Revealing The Causes Of Flatulence Death In Sows Based On 16S Sequencing And Study On Antibacterial Properties Of Preventive Probiotics

Gut microbes are a complex and diverse population,and their richness and diversity are closely related to host metabolism and health.Clostridium perfringens is a kind of conditional pathogen existing in the intestines of healthy people and animals.When the host’s own resistance declines or the external environment conditions are harsh,it will cause a large number of proliferation of Clostridium perfringens,thus secreting a large number of toxins,causing serious harm or even death to the host.The problem of flatulence of sows caused by Clostridium perfringens occurs from time to time.Because of its rapid onset and high mortality,it has caused huge losses to the pig industry.Since the implementation of the ban on antibiotics in China,the problem of sow flatulence caused by Clostridium perfringens has been increasing.Therefore,it is urgent to solve this problem.In this study,the intestinal contents and gastric contents of sows died of flatulence in a pig farm in Guangxi were used as samples to analyze the microbial diversity.At the same time,the culture omics method was used to find out the cause of death of sows.At the same time,combined with the requirements of the prohibition of resistance,the antibacterial properties of the probiotics preserved in our laboratory on Clostridium perfringens were studied,and the antibacterial substances were isolated and identified to provide a strain basis for the development of alternative resistance products.The specific research results are as follows:1.Analysis of gut microbiota in sows with flatulence deathThe analysis of intestinal microbial diversity of deflated sows showed that the intestinal microbial diversity was significantly different from that of the control group,and the intestinal microbial diversity was seriously lost.Through species composition analysis and species difference analysis,it was found that the abundance of unclassified Clostridium sensu stricto 1 in the intestine was 79.04 times higher than that in the ck group,and the abundance of Clostridium perfringens was increased by 106.14 times.A new type of clostridium was screened in the intestinal tract of deflated dead sows by the method of culturomics,which was named Clostridium sp.AYMIII8.Physiological and biochemical experiments showed that its glucose fermentation did not produce acid and gas,and gelatin liquefaction was positive,indicating that it belonged to clostridium group I,which may be unclassified Clostridium sensu stricto 1.At the same time,60 strains of Clostridium perfringens were screened and all belonged to type A.The above results indicate that the cause of sow flatulence death may be the excessive proliferation of Clostridium perfringens type A.2.Study on the antibacterial properties of Bacillus licheniformis B38 against Clostridium perfringensA strain of Bacillus licheniformis B38 with excellent antibacterial properties against Clostridium perfringens was screened from the probiotics preserved in the laboratory.The maximum inhibition zone diameter of Clostridium perfringens was31.14±0.90 mm,and it had good antibacterial effect on various Gram-positive bacteria.Hydrogen peroxide exclusion and protease digestion experiments showed that the antibacterial substances in the supernatant of B38 fermentation were not hydrogen peroxide and protein substances.The tolerance test showed that the antibacterial substances in the fermentation supernatant of B38 had excellent heat resistance,acid and alkali resistance,bile salt resistance,chemical reagent resistance and digestive juice resistance.3.The crude extraction and analysis of antibacterial substances in B38fermentation productsDifferent methods were used to extract B38 antibacterial crude products.It was found that freeze-drying method was the best method to extract antibacterial substances.The MIC of antibacterial crude products extracted by this method was 1.25 mg/m L.Through LC-MC analysis of B38 fermentation products,17 lipopeptide compounds were finally identified,including two new compounds with molecular formulas of C50H85N13O16 and C72H123N21O23.In summary,this study analyzed,isolated and identified the intestinal microorganisms of sows with flatulence death,and finally found that the cause of sow flatulence death may be caused by the excessive proliferation of type A Clostridium perfringens in the intestine.Based on this,48 strains of Bacillus preserved in our laboratory were used as probiotics to inhibit and screen Clostridium perfringens,and finally a strain of Bacillus licheniformis with excellent properties was obtained.Through LC-MC analysis,17 lipopeptide compounds were identified,and two new compounds C₅₀H₈₅N₁₃O₁₆ and C₇₂H₁₂₃N₂₁O₂₃ were identified.