| Lactobacilli as probiotic strains perform beneficial effects, maintaining the balance of the microflora in intestine and promoting animal health, which widely exist in the animal gastrointestinal tracts. In China, lactobacilli are often added to feed to promote the animal growth and health, especially in piglets. The lactobacilli can decrease the rate of diarrhea of piglets. Because the adhesion and colonization of lactobacilli to the intestines is the first step for performing probiotic effect, the adhesive ability is important. In my research, the effect and its mechanism of adhesion of lactobacilli were focused, studying the important role of the surface proteins involved in adhesion. Afterwards, the adhesive and colonization abilities of lactobacilli were evaluated in vivo and vitro in order to analyze the mechanism of adhesion of lactobacilli, which will lay foundation for improving the colonization of lactobacilli and isolating lactobacilli with high adhesive ability.Part one: Isolation and identification of lactoabacilli with good properties1. Isolation and identification of lactobacilli （Trial1）. In order to isolate lactobacilli, chyme and mucus from jejunum and ileum were collected from a healthy weaned piglet at age of28d. Eighty-five strains of lactobacilli were isolated from the pig intestines and identified by sequencing analysis based on16S rRNA gene, including1Lactobacillus johnsonii,54L. salivarius and30L. reuteri. The sequence were submitted in NCBI and blasted, the Genebank accession number were obtained. Meanwhile the lactobacilli were identified by sugar fementation tube, showing that the most lactobacilli were different. Among the85lactobacilli,36were from the chyme of jejunum,18from the mucus of jejunum,3from chyme of ileum and28from the mucus of ileum.2. Selection of lactobacilli by adhesion ability （Trial2）. In order to obatain lactobacilli with high adhesive ability,40lactobacillus strains were selcetd according the result of microscopy and evolutionary analysis. The ahesive ability of lactobacilli was evaluated using Caco-2cells in vitro. After lactobacilli and cells coincubated, the cells were Gram stained and observed under microscope, and the number of lactobacilli adhered to cells were caculated. The result showed that six strains possessed significantly higher adhesive ability than other strains （P<0.05）, including1L. salivarius and5L. reuteri. 3. The properties of lactobacilli with high adhesive ability （Trial3）. In this trial, the acid and bile tolerance, self coagulation and inhibitory effect against E. coli and salmanella of lactobacilli with high adhesive ability isolated from piglet were investigated. The results showed L. reuteri ZJ617and ZJ621with high adhesive ability had better acid and bile torlerance than lactobacilli with lower adhesive ability. Morever, lactobacilli with high adhesive ability possessed better self coagulation ability. At6h, the rate of coagulation of some strains were was up to80%, higher than that （28.5%） for commercial strain LGG （L. rhamnosus GG, LGG）. In order to study the inhibitory effect of lactobacilli, three assays （Competion, Exclusion and Displacement） were used. It is found that the strains with high adhesive ability could significantly inhibit the E. coli K88and Enteritidis Salmonella50335, while the strains with lower adhesive ability did not. Furthermore, the correlation between the adhesive ability of lactobacilli and its inhibitory effect were analyzed. The adhesive ability of lactobacilli showed positive correlation with its inhibitory effect against E. coli K88（r=0.68, P<0.05） and E. Salmonella50335（r=0.50, P<0.05）, but not in the Displacement assay. It is indicated that colonization of lactobacilli to the yong or newborn animal could show better probiotic effect against pathogens.Part two:Function of surface proteins involved in adhesion from lactobacilli with high adhesive ability1. Isolation and identification of surface adhesive protein from lactobacilli with high adhesive ability （Trial4）. Colonization of lactobacilli in the intestines of animal is the basis for performing benefical effect, in which the surface protein and other components from the surface of lactobcilli play an important role. In this trial, the adhesive surface proteins of lactobacilli involved in adhesion were investigated. A band of an approximately37kDa surface layer protein was detected from Lactobacillus strains ZJ616, ZJ617, ZJ621, ZJ623and LGG by Western blotting with Sulfo-NHS-Biotin labeled Caco-2and IPEC-J2cells, indicating that this protein has