Screening Of Marine Microorganisms That Inhibit Foodborne Pathogens And Study Of Corresponding Active Substances

Staphylococcus aureus,Listeria monocytogenes,Salmonella and Pseudomonas aeruginosa are common foodborne pathogens in food,and they usually cause vomiting,diarrhea,and abdominal pain,which can lead to death in serious cases,posing a great threat to human health and life safety.In addition,along with the widespread use of antibiotics,the emergence of drug-resistant strains of bacteria has brought great trouble to the prevention and control of foodborne pathogens.In this study,we proposed to screen microorganisms with significant inhibitory effects on foodborne pathogens from marine microorganisms and conducted in-depth studies on them,mainly as follows:In this experiment,a strain of Bacillus sp.with strong inhibitory effect on food-borne pathogens was isolated and screened from deep-sea sediments.The strain was named as Bacillus sp.YJ17 according to the morphology and 16 S r RNA sequence analysis.Subsequently,fermentation broth of Bacillus sp.YJ17 was acidified,dried,extracted with methanol and purified by reversed-phase high performance liquid chromatography(RPHPLC),and two components with significant antibacterial activities were obtained.The two components were analyzed by high energy collision-induced dissociation mass spectrometry(HCD-MS)and tandem mass spectrometry(HCD-MS/MS).The results showed that the two active agents belonged to family of fengycin and surfactin,and the amino acid sequence of purified fengycin and surfactin might be Glu-Orn-Tyr-Thr-GluVal-Pro-Gln-Tyr-Ile and Glu-Leu/Ile-Leu/Ile-Leu/Ile-Val-Asp-Leu/Ile,respectively.In addition,compared to the commercial food preservative nisin,the purified fengycin and surfactin produced by Bacillus sp.YJ17 showed unique advantages in inhibiting the growth of pathogenic bacteria with a broader inhibition spectrum.In order to investigate the mechanism of fengycin and surfactin produced by Bacillus sp.YJ17 to inhibit foodborne pathogens,this study was first conducted to observe their effects on P.aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus(MRSA)cells,respectively,by electron microscopy.The morphological and ultrastructural effects of P.aeruginosa PAO1 and MRSA cells were observed by electron microscopy.The results showed that after treatment with purified fengycin,P.aeruginosa PAO1 cells were abnormally aggregated morphologically,and the cell surface was uneven or even ruptured.After treatment with purified surfactin,MRSA cells clustered together,and the cell surface became rough and jagged.The fluorescence microscopy observation further showed that both purified fengycin and surfactin could cause the accumulation of reactive oxygen species(ROS)and cell membrane damage in P.aeruginosa PAO1 and MRSA cells.In addition,ROS scavenging-related enzymes such as catalase(CAT)and superoxide dismutase(SOD)activities were significantly reduced in P.aeruginosa PAO1 and MRSA cells after treatment with fengycin and surfactin,respectively.The above results indicated that the inhibition mechanisms of fengycin and surfactin was closely related with accumulation of ROS,which might be due to the decreased activity of CAT and SOD after treated with fengycin and surfactin,respectively.Since the yield of lipopeptides is usually low,the fermentation medium of Bacillus sp.YJ17 was optimized in this study to improve the yield of fengycin and surfactin.By adding different types of carbon and nitrogen sources and amino acids to the LB liquid medium,their effects on fengycin and surfactin production were determined separately.The results showed that the addition of carbon source glucose significantly increased the yield of both fengycin and surfactin.In addition,the addition of nitrogen sources such as peptone,yeast paste and sodium glutamate and amino acids such as glutamic acid and aspartic acid significantly increased the yield of fengycin and surfactin.Especially,peptone and yeast extract increased the yield of fengycin by about 14-16 times.In conclusion,two antibacterial active substances,fengycin and surfactin,were extracted from Bacillus sp.YJ17 in this study.Their purified products showed strong antibacterial activities against a variety of food-borne pathogens,indicating that they have good application prospects in the field of food preservation.