| With the intensification of fish,factory farming has increased significantly,which easily leads to a decrease in the abundance and diversity of fish intestinal flora,and frequent occurrence of intestine inflammation.Lactobacillus has the function of regulating the balance of animal intestinal flora,promoting animal growth and improving immunity.Lactobacillus plantarum LP-S25 is a strain of probiotic bacteria screened in the early stage of the laboratory,to better play to its probiotic effect,so that it can not only have a beneficial function,improve the structure of intestinal flora,but also partially kill the pathogenic bacteria in the intestinal of fish,improves the growth rate of fish while improving the immunity,and reduce the occurrence of intestine inflammation.In this study,LP-S25 was compound mutagenized and then evaluated the effect of the mutagenized strain in the culture of largemouth bass.This study is mainly divided into the following three parts:1.Compound mutagenesis of L.plantarum LP-S25.To obtain a high inhibition inhibition activity strain,the LP-S25 was subjected to compound mutagenesis,including 8 times of ultraviolet mutagenesis,3 times of nitrosoguanidine mutagenesis and 3 times of genome shuffling,and the three strains obtained after mutagenesis were named LP-UV8,LP-NT3 and LP-SM25,respectively.Compared with the starting strain LP-S25,the average inhibition diameters of these three strains against five pathogenic bacteria,namely Streptococcus agalactiae、Aeromonas hydrophila、Aeromonas veronii、Vibrio parahaemolyticus and Vibrio alginolyticus,were increased by 30.16%,56.18%and 75.77%,respectively.The strains LP-UV8,LP-NT3 and LP-SM25 still had high inhibition activity after 5 consecutive generations of transmission,indicating that these three strains have good genetic stability.The three strains were tested by infusion on nile tilapia and largemouth bass,and no mortality was observed in each group within 14 days,indicates a high level of security.2.Comparison of the inhibition performance of the starting strain LP-S25 and the final mutagenic strain LP-SM25.To study the inhibition performance of strains LP-S25 and LP-SM25,the supernatant was treated in different ways,and the inhibition effect was measured using Aeromonas veronii as the indicator bacteria,and the results showed that the fermentation broth and supernatant of had inhibition effect on Aeromonas veronii,and the bacteria itself had no inhibition.The inhibition substances produced by strains LP-S25 and LP-SM25 include proteins,organic acids and hydrogen peroxide.These antibacterial substances have good thermal stability,and loses antibacterial ability under neutral and alkaline conditions.3.Largemouth bass culture trials to evaluate the application of each strain.In order to evaluate the a pplication effect of the starting strain LP-S25 and the mutant strains LP-UV8,LP-NT3 and LP-SM25 in largemouth bass culture,6-week largemouth bass culture experiment was carried out.The experiment was divided into 5 groups:group C0:blank control;groups T1～T4:strain LP-S25,LP-UV8,LP-NT3 and LP-SM25 fermentation solution were added to the basal feed,and the final concentration of the strain in the feed was 1.0×10~7 CFU/g.The results showed that there was no significant difference in weight gain rate（WGR）and specific growth rate（SGR）between the groups at 3 weeks of breeding（P>0.05）.At 6 weeks,the WGR of the T4 group was significantly higher than that of the T1 and C0 groups（P<0.05）,and the WGR in the T2 and T3 groups was significantly different from the C0 group（P<0.05）,but not significantly different from that of the T1 group（P>0.05）.FCR in T2～T4 groups was significantly decreased compared with C0 and T1 groups（P<0.05）.It was shown that the mutant strain improved the inhibition ability and also promoted the growth of largemouth bass,and the final mutant strain LP-SM25 had the best growth promotion effect.Alkaline phosphatase（AKP）,acid phosphatase（ACP）,superoxide dismutase（SOD）,lysozyme（LZM）,amylase（AMS）and lipase（LPS）increased to different degrees in all test groups,with the highest values in T4 group at LZM,SOD,AMS and LPS,this indicates that the final mutagenic strain LP-SM25 can improve the activities of immune enzymes and digestive enzymes in largemouth bass.The thickness of intestinal muscle layer and the height of intestinal villi were increased to different degrees in each test group,with the highest thickness of the intestinal muscle layer in the T4 group and no significant difference in the height of the intestinal villi in the T2,T3 and T4 groups（P>0.05）.4.Effect of dietary supplementation LP-S25 and LP-SM25 on differential gene expression in the liver of largemouth bass.Since the liver is both an important digestive and immune organ,a RNA-Seq analysis of largemouth bass liver was performed to compare the mechanisms of differential effects of LP-S25 and LP-SM25 on growth and immune performance,the results showed that there were 35 differentially expressed genes in the C0 group compared with the T1group;48 differentially expressed genes in the C0 group compared with the T4 group;22differentially expressed genes in the T1 group compared with the T4 group.These differentially expressed genes were mainly enriched in Steroid biosynthesis and PPAR signaling pathway.3immune-related genes were selected from the differentially expressed genes,namely interleukin1β（IL-1β）,tumor necrosis factorα（TNF-α）and transforming growth factorβ（TGF-β）,and analyzed in different tissues of kidney,spleen and intestine using q RT-PCR.The results showed that the expression of TNF-α,IL-1βand TGF-βwas upregulated in the spleen,kidney and intestine of both T1 and T4 groups,with a higher degree of upregulation in the T4 group than in the T1 group.In summary,L.plantarum LP-UV8,LP-NT3 and LP-SM25,which were screened by mutagenesis,have good bacterial inhibition ability,safety and genetic stability.The fermentation broth and supernatant of strains LP-S25 and LP-SM25 have antibacterial effect,and the antibacterial substances produced include organic acid,hydrogen peroxide and protein.The antibacterial substances have good thermal stability and are sensitive to p H.The final mutagenic strain LP-SM25 improves growth performance,enhances immunity and improves intestinal tissue structure in largemouth bass.This is of great help in improving the immunity and health of animals. |
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