Studies On Swine-derived Lactobacillus Plantarum And Its Application As Probiotics

In the process of large-scale feeding,animals are prone to suffer from intestinal diseases,which are often manifested as intestinal microbial flora disorder and the proliferation of harmful bacteria,leading to animal disease and decreased production performance.The current solution is usually to use antibiotics for prevention.However,the long-term use of antibiotics leads to veterinary drug residues and bacterial resistance,which poses a great threat to human public health and safety.In addition,antibiotics are not completely absorbed and metabolized in animals.Instead,they are partly excreted into feces,which causes soil and water pollution over time.Therefore,it is urgent to develop antibiotic replacement strategies to reduce a series of problems caused by antibiotics.This study aimed to isolate and identify Lactobacillus plantarum from the intestinal tract of weaned piglets and explore its biological characteristics.And then to study the effects of Lactobacillus plantarum on intestinal inflammation and and if there is a correlation between gut microbiota and immune response with Dextran Sodium sulfate?Dextran Sodium Sulphate,DSS?induced colitis model in mice.Lastly,the probiotic effect was observed in weaned piglets,and the mechanism of improving the growth performance of piglets was revealed through changes in immune,antioxidant and faecal VFA indexes.The main research contents and results of this project are as follows:In the first part,a suspected lactobacillus with strong acid-producing capacity,which identified as Lactobacillus plantarum LPZ through physiological and biochemical analysis and 16S r DNA sequence analysis was isolated from the intestinal tract of35day-old healthy piglets.The biological characteristics of the strain were analyzed and the results are as follows:1. Results of growth curve of Lactobacillus plantarum LPZ strain showed bacteria was in the logarithmic stage after 2-9h culture,and the number of live bacteria increased rapidly.Between 9 and 12h,the growth of Lactobacillus plantarum LPZ entered the stable stage,and after 12h,that of Lactobacillus plantarum LPZ strain entered the decline stage.Results of p H curve showed the p H value of 0?10h decreased rapidly.The p H decreased from 6.8 to 4.4 at the beginning to 3.8 at 30h slowly,and it finally stabilized at around 3.7.2.The survival rate of Lactobacillus plantarum LPZ strain at p H 3.0 was 25.1%with p H 3.0 after 2 hours culturing,and it was 79.7%at 0.3%bile salt concentration.The survival rates at 50°C and 80°C after 20 min culturing were 86.8%and 1.2%,respectively.3.Lactobacillus plantarum LPZ strain has a broad spectrum of antibacterial activities,and has the strongest antibacterial effect against salmonella and E.coli O139.The strain is sensitive to chloramphenicol,erythromycin,penicillin,and is not sensitive to compound sulfamethoxazole,norfloxacin,amikacin,ciprofloxacin,and weak to gentamicin,cefazolin and ampicillin.In the second part,the effects of Lactobacillus plantarum on intestinal inflammation and and if there is a correlation between gut microbiota and immune response with Dextran Sodium sulfate DSS induced colitis model in mice.Twenty SPF grade female ICR mice?8-10 weeks old and with a body weight of 20±2.1g?were randomly divided into two groups,which named DSS group and LPZ group.Mice in the two groups were fed with the basic diet and the experimental diet for 1 week,respectively.The experimental diet was supplemented with 2 x 10¹⁰ CFU/kg Lactobacillus plantarum LPZ in the basal diet.On day 8,all mice were given 5%DSS to induce acute colitis by drinking water.Body weight of the mice was weighed on the morning of day 15,and the average daily gain was calculated.The mice were sacrificed after abdominal anesthesia and the length and weight of the colon was measured.Changes in body weight and disease activity index?DAI?were measured daily during the trial to determine the severity of colitis.Procedures of extracting and fixing,paraffin-embedding,and staining were done to evaluate epithelial loss score.TNF-?,IL-1?,Il-6,Ll-10,IL-17A levels in colon tissue were measured by multi-layer enzyme-linked immunosorbent assay.The fecal sample DNA was extracted for sequence amplification and sequencing.The research results are as follows:1. The body weight of LPZ group was significantly higher than that of DSS group?P<0.05?,and the length of colon was