Construction And Application Of Targeted Anti-biofilm Materials Based On Extracellular Polysaccharide Hydrolase Combined With Zein

Microorganisms can pose significant food safety risks that can greatly impact human health.Research indicates that most microorganisms exist in the form of biofilms,which are complex communities formed through the mass aggregation of microbial cells and adhesion with extracellular secretions such as polysaccharides,proteins,and DNA.Biofilms pose a significant threat to the food industry.They enable bacteria to withstand the pressure of food sterilization technology and resist the invasion of bacteriogenic substances and other adverse conditions.Moreover,biofilms can easily cause secondary contamination of food,accelerate food deterioration.Therefore,modifying food processing-related materials and developing targeted antibiofilm materials can effectively reduce microbial attachment and prevent the formation of biofilms.Extracellular polysaccharides are essential components of bacterial biofilms and play a vital role in biofilm formation and bacterial drug resistance.These hydrolases are highly specific and can effectively target exopolysaccharides in biofilms,clearing them of food-borne pathogens.Innovative materials developed using these hydrolytic enzymes can effectively inhibit the growth of biofilms on the material’s surface by preventing bacterial adhesion at the source.Therefore,in this study,the researchers targeted the extracellular polysaccharides(Pel,Psl,algin,PNAG,and cellulose)at the core of biofilm formation in common pathogenic bacteria.Bioinformatics technology was used to extract the hydrolase sequence information targeting different extracellular polysaccharides,based on the extensive gene sequence data available in the NCBI database.Using the prokaryotic expression system of Escherichia coli,high-purity extracellular polysaccharide hydrolases PelAN and PslGN were obtained.Building on this achievement,a novel anti-biofilm material(PelAN-PslGN-Zein)was developed,incorporating Zein.Subsequently,PelAN-PslGN-Zein was further enhanced by the addition of Thymol,resulting in a new material(PelAN-PslGN-Thymol-Zein)with dual functions of sterilization and preservation,as well as biofilm resistance.The study’s specific research content and outcomes are detailed below:1.Bioinformation mining of bacterial biofilm core extracellular polysaccharidetargeting hydrolaseBased on the abundant gene sequence information in NCBI database,153 hydrolases and their sequence information targeting the extracellular polysaccharide of biofilm core of common pathogenic bacteria,Pel,Psl,algin,PNAG and cellulose,were screened.Among them,there are 30 hydrolases targeting Pel exopolysaccharide,belonging to the Glycoside-hydrolase family GH114;a total of 25 hydrolases targeting Psl exopolysaccharides belong to the glycosidic hydrolase superfamily;there are 33 hydrolases targeting algin exopolysaccharides,belonging to the algin lyase superfamily;there are 30 hydrolases targeting PNAG extracellular polysaccharides,belonging to the Glycoside-hydrolase family GH13;there are 35 hydrolases targeting cellulose exopolysaccharides,belonging to the Glycosyl hydrolases family GH 8.Phyre2,SwissModel and other bioinformation tools were used to analyze the physical and chemical properties,genetic evolution,functional domain and three-dimensional structure of these hydrolases.The results showed that these hydrolases have the potential to target the core extracellular polysaccharides of biofilm,which needs to be further developed and applied.This study provides the most comprehensive sequence composition and biological information of core extracellular polysaccharide hydrolase in biofilms so far,laying a solid data foundation for precise prevention and targeted control of biofilms.2.Preparation and preliminary application of PelAN-PslGN-Thymol-Zein novel anti-biofilm materialsZein has been widely studied for its excellent film forming ability and hydrophobic properties,making it an ideal material for food preservation packaging when combined with Thymol.However,due to its high protein content,zein is susceptible to bacterial biofilm contamination,which limits its application in the field of food storage and fresh-keeping.Thus,there is a need to enhance zein’s anti-biofilm properties and expand its potential in food preservation.To address the issue of bacterial biofilm contamination on zein-based food packaging,this study focused on obtaining highpurity extracellular polysaccharide hydrolases PelAN and PslGN.Using the E.coli prokaryotic expression system and gel