Screening Of High-efficiency Antibacterial Strains Of Rhamnolipid And Optimization Of Fermentation Parameters

Antibiotics are commonly used bacterial disease prevention and control drugs in aquaculture,but with the emergence of drug resistance in recent years,the prevention and treatment effect of antibiotics is getting worse and worse,posing great risks to human health and food safety.Therefore,finding efficient and safe antibacterial drugs has become a new research goal and hotspot.Rhamnolipid is an amphiphilic biosurfactant with good emulsification,reduced interfacial tension,non-toxicity,easy degradation and antibacterial properties.It can be widely used in petroleum,food,agriculture and medical industries.Has a wide range of application prospects.The purpose of this study was to screen a highly efficient antibacterial rhamnolipid producing strain to provide theoretical and technical support for the development of rhamnolipids as an antibacterial agent for aquaculture.1.Improvement of screening technology of rhamnolipid strain and screening of high-yield rhamnolipid producing strainIn this paper,325 strains were isolated from the oil-soil soil by enrichment culture and gradient dilution plate method,among which 11 strains produced color reaction on CTAB plate;4 strains had good emulsifying property,and the strain A39-1 was emulsified.The index can reach 70.8±1.49 %;6 strains have good oil drainage,the oil discharge of strain A39-1 is 60.81±0.64 mm,and the concentration of rhamnolipid in fermentation broth is 8.46 g/L;And acid precipitation test to verify the presence of glycolipids and lipopeptide surfactants in the fermentation broth of 6 strains;strains A10,A11,A39-1,A39-2,A40-1 and confirmed by thin layer chromatography experiments combined with the above experiments A40-2 is capable of producing rhamnolipid.Finally,through morphological characteristics and 16 r RNA analysis,strain A10 was identified as Pseudomonas stutzeri,strains A11 and A40-1 were identified as Pseudomonas benzenivorans,strain A39-1 was identified as Pseudomonas mendocina,strain A39-2 was identified as Pseudomonas nitroreducens,and strain A40-2 was Pseudomonas fulva.In this study,the traditional CTAB plate method was combined with the punching method,and the technical route of the original screening bacteria was modified.The strain was first fermented and then the improved CTAB plate method was used to screen the strain of rhamnolipid,and the strain was expanded.A total of 6 strains of rhamnolipid producing strains were screened,which were the first to detect the production of rhamnolipid strains.It has not been reported at home and abroad.The highest yield of strain A39-1 Pseudomonas mendocina is 8.11 g/L.The improved screening technique improves the efficiency of screening rhamnolipid strains and provides a useful technique for rapid screening of rhamnolipid strains.Six strains of rhamnolipid producing strains were screened from the edible oil soil,which provided a theoretical basis for the recycling of food waste oil and as a carbon source for fermenting rhamnolipid.2.Preliminary study on antibacterial properties and mechanism of rhamnolipidThis project initially explored the antibacterial properties and mechanisms of six strains of rhamnolipid produced by four strains of common pathogens in aquatic products.The Oxford Cup method measured the fermentation product of strain A39-1,rhamnolipid,which had good antibacterial effect on 4 pathogens commonly found in aquatic products.The results showed that the minimum inhibitory concentration(MIC)of Vibrio parahaemolyticus,Vibrio harveyi,Streptococcus agalactiae and Pseudomonas fluorescence was 15.63 mg/L,7.8 mg/L,7.8 mg/L,and 7.8 mg/L,respectively.The effect of rhamnolipid on biofilm pre-formation was studied by crystal violet staining.The results showed that as the concentration of rhamnolipid gradually approached the MIC concentration,the formation rate of bacterial biofilm was lower.When the concentration of rhamnolipid reached 6mg/L,it began to exert strong inhibitory effect on the biofilm formation of Vibrio parahaemolyticus and Streptococcus agalactiae,and continued to increase the inhibition effect of rhamnolipid concentration on bacterial biofilm;When the rhamnolipid concentration reached 4mg/L,it began to have a strong inhibitory effect on the biofilm formation of Vibrio harveyi and P.aeruginosa,and continued to increase the concentration of rhamnolipid against Vibrio harveyi.The inhibitory effect of the biofilm is weakened.The effect of rhamnolipid on the formed biofilm was studied by crystal violet staining.The results showed that except for Vibrio harveyi,rhamnolipids against Vibrio parahaemolyticus,Streptococcus agalactiae and Pseudomonas fluorescence.The formed biofilm has a strong destructive effect and the biofilm damage effect to Vibrio parahaemolyticus is most significant.When the rhamnolipid concentration is 8 mg/L,the biofilm formation rate of Vibrio parahaemolyticus reaches a minimum of 10.3±2.11%.The rhamnolipid had the worst effect on the biofilm formation of Vibrio harveyi.When the rhamnolipid concentration was 6 mg/L,the biofilm formation rate was as high as 75.5±1.14%.The experimental results show that rhamnolipid has good antibacterial properties and it has great significance to develop antibacterial preparations for aquaculture.The strain A39-1 is a first-time discovery of rhamnolipid,which has high antibacterial properties.It has not been reported at home and abroad,and the biosafety level is: Grade 4.3.Optimization of Fermentation Parameters of Pseudomonas mendocina Strain A39-1In this study,single factor method and response surface method were used to optimize the fermentation medium composition and fermentation conditions of strain A39-1.Under the condition of shake flask fermentation,the optimal fermentation medium components and culture conditions of strain A39-1 were screened by single factor method,and the seven main influencing factors affecting the fermentation of rat rhamnolipid were determined.According to the PB design test results,the three most significant factors affecting the production of rhamnolipid were selected,and the steepest climbing path method was used to determine the response center point and the optimal concentration range of the three factors.Finally,the optimal media formulation was obtained by Box-Behnken design and response surface analysis.The optimized results showed that the optimal medium and optimum fermentation conditions for strain A39-1 Pseudomonas mendocina: sunflower oil 50 g/L,sodium nitrate 12.15 g/L,potassium dihydrogen phosphate + disodium hydrogen phosphate 6 g/L,magnesium sulfate 0.48 g/L,calcium chloride 0.05 g/L,trace element 2 mL/L,p H 6.8,temperature37 °C and rotation speed 186.07 r/min.The yield of rhamnolipid was 8.11 g/ before optimization.L,the optimized rhamnolipid production was 9 times higher than before optimization.In this study,six strains of rhamnolipid producing strains were screened from the oily soil of the catering industry.Among them,the strain A39-1Pseudomonas mendocina was the first to be found to produce rhamnolipid,which has good antibacterial properties.It is reported that the improved CTAB plate method contributes to the screening of strains and expands the screening range of strains.The antibacterial mechanism of rhamnolipids on four pathogens was preliminarily investigated and optimized by medium components and culture conditions.The yield of rhamnolipid produced by strain A39-1 was increased by 9 times,and the measured rhamnolipid concentration was 72.84 g/L.