significantly longer than that of DSS group?P<0.05?.While the DAI and histological score of LPZ group was significantly lower than that of DSS group?P<0.05?,2. The levels of IL-1?,IL-6,IL-17 and TNF-?in the Lactobacillus plantarum LPZ group were lower,while IL-10 was higher than those in DSS group?P<0.05?.3. Lactobacillus plantarum LPZ significantly increased the Phytophthora and Bacillus strains at phyla level,and significantly reduced the abundance of Lactobacillus at genus level.Significantly improve kind level of Staphylococcus xylosus,Bifidobacterium animalis,Turicibacter,intestinal Lactobacillus intestinalis and rat Lactobacillus murinus,Gemmobacter?intermedius and eats Lactobacillus Prophage abundance of Lj928.In the third part,Ninety-six healthy and similar weights?26±2d?piglets were selected and randomly divided into 3 treatments according to the principle of body weight and male and female.Each treatment consisted of 4 replicates?pens?,with 8piglets in each replicate.The experiment started at the age of 26 days and ended at the age of 54 days with a duration of 28 days.Piglets in control group were fed basic diets,those in the antibiotic group were fed basic diets with compound antibiotics?terramycin calcium 200 mg/kg effective ingredients,active ingredients kitasamycin 50 mg/kg?,and those in LPZ groups were fed basic diets with 800 mg/kg 1 x 10¹⁰ cfu/g Lactobacillus plant LPZ.Blood sample was collected from piglets that was close to average weight by vena on the morning of the 15th and 29th days after the start of the experiment to measure immune and antioxidant indexes.At 3:00 PM on the 1st and 28th day of the experiment,2piglets were randomly selected from each replicate to collect the rectal contents,which stored in the refrigerator at-20?with 50 m L spiral-tube under sterile anaerobic condition.The contents of acetic acid,propionic acid and butyric acid in feces was examined using gas chromatography.The main research results are as follows:1.Compared with control group,Lactobacillus plantatus LPZ can significantly increase ADG and FCR?P<0.05?,while ADG,ADFI,and FCR were not different between LPZ group and antibiotics group?P>0.05?.2.Compared with the control group,diarrhea index and diarrhea rate of piglets in LPZ and antibiotic groups decreased significantly?P<0.05?.However,these two parameters in LPZ group were significantly higher than those in antibiotic group?P<0.05?.3. Serum Ig A and Ig G levels of piglets in LPZ group on the 14th and 28th days were significantly higher than those in control group and antibiotic group?P<0.05?.Compared with control group,T-AOC and SOD in the blood of piglets in LPZ group on day 14 and28 were significantly increased?P<0.05?,and MDA in the blood were significantly decreased?P<0.05?.Compared with piglets in antibiotic group,the MDA level of piglets in LPZ group decreased significantly on the 14th day?P<0.05?,while the difference in other indexes were not significant?P>0.05?.4. Compared with control group,Lactobacillus plantarum LPZ significantly increased butyric acid level in feces of piglets on the 14th and 28th days?P<0.05?.Compared with antibiotic group,acetic acid level in feces of piglets on day 14 and day 28were significantly increased?P<0.05?,and the contents of acetic acid,propionic acid and butyric acid on day 28 were significantly increased?P<0.05?.In conclusion,we can conclude that:?1?Lactobacillus plantarum LPZ strain has the ability of rapid growth and lactic acid production.It could crosse the stomach and survive in the intestinal tract,and withstands50?ambient temperature.Lactobacillus plantarum LPZ strain has broad-spectrum antibacterial activity,and has the strongest antibacterial effect against salmonella and E coli O139.?2?Lactobacillus plantarum LPZ could prevent intestinal injury caused by inflammation in mice,reduce symptoms of DSS-induced colitis,and significantly relieve inflammation.Meanwhile,it could improve the diversity of microbial species in colon,maintain homeostasis of intestinal microbial flora,and improve intestinal health of animals.?3?Lactobacillus plantarum LPZ strain could significantly improve immunity and antioxidant capacity of piglets,promote changes in intestinal fatty acid composition,facilitate growth of beneficial microbes in intestine and growth performance of weaned piglets,reduce diarrhea rate.It has the potential to replace feeding antibiotics and has a good clinical application prospect and popularization value.