Photodynamic Inactivation Mechanism Of Pseudomonas Fluorescens And Its Biofilm Based On Octyl Gallate And Blue Light

P.fluorescens is a common food-borne spoilage bacteria that can grow and multiply rapidly under low temperature conditions,causing spoilage of aquatic products and meat.To make matters worse,it can adhere to the surface of food and form a biofilm,making it more difficult to remove,seriously affecting food quality and hygiene.Usually,people choose to use antibiotics to solve the above problems,but the abuse of antibiotics will lead to the emergence of bacterial resistance.Therefore,it is urgent to develop a safe and effective method to kill P.fluorescens and eradicate its biofilm.Studies have shown that natural plant extracts,such as phenolic acid compounds,have excellent biological activities such as antioxidant,antibacterial and anti-inflammatory.Compared with phenolic acids,phenolic esters have stronger biological activity.Studies have shown that gallic acid alkyl esters（GACs）show better activity than gallic acid in antibacterial,anti-biofilm,antioxidant and anti-inflammatory effects.Different lengths of alkyl chains in GACs have significant differences in their biological activities,especially the antibacterial activity.Among them,octyl gallate（OG）with an alkyl branched chain of 8 carbons in its molecular structure exhibits the best affinity with bacterial membranes and the best antibacterial activity.At the same time,OG is also allowed to be added to food as a functional additive by the U.S.Food and Drug Administration（FDA）and the World Health Organization（WHO）.Based on this,this study developed an ultra-efficient photodynamic inactivation system combining OG with blue light（420 nm）irradiation,and discovered for the first time that OG has dual effects of bacteriostasis and photosensitivity.The OG-mediated PDI technology was applied to efficient sterilization and removal of biofilms,and the antibacterial mechanism of OG-mediated PDI was further elucidated.Finally,combining PDI technology with nano-electrospinning technology,a green and efficient photosensitive nanomaterial was developed and applied in the storage and preservation of Chinese giant salamander,showing their broad application as photodynamic antibacterial materials in the field of food safety.The main findings of this paper are as follows:1.The antibacterial activities of GA and GACs were preliminarily screened.P.fluorescens（G+）and S.aureus（G-）were selected as experimental strains,and the MICs and MBCs value of GA and GACs were determined.Combined with the oil partition coefficient,the influence of the hydrophobicity of GACs on the inhibitory activity of the above target bacteria was analyzed.The results showed that OG exhibited excellent antibacterial activity against these two experimental bacteria,with MIC and MBC values of 0.1 m M,3.2 m M and 0.05 m M,0.1 m M,respectively.Subsequently,the uptake levels and rates of bacterial uptake of GACs with different alkyl chain lengths were compared by analyzing the bacterial affinity for GA and GACs,among which OG had the fastest uptake to both P.fluorescens and S.aureus(90.6 min^-1 and124.5 min^-1).The results showed that the affinity of bacteria to OG was the best,which further confirmed that the affinity of bacteria to GACs was closely related to the antibacterial activity of GACs.Finally,rapid killing of P.fluorescens and S.aureus by OG at low concentrations（0.4 m M and 0.1 m M）was achieved by effectively combining OG with blue light irradiation（15 min and 10 min）.2.The antibacterial mechanism of OG-mediated PDI against P.fluorescens was studied.OG can interact with the bacteria cell membrane due to its high affinity to the membrane in terms of its hydrophobic portion to exert itself an excellent antimicrobial ability.By measuring the uptake of OG by P.fluorescens under blue light irradiation,it was found that blue light irradiation is beneficial to promote the penetration of OG into cells faster and more,and a large amount of OG accumulated in cells will photocatalyze when irradiated with blue light.Redox reaction occurs,electron transfer occurs to generate hydrogen peroxide（H₂O₂）,and H₂O₂ is subsequently photolyzed by blue light to generate hydroxyl radical（^●OH）.Therefore,the produced^●OH will attack multiple targets in bacterial cells,such as cell membranes,lipids and proteins,making it difficult for bacteria to develop drug resistance.Therefore,the synergistic effect of OG and blue light can be considered as a low-risk,safe and efficient bactericidal method due to the multi-target attack mode of the ROS generated.3.The effect of OG-mediated PDI on P.fluorescens biofilm was investigated.The ability of OG to synergize with blue light irradiation to inhibit biofilm formation was first investigated.Under sublethal pre-treatment（0.1 m M+15 min BL）,the amount of biofilm formation was reduced by 84%,effectively inhibiting the formation of biofilm.The scavenging effect of OG synergistic blue light irradiation on mature biofilms was also studied.The results showed that 55%of mature biofilms were effectively removed after 0.4 m M OG synergistic blue light irradiation for 90 min.Therefore,OG-mediated PDI can also effectively remove biofilms and reduce their biomass.Finally,by analyzing the number of viable bacteria in the biofilm and supernatant,it was found that PDI not only reduced the biomass of the biofilm,but also decreased the number of viable bacteria in it.The number of viable bacteria in the biofilm and supernatant decreased by 68%and 79%,respectively.The above results demonstrate that OG-mediated PDI not only has an efficient killing effect on planktonic bacteria,but also inhibits the formation of biofilms and effectively removes mature biofilms.4.The effect of OG/βCD-IC NFs as a novel photodynamic antibacterial material on the preservation of Chinese giant salamander was studied.OG/βCD-IC NFs is a photodynamic bacteriostatic material,which can achieve photodynamic sterilization by releasing OG in solution environment.At the same time,OG/βCD-IC NFs were coated on the surface of Chinese giant salamander,which could effectively inhibit the growth of microorganisms on the surface of giant salamander,maintain the fresh flavor of giant salamander meat to a certain extent,and prolong the shelf life.It shows its broad application prospect as a new photodynamic antibacterial material in the field of food safety.