filtration chromatography technology,these hydrolases were successfully obtained.Furthermore,crystal purple experiment and confocal microscopy(CLSM)confirmed their ability to effectively clear biofilms.To improve the anti-biofilm properties of zein-based food packaging,novel materials were constructed using PelAN-Zein,PslGN-Zein,and PelAN-PslGN-Zein composites.Single zein was used as the control.Results showed that the PelAN-PslGN-Zein composite exhibited the most effective anti-biofilm properties.Inhibition rates of biofilm formation for Pseudomonas aeruginosa PA14,PAO1,Pseudomonas putiodus21624,and Pseudomonas fluorescein 13525 were 91.68%,91.58%,79.68% and 77.75%respectively.To further enhance the anti-biofilm properties of PelAN-PslGN-Zein,Thymol was added to create a new composite material,PelAN-PslGN-Thymol-Zein.Results showed that this new material had high inhibition rates against four Pseudomonas biofilms,reaching 92.72%,97.34%,97.86% and 89.47%,while ThymolZein only had inhibition rates of 85.56%,68.55,67.42% and 78.79% against the same biofilms.This study successfully designed a composite material with both sterilization and preservation capabilities and demonstrated its potential application in the field of food storage and preservation.Further research is needed to confirm its practical effectiveness.3.Application of PelAN-PslGN-Thymol-Zein anti-biofilm materials in Salmon and BasaThe storage and preservation of fish is a crucial concern in the field of food science,particularly for high-consumption fish like salmon and tilapia.This study aimed to investigate the effectiveness of the newly constructed PelAN-PslGN-Thymol-Zein material for the storage and preservation of these two fish species,with traditional zein used as the control group.Quality characteristics such as color difference,texture,water content,p H,total volatile base nitrogen,and K value,as well as microbial changes such as total bacterial count,coliform bacteria,pseudomonas,and microbial diversity,were studied over a 5-day storage period at 4 ℃.The results of quality characteristics showed that during the 5-day storage period,the color change of fish samples in the experimental group was slower than that in the control group,and the hardness and elasticity decreased significantly.The color difference values of Salmon and Basa fish in the control group were 1.2 times and 1.1 times of that in the experimental group,respectively.In the control group,the hardness of Salmon and Basa decreased by 65.76%and 60.79%,while in the experimental group,the hardness of Salmon and Basa only decreased by 50.67% and 39.72%.In the control group,the elasticity of Salmon and Basa decreased by 31.55% and 46.37%,while in the experimental group,the elasticity of Salmon and Basa fish decreased by only 14.00% and 42.55%.Low field NMR test found that the free water content of Salmon and Basa in the control group was 5.00%and 3.00% higher than that in the experimental group,indicating that the material can effectively prevent the bound water and not easy to flow water to free water transformation,to ensure the quality of aquatic products.The K value of Salmon and Basa in the experimental group was 11.00% and 14.00% lower than that in the control group.The results of TVB-N determination showed that the material could prolong the shelf life of Salmon by 1 day.During the 5-day storage period,the total colonies of Salmon and Basa fish in the experimental group were 0.7 log CFU/m L and 1.5 log CFU/m L lower than those in the control group,and the total coliforms of Salmon and Basa in the experimental group were 0.2 log CFU/m L and 1.0 log CFU/m L lower than those in the control group.In the experimental group,the total number of pseudomonas colonies in Salmon and Basa fish was lower by 1.0log CFU/m L and 1.5log CFU/m L.The analysis of microbial diversity showed that the chao1 diversity index of Salmon and Salmon in the experimental group was significantly lower than that in the control group.This study comprehensively evaluated the storage and fresh-keeping effect of this new material on salmon and Basha through six classic quality indicators and four microbiological indicators,laying a solid foundation for the application of this material in the food field.To sum up,this study discovered a large class of enzyme molecules with antibiofilm activity through bio-information mining,and developed new composite materials with both storage and anti-biofilm properties on this basis,providing new ideas and new technologies for promoting the high-tech development in the field of aquatic products storage and preservation.