Selection Of Lactic Acid Bacteria Producing Biofilm From Swine And Study On Their Biological Characteristics

At present,with the intensive and large-scale development of China's pig industry,the frequent occurrence of bacterial diseases has caused the abuse of antibiotics,leading to the increase of drug resistance of pathogenic bacteria in animal body and drug residues in animal products,which has affected the production and development of pig industry and human health,probiotics originally emerged as an alternative to antibiotics.Lactic acid bacteria,the main probiotics,can inhibit the growth of harmful bacteria,regulate the function of animal gastrointestinal tract and promote the growth of animal organisms.Therefore,it has gradually become a research hot spot.In addition,bacterial biofilm,as a protective growth mode of bacteria itself,can enhance the resistance to the external environment,it has been reported that feed additives prepared by biofilm probiotics have stronger heat resistance,acid resistance and resistance to freezing and drying.However,little research has been conducted on biofilms of probiotics at home and abroad.In this study,the lactobacilli in pig feces were separated by plate coating method.108suspected lactobacilli with positive gram and negative touch enzyme were preliminarily screened by morphological observation,physiological and biochemical identification,the antibacterial properties and biofilm formation ability were determined by the Oxford Cup method and the crystal violet staining method respectively.The results showed that five isolates had both antibacterial properties and biofilm forming ability.After the molecular biology identification of 16S rDNA,4 strains were Lactobacillus johnsonii(L-8,L-11,L-18,L-76),and1 strain was Lactobacillus amylovorus(L-102).In order to obtain probiotic lactic acid bacteria,their acid-producing ability,tolerance to pH,bile salt,trypsin and high temperature,cell adhesion,broad-spectrum bacteriostasis,drug sensitivity,growth activity and safety were tested respectively.The results showed that the pH values of supernatant of each strain were between 4.1 and 4.4 after 24 hours of culture;in pH2.0 environment,with the extension of time,the viable counts of L-8,L-11 and L-18 bacterial solution decreased,while the viable counts of L-76 and L-102 bacterial solution remained unchanged;after being treated with 0.4%bile salt,the viable counts of L-76 and L-102's bacteria fluid could reached 10~3CFU/ml and 10~4CFU/ml,respectively,while L-8,L-11 and L-18 could hardly survive;after testing,the adhesion rates of five lactic acid bacterias to porcine intestinal epithelial cells was between 77.04%and 79.76%,and the five lactic acid bacterias had inhibitory effect on pathogenic bacteria such as Escherichia coli,Salmonella,Staphylococcus aureus and Aeromonas vivax;The viable counts of L-8's bacteria fluid decreased to 10~4 CFU/ml at 60?,and the viable counts of L-11,L-18,L-76 and L-102's bacteria fluid could still reach 10~7 CFU/ml;in the drug sensitivity test,L-76 and L-102 were resistant to 8 drugs,and the drug resistance was stronger than L-8,L-11 and L-18;in the growth curve test,L-76 entered logarithmic phase after 5 hours of culture,and its growth rate was relatively fast.In conclusion,the above five strains of lactic acid bacteria have certain acid production,trypsin tolerance,cell adhesion,broad spectrum antibacterial activity and growth activity,and the biological characteristics of L-76 and L-102 were relatively good;by simulating the acute poisoning experiment in animals,the results showed that the growth status of mice was normal after intragastric and intraperitoneal injection of 10~8 CFU/ml L-76 and L-102 bacterial solution respectively,indicating that L-76 and L-102 were both safe.This experiment further studied the bacteriostatic activity of the supernatants of lactic acid bacteria L-76 and L-102 in biofilm and plankton to pathogenic bacteria.The results showed that the growth of pathogenic bacteria was inhibited after co-culture with the supernatant of lactic acid bacteria in biofilm and plankton;the supernatants of lactic acid bacteria in two states were treated with different pH(3,5,7,9),enzymes(catalase,pepsin,trypsin,protease K)and temperatures(40,50,60,70,80 C)to detect the inhibitory effect on pathogenic bacteria,the results showed that supernatants'inhibition zone diameter of biofilm and planktonic lactic acid bacteria L-76 and L-102 decreased with the increase of pH value,indicating that their inhibitory activity decreased,and when pH was 7.0 and 9.0,they had no inhibitory activity;after the action of catalase and different temperatures,there was no significant difference in the diameter of inhibition zone between the supernatants of biofilm and planktonic lactic acid bacteria L76and L-102,indicating that catalase and temperature had little effect on the antibacterial activity of lactobacillus'supernatants under the two conditions;after the action of protease,the inhibitory zone diameters of L-76 and L-102 supernatant on the indicator bacteria were significantly reduced in both conditions,and compared with plankton,the inhibitory zone diameter of biofilm lactobacillus supernatant was smaller,which indicated that protease had a greater influence on the antibacterial activity of biofilm lactic acid bacteria's supernatant.In summary,the antibacterial activity of the supernatants of biofilm and planktonic lactic acid bacteria showed ph dependency,temperature stability,catalase stability and protease sensitivity,and the antibacterial activity of biofilm lactic acid bacteria's supernatants was more sensitive to protease than that of planktonic lactic acid bacteria,the antibacterial components in the supernatant of biofilm and planktonic lactic acid bacteria may contain organic acids,proteins,polypeptides and other substances,and the activity or content of antibacterial substances in the supernatant of biofilm lactic acid bacteria is higher than that of planktonic lactic acid bacteria.The inhibition of lactic acid bacteria L-76 and L-102's supernatants on pathogenic bacteria under two conditions was studied by scanning electron microscopy,the results showed that the morphology of pathogenic bacteria changed or even died after co-culture,and compared with the plankton state,the supernatant of biofilm lactobacillus had a slightly greater effect on the morphology of the indicator bacteria,which proved that the supernatant of lactic acid bacteria contained antimicrobial substances in both conditions,and the activity of bacteriostatic substances in the supernatant of biofilm lactobacillus was slightly higher or the content was slightly higher.