specific binding to Caco-2cells and IPEC-J2cells and is involved in adhesion. According to the Mass spectral analysis for the adhesion-related protein of L. reuteri ZJ617, this protein showed100%homology to that of the glyceraldehyde-3-phosphate （GAPDH） of L. reuteri JCM1112[GenBank accession number YP001841377]. This protein is hydrophobic and has transmembrane region, ranging from149th to164th amino acids, with special amino acid sequence was Ala- Gly-Ser-Cys-Thr-Thr-Ser-Cys-Leu-Ala-Pro-Met-Ala-Lys-Val-Leu2. GAPDH obtained by E.coli expression system （Trial5）. To validate the role of GAPDH of L. reuteri involved in adhesion and displayed on the surface layer of lactobacilli, the GAPDH protein was artificially expressed in Escherichia coli and purified for antibody preparation. The GAPDH protein was purified by affinity chromatography using anti-His-tag Ni+agar. This protein obtained had the purity of96%, and the antibody was sucessfully obtained.3. Function of surface proteins involved in adhesion from lactobacilli and its location on the lactobacilli （Trial6）. The adhesive ability and inhibitory ability against pathogens decreased significantly for L. reuteri ZJ617and ZJ621treated by LiCl or blocked by an anti-GAPDH rabbit antibody compared with the untreated strains （P<0.05）. Immunoelectron microscopic and immunofluorescence analyses successfully proved that GAPDH is located on the surface layer of L. reuteri ZJ617.Part three:The colonization and probiotic effect of L. reuteri with high adhesive ability in mice and new-born piglet1. The colonization of L. reuteri in mice intestine and discharged in feces （Trial7）. In order to verify the effect of lactobacilli with high adhesive ability in vitro, and study its safety and colonlization in the animal intestine,80healthy six-week-old male BALB/c were used. The mice were divided into four group based on body weight, i.e., Control, ZJ615, ZJ617and ZJ621group. To evaluate the number of lactobacilli adhesion in the mice intestines, the strains with streptomycin resistance were selected. The feeding trial lasted for13days, the lactobacilli were chronically administered to mice at16:00-17:00during the first3days, then5mice in each group were slaughtered on the1st,3rd,6th and10th day since the oral administration stopped to detect the colonization of lactobacilli in jejunum, ileum and colon and the lactobacilli in feces.The lactobacilli did not affect the mice health. Compared to control and ZJ615with low adhesive ability, ZJ617and ZJ621with high adhesive ability could colonize better in the ileum and colon of mice, and the lactobacilli was also detected in the feces of mice.2. The colonization of L. reuteri in piglet intestine and discharged in feces and its effect on the performance, improvement of intestine intestinal development in piglet （Trial8）. In order to investigate the colonization of L. reuteri with high adhesive ability in piglet and its probiotic effect,50newborn piglets with half male were divided into5group based on the bodyweight, gender and litter, Control, ZJ615, ZJ617, ZJ621and LGG group （positive control）. The lactobacilli were chronically administered to piglets for14days since the age of three days. The dose was from103to109cfu and gradually increased per two days, adding up to2.22×109cfu per piglet. The feeding trail lasted for25days, and piglets were weaned at21st day. Durig the trail, the feces were collected from anus at13th,17th,21st, and25th day to evaluate the numeber of lactobacilli discharged in feces. On25th day,4piglets from each group were slaughtered, the jejunum, ileum, colon and blood were collected to evaluate the the colonization of lactobacilli and its protiotic effect. The L. reuteri had no effect on daily weight gain, but could significantly reduce the rate of diarrhea of piglet and improve the villus height and lymphocytes （P<0.05） of jejunum and ileum. Meanwhile, ZJ617and ZJ621could significantly colonize in the ileum and colon of piglets.In summary, the lactobacilli with high adhesive ability were isolated and identified from the present study, which performed fine resistance to acid and bile and showed probiotic properties. The adhesion protein, GAPDH was identified from the surface of lactobacilli, involved in adhesion to the epithelial cells and played an important role in inhibiting the pathogens. In vivo assays, the L. reuteri strains with high adhesive ability could colonize permanently in the intestines of mouse and piglets, and effectively decrease the diarrhea and promote the animal health. |
PDF Full Text Request