Study On The Inhibitory Effect And Mechanism Of Spices On Putrefying Bacteria Isolated From Dry Salted Duck

Dry salted duck also known as the tribute duck,which is a unique cured meat product in China.It is loved by consumers both at home and abroad for its thin skin、.tender meat and flavor characteristic.However,the traditional workshop-style production methods of dry salted duck also cause problems such as difficulty in achieving hygiene management and unstable product quality,which made ducks vulnerable to microorganism and spoilage,severely shorten the shelf-life of ducks.Traditional chemical preservatives have been questioned for their potential harm to the human body.Finding safer food preservatives from natural sources has become a new research hotspot.As a flavoring plant commonly used in everyday life,the spices has also attracted the attention of many researchers.Therefore,we used the spoiled duck to isolate the main spoilage bacteria,and the spoilage organisms in ducks were identified through physiological and biochemical experiments.And molecular biology methods also used to determine the main spoilage organisms in dry salted duck.We used the isolated spoilage bacteria from the dry salted duck to study the optimum extraction method and antibacterial stability of the spice extracts.Finally,the antibacterial mechanism of the spices extracts against spoilage organisms in dry-salted duck was studied by scanning electron microscopy and SDS-PAGE etc.It can provide theoretical basis of spice extracts used to prevent the spoilage of dry salted duck and other foods.The main findings are as follows:(1)Five strains of spoilage bacteria were isolated from the spoilage ducks.Physiological and biochemical tests were performed on 5 strains of spoilage bacteria.,the experiment results combined with 16 S rDNA sequencing results were used to identify strains.The results showed that the five strains of spoilage bacteria were Enterococcus faecalis 1,Staphylococcus simulans 2,Staphylococcus sciuri 6,Staphylococcus gallinarum 7,Bacillus cereus y2,and Staphylococcus predominate in spoilage organisms.(2)Antibacterial activities of 16 kinds of spices were screened.The results showed that clove and licorice had the best antibacterial effect on putrefying bacteria isolated from dry salted duck.The extraction time,ethanol concentration and solid liquid ratio were selected to optimize the extracting methods of antibacterial substances from cloves and licorice.The experiment results showed that the optimum extraction conditions of clove was: ethanol concentration 70%,extraction time 2 h,extraction material ratio(mg/mL)3:20,and the optimum extraction conditions of licorice was: ethanol concentration 70%,extraction time 6 h,extraction material ratio(mg/mL)3:20.The effects of various production conditions on the antibacterial activity of spices were simulated by high temperature treatment,UV irradiation,and the pH of the medium to study the antibacterial stability of the spices under different conditions.The experiment results showed that the high temperature and UV treatment almost do not affect the antibacterial activity of the spices.The sterilization methods commonly used in production and processing do not affect the antibacterial properties of the spices.The spices does not need to be protected from light when it is applied to prevent spoilage of ducks or other foods.The best medium pH of extracts of clove and licorice on different spoilage organisms are different.(3)The minimum inhibitory concentration(MIC)of the extracts of clove and licorice against 5 spoilage bacteria were determined by the inhibition zone method,and the experiment results showed that clove and licorice could inhibit the spoilage bacteria in ducks at the lowest concentration of 6.25 mg/mL and 12.5 mg/mL..The growth curve experiment results showed that the treatment of spice extracts has a significant inhibitory effect on the proliferation of bacteria,and the peak of bacterial proliferation is significantly reduced.The coomassie brilliant blue staining method was used to determine the protein concentration of the bacterial culture solution.We found that the addition of clove and licorice extracts increased the concentration of extracellular proteins,and the treatment of spices changed the permeability of the cell membrane and caused the leakage of intracellular substances.We also observed the changes of the whole protein band of bacteria,SDS-PAGE results showed that the treatment of spices reduced the expression of bacterial proteins and interferedwith the normal physiological activities of the bacteria.The extrinsic feature of spoilage bacteria was observed by SEM.The results showed that after treatment of extracts of spices,the morphology of the bacteria changed,and the cell surface was wrinkled and the integrity of the cell membrane was destroyed.The spices extracts can cause the effect of bacteriostasis.by affecting the structure and function of bacterial cell membranes and cell walls.