| In recent years,with the rapid development of aquaculture,aquatic animal diseases have become frequent,and the addition of probiotics to feed to prevent diseases and improve the immunity of the organism has become a new research hotspot.Lactobacillus plantarum is a relatively common probiotic,which has probiotic effects such as synthesizing vitamins and amino acids,promoting intestinal digestion,enhancing host immunity,maintaining the microecological balance of intestinal flora and resisting pathogenic bacteria from invading the intestinal tract.Lactobacillus plantarum has been approved for use as a feed additive by the Ministry of Agriculture and Rural Development in China.However,the digestive systems of aquatic animals and livestock animals are vastly different,and the colonisation pattern of Lactobacillus plantarum may differ between them.In order to use Lactobacillus plantarum more scientifically and precisely in aquaculture production,this study therefore investigated the colonisation pattern of Lactobacillus plantarum in AA broiler and largemouth black bass by constructing Lactobacillus plantarum LP-37 with GFP tags,and analysed the The differences in the colonization of Lactobacillus plantarum in aquatic animals and livestock animals were analyzed.This study can provide basic data for promoting the application of Lactobacillus plantarum LP-37 in aquaculture and expanding the application of Lactobacillus plantarum LP-37,and can provide a scientific basis for exploring a green and sustainable development pathway for the aquaculture industry in China.The main elements of the study are as follows.（1）Acquisition of recombinant strains.In this study,GFP green fluorescent protein was selected as the target gene and the Pldh L promoter was used as the sustained expression promoter to construct a recombinant plasmid by ligating it with the vector p SIP403.The recombinant plasmid was identified correctly,concentrated and electrotransferred into Lactobacillus plantarum LP-37 to construct a recombinant strain.The luminescence of the recombinant strain was observed under fluorescence microscope,and it was found that the recombinant strain was able to excite strong green fluorescence,which indicated that GFP was well expressed in the recombinant strain.The luminescence of the recombinant strains was observed under fluorescence microscopy after 8 passages without antibiotics,and the intensity and luminescence of the recombinant strains did not decrease significantly.The results indicated that the recombinant strain had good and stable heritability under antibioticfree conditions.（2）The colonization pattern of recombinant strains in largemouth bass（Largemouth bass）.By feeding the recombinant strains to largemouth bass and setting different feeding days,the concentration of Lactobacillus plantarum in the intestine and faeces were counted to investigate the colonisation pattern of recombinant strains in largemouth bass and the effect of feeding time on the concentration of recombinant bacteria in largemouth bass.The study showed that the concentration of Lactobacillus plantarum in the largemouth bass was highest in the faeces,followed by the hindgut,and intermediate in the foregut and midgut.The highest concentration in the faeces was 1.38 x 105 CFU/g,while the rest of the parts had similar concentrations with no significant difference,the highest concentration reached 4.67 x104 CFU/g.The concentration of Lactobacillus plantarum in all parts showed a continuous decreasing trend after stopping feeding,the longer the feeding time of L.plantarum,the higher the concentration of the strain after stopping feeding,and the best result was achieved after 7 days of continuous feeding.Our recommendation is to keep feeding the recombinant bacteria every four days for two days after the first seven days of continuous feeding to ensure a high concentration of the strain in the largemouth bass and at the same time to save as much as possible.（3）The colonization pattern of recombinant strain in AA broiler chickens.By feeding the recombinant strain to AA broilers and setting different feeding days,the changes in the concentration of Lactobacillus plantarum in the intestine and faeces were counted to study the colonisation pattern of Lactobacillus plantarum LP-37 in AA broilers and the effect of the feeding time on the concentration of Lactobacillus plantarum in AA broilers.The concentrations of Lactobacillus plantarum in AA broilers were the highest in faeces,followed by the cecum,duodenum,jejunum and jejunum.The concentration of Lactobacillus plantarum in the faeces was the highest,the cecum the second highest and the duodenum,jejunum and ileum the second highest.The highest concentration of L.plantarum in faeces was 4.46 x 107 CFU/g,the highest concentration of L.plantarum in cecum was 2.98 x 106CFU/g,and the highest concentration of L.plantarum in duodenum,jejunum and ileum was similar,with a maximum of 2.29 x 105 CFU/g.The concentration of L.plantarum in all sites tended to decrease gradually after stopping feeding,and the longer the L.plantarum was fed,the higher the concentration of L.plantarum in duodenum,jejunum and ileum.The longer the feeding time of the recombinant bacteria,the slower the concentration of recombinant bacteria decreased after stopping feeding.Increasing the feeding days could prolong the residence time of recombinant bacteria in AA broilers,but the effect of increasing the feeding time on increasing the residence time of Lactobacillus plantarum gradually weakened as the feeding time increased.The concentration of recombinant bacteria in AA broilers reaches a high level after 3 consecutive days of feeding and decreases more slowly when feeding is stopped.Our production advice is to feed Lactobacillus plantarum continuously for 3 days at the beginning and then to maintain the frequency of feeding Lactobacillus plantarum every 4 days for 2days to ensure a high concentration of the strain in AA broilers and to minimise the dosage.In conclusion,this study can help to establish a scientific method of feeding Lactobacillus plantarum and provide important theoretical and technical support to optimise the application of Lactobacillus plantarum as a new feed additive. |
PDF Full Text Request