Understanding the tolerance responses of P.fluorescens detached and adhered biofilms treated with disinfectant under the conditions of food processing environment were essential for providing important information for systematically revealing mechanism of tolerance response of biofilm to disinfectants,which can provide an experimental basis for the development of effective and novel strategies to control biofilm.Hence,in this paper,meat-borne P.fluorescens was taken as the research strain and stainless steel surface was the adhesion surface.This study aimed to evaluate the performance of planktonic cells and detached and adhered biofilms formed by P.fluorescens exposed to acidic electrolyzed water(AEW)and then investigate the morphophysiological differences,such as morphology,cell membrane properties and EPS,between detached and adhered biofilms.The proteomics techniques have been used to investigate the regulatory and differential proteins of three sample types of P.fluorescens under disinfectant treatment,which can reveal the tolerance response mechanism of P.fluorescens.The context and results were shown as following:1.Tolerance difference of P.fluorescens planktonic cells,detached biofilms and adhered biofilms to acidic electrolyzed waterMeat-borne P.fluorescens was the research strain and the food grade 304 stainless steel was used as the contact surface.The planktonic cells were used as control,and detached and adhered biofilms were used as the treatment group.The performance of three sample types exposed to AEW was evaluated using the plate counting method,and the morphological characteristics of the cells were investigated by scanning electron microscopy(SEM)and particle size distribution analysis.The results showed that AEW had good bactericidal effect on P.fluorescens,and the increasing concentration of AEW played significant role in the survival of bacteria,which could effectively control P.fluorescens,but the effect of AEW on two kinds of biofilms was weakened.The adhered biofilms presented the strongest resistance and tolerance.The Weibull model was used to fit the bactericidal curve,and the fitting parameters were R2>0.90,indicating that the model fits well to the bactericidal kinetics curve of AEW-treated P.fluorescens.The morphology of the cells changed significantly after treatment,especially the planktonic cells which exhibited surface indentations and cell distortions,which indicated the loss of typical structure.However,the cells in biofilms exhibited a small number of surface indentations and cell distortions and the EPS was significantly reduced.The particle distribution of biofilms showed a bimodal distribution,and planktonic cells showed a single peak distribution.The particle size of the cells decreased significantly after treatment of AEW.2.Research on the characteristics and extracellular polymeric substances of AEW-tolerant bacteriaAccording to chapter one,20 and 60 mg/L AEW were used to treat detached and adhered biofilms for 10 min.The cell membrane characteristics and proton driving force of the cells after AEW treatment were investigated.The chemical analysis methods and ATR-FTIR and Raman spectroscopy were combined to explore the changes in the composition and content of EPS.The results showed that AEW disrupted the integrity of the cell membrane and the increasing concentration of AEW played significant role.Damaged cell membranes cause leakage of intracellular substances,making intracellular structures exposed to the external environment,which causes a large amount of effective ingredients of AEW to enter the cells.The intracellular environment was then acidified,reducing membrane potential,intracellular ATP and pH,and interference intracellular metabolic process.The protein and carbohydrate content of EPS decreased significantly after AEW treatment.When added to AEW,EPS had a significant negative impact on the physicochemical properties of AEW,indicating that EPS played an important role in hindering lethal effect of AEW.More EPS secretion is one of the reasons for the stronger tolerance of adhered biofilms.ATR-FTIR and Raman results show that the spectrogram of the adhered biofilms is significantly different from the detached biofilms and planktonic cells.The adhered biofilms have more abundant protein and polysaccharide peaks,and the disappearance and weakening of some characteristic peaks after AEW treatment verified the consumption of EPS in the chemical analysis.3.Research on the proteomics of response of Pseudomonas fluorescens to acidic electrolyzed waterThe proteomics techniques were used to explore differential proteins of three sample types after AEW treatment.The result showed that there are 117 differentially expressed proteins(DEPs)related to the tolerance of adhered biofilms to AEW,and 166 of those were correlated with the tolerance of detached biofilms to AEW.The number of DEPs between planktonic cells control and detached biofilms control or adhered biofilms control were 577 and 484.The GO functional annotation showed that DEPs were distributed in metabolic process,cellular component organizations or biogenesis and single-organism in the category of cell components,distributed in binding and transporters activity in the category of molecular function,and distributed in organelle,cell and macromolecular complex in the category of cellular component.Pathway analysis showed that the DEPs of two kinds of biofilms were enriched in tolerance-related pathways,mainly in the two-component system,ABC transporter and ribosome,which participate in the efflux of the disinfectants and the chemotaxis;while the DEPs between biofilms and the planktonic cells are mainly enriched in the biosynthesis of metabolites,the metabolism of microorganisms in different environments,and ABC transporters,which were involved in cell motility,reproduction and biofilm formation.