Antimicrobial Activity And Application Of Secondary Metabolites From Actinidia Chinensisendophytic Bacteria

Nowadays,the loss of fruits and vegetables due to diseases is one of the difficulties faced in the world.Traditional chemical fungicides have been abused,leading to increased resistance to pathogenic bacteria.In addition to the harmful effects of pesticides on fruits and vegetables on human health,the safety of chemical fungicides has been widely questioned.Plant endophytes have a special living environment and can synthesize natural products that are the same or different from the host plants.It is one of the important ways for us to obtain the source of new compounds.Its metabolites contain a range of structurally diverse antibacterial active compounds,providing a new approach to the development of new,safe and efficient natural antibacterials.In this study,two endophytic bacteria MR-1 and ML-1 with broad-spectrum antibacterial effects were isolated and screened from the roots,stems,leaves and fruits of healthy kiwifruit.In order to study the role of endophytic bacteria in postharvest storage of cherry tomatoes,the inhibitory rate of endophytic bacterial MR-1 fermentation filtrate against indicator bacteria was determined by using the method of perforation as the indicator strain.The single factor experiment was used to optimize the inhibitionrate of endophytic bacteria MR-1 fermentation filtrate.The medium components and fermentation conditions were used.On the basis of this,the fermentation conditions were optimized by response surface.The effect of MR-1 fermentation filtrate on the storage and preservation of cherry tomatoes at room temperature?25??was studied,and the antibacterial active substances were applied.Initial separation and identification.The specific research results are as follows:A total of 18 endophytic bacteria were isolated from the roots,stems,leaves and fruits of healthy kiwifruit plants by grinding.Three post-harvest pathogenic bacteria including Plasmodium sinensis,Capsicum annuum L.and Sclerotinia sclerotiorum were used as antagonistic bacteria.Two strains of endophytic bacteria MR-1 and ML-1 with potential broad-spectrum antibacterial effects were screened by plate-to-sputum methed.The antibacterial zone of endophytic bacteria MR-1 against three pathogenic bacteriais greater than 5cm,and the endophytic bacteria ML-1 is more than 5cm against the pathogen of Capsicum annuum and R.sclerophylla,and the antibacterial zone against V.More than 4cm.The strains MR-1 and ML-1 were identified as Bacillus amyloliquefaciens by cell morphology,colony morphology observation,physiological and biochemical assay and 16 S r DNA sequence analysis.Bacillus amyloliquefaciens MR-1 and ML-1 have strong inhibitory effects on the postharvest pathogenic bacteria of eight fruits and vegetables tested.The inhibition rate of eight pathogenic bacteria wasdetermined by perforation method.The inhibition rate of Botrytis cinerea,Brassica campestris and Capsicum annuum was the highest 48%?42.22%?38.47%,and the inhibition rate against Plasmodium was 16%.The strain ML-1 had the strongest inhibitory effect on the pathogen of Brassica campestris,Capsicum annuum,and Alternaria,and the inhibition rates were 37.5%,36%,and 29.17%,respectively.The lowest inhibitionrate against Plasmodium was 15.38%.The strains MR-1 and ML-1 showed broad-spectrum antibacterial activity,and the antibacterial effect of endophytic MR-1 was better than that of endophytic ML-1.2.Single factor test to obtain the optimal medium group distribution ratio?glucose 5g/L,tryptone 10g/L,yeast extract 5g/L,potassium dihydrogen phosphate 5g/L?,inhibition rate than the basic medium 11.32% higher.On the basis of this,the single-factor test was optimized for the culture conditions,and the four-factor and three-level response surface optimization was carried out with fermentation temperature,initial pH,rotation speed and liquid loading?fermentation temperature 30.00?,initial p H7.00,liquid loading 150.00 m L?.The rotation speed is 143.80r/min and the fermentation time is 40.0h).After optimization,the inhibition rate is increased by 19.34%.3.Study on the stability of antibacterial active substance of fermentation filtrate:It has good thermal stability,and the temperature is unchanged at-78¹00? for 45min.The inhibition rate is not obvious.After autoclaving at 121?,the antibacterial active substance Inactivated;sensitive to acid environment,insensitive to alkaline environment,p H 6¹0,no significant change in inhibition rate,pH3⁵,inhibition rate decreased,inhibition rate of p H3 was 64.22% in control group;continuous irradiationbyUV light for 210 min The inhibition rate did not change significantly;after pepsin treatment,the inhibition rate was only 15.58% of the control group,but it was stable to papain,alkaline protease,trypsin and neutral protease.4.“Zhuzihong” cherry tomato was used as the test fruit and vegetable.The optimized endophytic MR-1 fermentation filtrate was used as the treatment group,the chemical preservative was used as the positive control group,and the sterile water was treated as the control group CK.The effect of post-harvest preservation.The experiment showed that after 18 days of storage,the treatment group significantly reduced the rot rate and weight loss rate of cherry tomatoes,which was reduced by 42.07% and 58.68%,respectively,compared with the control group CK.During the whole storage period,the treatment group reduced the loss of vitamin C,soluble solids and titratable acid of cherry tomatoes,and better maintained the nutritional value and sensory quality of the fruit.Effectively reduces the accumulation of malondialdehyde?MDA?and maintains the integrity of the cell membrane.Significantly increased the activity of antioxidant enzymes POD,SOD,CAT.5.The column was packed with large pore size adsorption resin D101,and the column was washed with 70% ethanol aqueous solution as the eluent and the flow rate of 1m L/min to obtain 7 active tubes with antibacterial action.The liquid chromatography was further separated.Identify the active components.Six compounds wereobtained with mass fractions of 104?139?230?163?218 and 262,respectively.The molecular formulas were C₅H₁₃NO?C₅H₆N₄O?C₅H₁₁N₉O₂?C₅H₁₅N₃O₂?C₈H₂₃N₇?C₁₁H₂₈N₄O₂.