Recombinant Expression Of Salmonella Phage SLMP1 Lysin And Application Of Phage In Aquatic Product

Salmonella is a gram-negative bacillus which has similar biochemical reactions and antigen structures and parasitize in the intestine of humans and animals.It is mainly transmitted to humans through poultry,meat,milk,eggs,aquatic products,and etc.Salmonella infection can cause the symptoms of vomiting,diarrhea,fever,etc.,even coma or death in some severe cases.Due to the nutrition and high moisture content of aquatic products,the bacteria will grow and cause the corruption of aquatic product and shorten the shelf life if aquatic products are stored under improper conditions.If the aquatic products are contaminated with foodborne pathogens,they will pose a risk to human health and cause huge economic losses to the country.The traditional control technology of foodborne pathogens is mainly based on chemical sterilization.However,the safety of chemical preparations has gradually been concerned by people.Therefore,there has important practical significance to develop safe and effective bio-bacteriostatic agents.Therefore,it is necessary to develop more safe and effective alternative biocontrol methods for foodborne pathogens.One of the promising approaches for food safety is the use of lytic bacteriophage（phage）to reduce foodborne pathogens.This biocontrol strategy using phages is promising because it has many advantages such as safe,high efficiency and specificity,no effects on food properties,etc.In the present study,the recombinant lysin derived from Salmonella bacteriophage SLMP1 was expressed,and its bactericidal properties were analyzed.The biocontrol of Salmonella in raw salmon fillets and scallop adductors using bacteriophage SLMP1 was studied.In addtion,the Salmonella control and freshness preservation of raw salmon using SLMP1 combined with bacteriostatic agents was analyzed.The research contents and main results were as follows:1.The genomic DNA was extracted from the phage and the full-length fragment of lysSP1 gene was amplified by PCR.The amplified fragment was inserted into the prokaryotic expression vector pET-28a（+）and transformed into Escherichia.coli DH5a.The recombinant plasmid was extracted and confirmed,which was named as pET28a-lysSP1.The recombinant plasmid was transformed into E.coli BL21 and successfully expressed.The lysin was partially soluble expressed and the expression level was highest when the expression bateria were induced at 37℃for 4h by the addition of IPTG to a final concentration of 1 mM.The recombinant lysin LysSP1 was obtained by the steps ofultrasonication,purification through Ni-NTA protein purification column and desalting.The protein concentration of lysin was tested using the Coomassie brilliant blue kit and was diluted to 0.1 mg/mL for further bactericidal properties analysis.2.The optimal conditions of recombinant lysin LysSP1 were analyzed.The results showed that bactericidal effect was best under the condition of temperature 45°C,pH 9.0 with additional EDTA of 5 mmoL/L.When the ion concentration was 0.1 mmoL/L,Fe³⁺and Mg²⁺could inhibit the enzyme activity.When the ion concentration was 1 mmoL/L,Zn²⁺,Mn²⁺,Fe³⁺,Mg²⁺,and Ca²⁺all had inhibitory effects on lytic enzymes,among which Fe³⁺had the greatest effect on enzyme activity,followed by Mn²⁺.This lysin had different bactericidal effects on S.typhimurium,S.enteritidis,S.paratyphi A,S.paratyphi A,E.coli,E.coli O157,Shigella boydii,and S.dysenteriae,Listeria monocytogenes,Staphylococcus aureus.3.The effectiveness of phage SLMP1 to reduce Salmonella on contaminated raw salmon fillets and scallop adductors were evaluated.With the increase of phage dose,the Salmonella counts in salmon fillets and scallop adductors decreased.The best bactericidal effect was observed at the phage dose of 10⁸ PFU/g,and Salmonella can be reduced from the initial concentration of 10² CFU/g to below the detection limit of1 CFU/g after storage at different temperatures at this phage dose.When the initial concentration is 10⁴ CFU/g,the number of Salmonella is reduced by 1.5³.0 log₁₀ CFU/g.In the process of experiment,phage SLMP1 was stable in salmon fillet and scallop adductors.4.To control Salmonella and spoilage of raw salmon,nisin and sodium diacetate were selected to combine with Salmonella phage by the experiments of minimal inhibitory concentration test and stability of phage.The raw salmon fillets（artificially contaminated with Salmonella10⁴ CFU/g）treated with phage（10⁸ PFU/g）,nisin（0.1mg/g）,sodium diacetate（2.0 mg/g）and their combination at 4±1℃were evaluated.The results showed bacteriostatic agents containing phage could significantly（p<0.05）reduce Salmonella counts in the samples.The bacteriostatic agents containing nisin could significantly inhibite the growth of aerobic colony counts.And the bacteriostatic agents containing sodium diacetate could significantly inhibite the increase of TVB-N.After 14 day’s storage,the combination treatment of phage,nisin and sodium diacetate could reduce Salmonella counts below the detection limit 1 CFU/g,as well as,the aerobic colony counts and TVB-N were reduced by 2.5 log₁₀0 CFU/g and 13.73 mg/100 g compared with control group.The results indicated the combination of phage,nisin and sodium diacetate had great potential to be as a good effect in control of Salmonella and spoilage of raw salmon.The Salmonella phage SLMP1 and prepared recombinant lysin in this study have a significant bactericidal effects on Salmonella,which are promissing green and natural bactericidal agents.This study establishes a foundation for further promotion of application of phage SLMP1 and provides a new technology for safety control and fresh preservation of aquatic product.