Two Kinds Of Plant Polyphenols In Vitro Of Shewanella And Pseudomas And Anti-Biofilm Research

Seawater fishes is becoming popular in people’dining-table, due to its delicious flavor and tender taste rich of amino acids essential for human and rich vitamins. However, because they contain abundant moisture,protein and fatty acids, especially higer content of unsaturated fatty acid, which it easy to corrupt. Meanwhile, the activity of microbial reproduction causes for the quality deterioration of aquatic products during the storage, especially special spoilage bacterial. There are studies have shown that the fish, shellfish of temperate waters specific spoilage organism is S. putrefaciens and P.aeruginosa. Plant Polyphenols are widely found in fruits, vegetables and others foods containing flavan tannins, phenolic acids, flavonoids and so on. They are wide source of bacteriostatic and can also inhibit the formation of biofilm.The S. putrefaciens and P.aeruginosa are the most common spoilage bacteria reported in fish and fish products and were aimed in the study. The antibacterial of plant extracts on S. putrefaciens and P.aeruginosa were studied. The effects of tea polyphenols (TP) and grape seed extract (GSE) on the characters, membrane permeabilization of bacteria were discussed. And a possible mechanism of the TP and GSE antibacterial ability was discussed through bacterial membrane damage. The inhibition of sub-MIC TP and GSE on the biofilm though the reduction of EPS and metabolism were also discussed. The main results obtained in this study were as follows:1. Six plant extracts were investigated for five methods evaluation of the antioxidant activity. TP and GSE showed higher antioxidant properties in six plant extracts. Total phenolic, the DPPH and reducing power exhibited a concentration dependent activity. The highest content of total phenolic was found in TP (613.95mg GAE/g), followed by GSE (499.69mg GAE/g), The IC50of DPPH was5.05μg/mL and6.63μg/mL for TP and GSE respectively; The IC50value of reducing powder of TP and GSE was about33.10mg/mL and74.40mg/mL, which were lower than VC and BHT. The ratio of Chelated iron and O2free radical scavenging of TP and GSE were above50%when the concentration were1mg/mL. Additionally, six plant extracts can inhibit S. putrefaciens and P.aeruginosa,TP and GSE showed strong antibacterial activity with minimum inhibitory concentration (MIC) value of TP (1mg/mL),GSE(2mg/mL), rosemary extract(32mg/mL), liquorice extract(32mg/mL), Houttuynia cordata extract (16mg/mL) and Ginger Extract(32-64mg/mL). The Results also confirmed the positive correlation between antioxidant capacity and bacteriostatic activity of plant polyphenols. 2. TP and GSE showed strong antibacterial activity with MIC lmg/mL and MBC16mg/mL of TP, MIC2mg/mL and MBC32-64mg/mL of GSE. When TP and GSE at concentration of2MIC was added, the growth of S. putrefaciens and P.aeruginosa was repressed significantly, especially at the initial48h. The population of S. putrefaciens and P.aeruginosa decrease about4-5Log CFU/mL after48h for the TP and GSE addition. The bactericidal activity of TP and GSE carried out on the viable counts of S. putrefaciens and P. aeruginosa both at25℃and4℃. The population of S. putrefaciens and P.aeruginosa decrease about5and3Log CFU/mL after96h for the TP and GSE addition respectively at25℃. The addition of TP and GSE significantly reduced the population as the length of incubation time. Additionally, the bactericidal effect both of TP and GSE against two spoilage bacterial was stronger at25℃than at4℃(P<0.05). Among the four monomer, the order of bactericidal activity were EGCG>ECG>EGC>EC. The TVC, HbS-producing bacterial and P.aeruginosa in the large yellow croaker matrix reduced3,3,2Log CFU/mL for treatment with TP and2,2,1Log CFU/mL for GSE. The bactericidal effect both of TP and GSE against two spoilage bacterial was stronger at25℃than at4℃(p<0.05).3. The enzyme activity of Na+/K+-ATP and AKP in the suspension of S. putrefaciens and P.aeruginosa gradually increased with the time extension in the TP treated samples, indicating permeability of cell membrane increased. But the absorption value of260nm did not changed significantly meaning the nuclear membrane not damaged. TEM images revealed that treatment of TP might leads to damage and deformation of cell. Thus, the antimicrobial mechanism of TP was attributed to disruption of cell walls and out membrane, the increase of the membrane permeability and the change of cell morphology, finally caused cell death.4. Both S. putrefaciens and P.aeruginosa can form biofim, which2-3days were fast film-forming period, five days later formed the mature biofilm. Sub-MIC TP and GSE can inhibit the biofilm formation of S. putrefaciens and P.aeruginosa. The biofilm reduction caused by varying levels of TP and GSE exhibited an obvious dose-effect correlation. Biofilm reduction caused byTP and GSE decreased with the time of addition (before12h), the inhibition reached60～80%. And applying TP and GSE after12～24h or later had no inhibitory effect against biofilm formation.In addition, sub-MIC TP and GSE can not eliminate the matured biofilm. Metabolic activity and EPS of the biofilms decreased considerably after incubation with the sub-MIC of TP and GSE.