Research Of The Development And Sterilization Mechanism Of Microchip Pulsed Electric Field System

Juices are susceptible to microbial infection and spoilage,resulting in economic losses.Traditional thermal sterilization method can effectively prevent the spoilage of juice caused by microorganisms,but it also causes the damage of flavor and nutritive substances.In order to obtain safe,high quality juice products,this project developed a new microchip pulsed electric field(MPEF)sterilization technology.For the first time,the microchip is used in the field of juice sterilization of pulsed electric field(PEF),and sterilization under low voltage was achieved.To explore the feasibility of this technique,the blueberry juice was used as the object to study the effect of this technology on the spoilage microorganisms.In addition,the underlying lethal mechanism of MPEF on Pichia sp.was analyzed at cellular and molecular level,and the effect of this technology on the quality of blueberry juice was investigated.The main results were as follows:1.The main spoilage microorganisms in blueberry juice were identified by morphological,spoilage verification and sequence analysis,including Pantoea sp.,Meyerozyma spp.,Cryptococcus sp.,Pichia sp.,Burkholderia sp.,Aurcobasidium sp.,Penicillium sp.,Cladosporium sp.,among them,Cryptococcus sp.,Meyerozyma spp.and Pichia sp.were specific spoilage organisms(SSO).2.COMSOL software was used to simulate the capability of inactivating microorganisms of microelectrode structure.Appropriate electrode and packaging materials were selected to process microchips and packaged into micro-treatment rooms.According to the sterilization effect on Escherichia coli of the treatment rooms and the problems exposed in the experiment,the optimized treatment room was achieved and the experimental platform was established.The inactivation effect of MPEF on microorganisms was enhanced with the increasing of voltage(100-500 V),pulse number(20-100)and pulse duration time(50-250 ?s),besides,the type of microbes,growth period,pH and conductivity also affected the inactivation effect.Under the conditions of 400 V,200 ?s and 80 pulses,MPEF obtained a good lethal effect on Escherichia coli,Staphylococcus aureus.Saccharomyces cerevisiae and the spoilage microorganisms in blueberry juice.3.MPEF treatment can result in normal survival,sublethal or death of Pichia sp.under different processing conditions.The maximum rate of sublethal injury cells was up to 27.2%under 200 V.When the voltage was increased to 400 V,the sublethal rate of the Pichia sp.was close to 0.MPEF can cause the damage of cell morphology and ultrastructure,lead to a decrease in cellular enzymes,antioxidant enzyme activity and cell membrane fluidity,and destroy the structure of proteins and nucleic acids.Beisdes,the leakage of intracellular protein,nucleic acid,K+,Mg2+ and Ca2+-ATPase gradually increased as the increasing of voltage.Flow cytometry analysis showed that MPEF had significant effect on membrane potential and not obvious influence on non-specific esterase.The damage to cell membranes,leakage of contents,denaturation of proteins,reduction of enzymatic activity,changes in nucleic acids,and destruction of mitochondria can contribute to the death of Pichia sp.after MPEF treatment.4.829 significant difference proteins and 794 significant difference genes were obtained by measuring the proteome and transcriptome of Pichia sp.after MPEF treatment.This treatment has significant effect on the transcriptome and proteome of Pichia sp.,association analysis showed that there was a certain correlation between transcriptome and proteome.The genes involved in membrane integrity,organelles,nucleolus,protein transcription and translation,ribosomes,and enzyme activity of Pichia sp.were changed significantly after MPEF treatment.Destruction of cell structure,gene damage,weakened biosynthesis and disordered metabolic might be the main reasons for the death of Pichia sp.caused by MPEF treatment.5.Under MPEF treatment at 350 V,total plate count,total yeast and molds were totally inactivated,while anthocyanin content increased slightly.Flavor,Vitamin C,total phenols,titratable acid,color indicators had no significantly changes.During 40C,30 days storage,MPEF-treated blueberry juice had higher edibleness and quality than thermal-treated blueberry juice.