| Probiotics are defined as living microorganisms that,when administered in adequate amounts,confer a health benefit to the host.Probiotics usually refer to the beneficial bacteria,which are widely developed and applied due to their simple culture mode,rapid propagation speed and strong environmental tolerance.However,probiotic bacteria also have their fatal weaknesses,such as gene transfer,which easily leads to the spread of resistance genes.Fungi have the characteristics of genetic isolation,effectively avoiding the transfer of resistance genes.Beta-glucan in the fungal cell wall has been shown to be a highly beneficial biomass that regulates the immune system.In addition,fungi can serve as a good vaccine delivery platform,with the natural conformation of genetic engineering product expressed by the fungus as a vector.In conclusion,fungi have the potential to be exploited as excellent probiotics.Although the effects and mechanisms of probiotics are complex,there are mainly the following aspects.First,because the primary purpose of our use of intestinal probiotics is to increase the weight of livestock and improve feed conversion ratio,improving growth performance is an important indicator for evaluating probiotic effects.Secondly,the development of the digestive system directly affects the absorption and utilization of nutrients by livestock and poultry.Therefore,the influence of probiotics on the development of the digestive system is also a factor in evaluating the quality of probiotics.Thirdly,the health of livestock is primarily determined by the immune system,and probiotics should be able to enhance or coordinate the host’s immune response.Finally,the intestinal flora affects the body’s absorption of nutrients and its own metabolic levels.Probiotics should be able to maintain the balance of the intestinal flora and even promote the development of the intestinal flora in a beneficial direction.At present,probiotic strains are mainly isolated from the environment,and it is rare to obtain probiotic strains by random mutagenesis or targeted genetic modification.Earlier,a strain named Diutina rugosa(D.rugosa)SD was isolated from chicken feces in our laboratory.Preliminary experiments showed that D.rugosa SD could not be colonized into the intestinal tract or tolerate the adverse intestinal environment of chicks.Based on this strain,probiotic strains with strong intestinal colonization ability were obtained by mutagenesis screening,and their specificity and probiotic mechanism were further studied in this study.1.Screening of intestinal colonization strains and analysis of strain-specificityIn this study,the survival rate of 50%~70% was used as the standard to determine the optimal conditions for UV and NTG mutagenesis.Finally,the optimal mutagenesis conditions we screened were: 15 J,0.4 min and 50 mg/m L,40 min.According to the optimal mutagenesis conditions,twenty-three strains of mutant strains were screened by UV and NTG complex mutagenesis and colonization(12 h)in the intestines of chicks.Twenty genetic stability tests were performed and we finally obtained a genetically stable mutant strain.Through evolutionary analysis,it was found that the mutant strain had the closest relationship with the type strain D.rugosa ATCC10571(bootstrap value 100%)and was named D.rugosa SD-17.The biological characteristics of D.rugosa SD-17 have significant changed,proliferation ability of D.rugosa SD-17 has been significantly enhanced,D.rugosa SD-17 showed significant depression in the center of the colony on the YTD solid medium,the cell wall of D.rugosa SD-17 was found to be significantly thickened by transmission electron microscopy,and the content of β-glucan extracted from the cell wall by the classical acid-base method was found to be significantly increased.2.Research on the probiotic potential of D.rugosa SD-17To further explore the probiotic potential of D.rugosa SD-17,we studied the tolerance of D.rugosa SD-17 using an experimental model of the intestinal environment simulated by artificial gastric juice and artificial bile.The results showed that the survival rate of D.rugosa SD-17 was still close to 50% after treatment for 8 h in gastric acid solution(p H=2).In addition,0.3% bile salt solution had no significant effect on the reproduction of D.rugosa SD-17.D.rugosa SD-17 grew well in the gut temperature(37 ℃ ~ 41 ℃).The cell adhesion test showed that the adhesion rate of D.rugosa SD-17 to Caco-2 cells was 61.5%,which was significantly higher than that of the standard strain.The chickens were administrated with D.rugosa SD-17 and the contents of the intestines were collected.It was found that 10% of D.rugosa SD-17 could be isolated from the ileum after 16 hours of gavage,indicating that D.rugosa SD-17 colonized in the ileum of the chicks briefly.In conclusion,D.rugosa SD-17 is well tolerated in the simulated intestinal environment and can be temporarily colonized in the intestinal tract of chicks.3.Study on the probiotic effect of D.rugosa SD-17 on chicksThe safety of D.rugosa SD-17 on chicks was evaluated by acute toxicity experiments and chronic infectivity experiments.The results showed that neither the acute toxicity test nor the chronic infection test caused death.In addition,the chicks had a good mental state,normal intake,no pathological changes in the appearance and internal organs,and normal blood cell indexes.These results suggest that D.rugosa SD-17 was not pathogenic to chicks.The addition of D.rugosa SD-17 to fodder resulted in the increase of the weight of chickens,the improvement of feed conversion ratio,and the development of villi in the ileum.It is worth noting that the ileum is also the main colonization site of D.rugosa SD-17,indicating that the strain has a certain stimulating effect on ileal epithelial cells.Peripheral blood lymphocytes were stimulated with Con A and found that the lymphocyte proliferation ability of D.rugosa SD-17 group was significantly improved.Lymphocyte subsets were detected by flow cytometry and it was found that D.rugosa SD-17 significantly increased the content of CD3+CD4+(T helper,Th)cells.In addition,Quantitative Real-time PCR was used to detect the marker cytokines on the surface of Th cells.It was found that D.rugosa SD-17 promoted the expression of Th1 cytokine,but had no effect on the expression of Th2 cytokine.These results indicate that D.rugosa SD-17 has a certain immunostimulatory effect on chicks.4.Study on the effect of D.rugosa SD-17 on intestinal flora of miceIn this study,16 S r DNA sequencing was used to analyze changes in ileal flora,cecal flora,colonic flora and fecal flora to reveal the mechanism of action of D.rugosa SD-17 on the intestinal environment of mice.Firstly,administration of D.rugosa SD-17 to mice caused a significant increase in the abundance of ileal flora.Secondly,the stimulation of D.rugosa SD-17 caused changes in the content of the Firmicutes and Bacteroidetes in the mouse fecal flora: the proportion of Firmicutes increased and the proportion of Bacteroidetes decreased.It is beneficial to increase the absorption and utilization of energy in the feed.Finally,the stimulation of D.rugosa SD-17 caused a significant increase in the proportion of Lactobacillus.These results preliminarily showed that the stimulation of D.rugosa SD-17 caused the adjustment of the intestinal flora structure of mice and increased the proportion of beneficial bacteria.5.Study on the effect of D.rugosa SD-17 on metabolic pathway in miceBased on the sequencing of mouse intestinal flora,we used the KEEG database for enrichment analysis to reveal the effect of D.rugosa SD-17 on the metabolic level of mice.We enriched five specific metabolic pathways from the colonic flora,involving cell adhesion molecules,cytoskeleton regulation,viral infection,and cardiomyopathy.We selected the cell adhesion molecule pathway for analysis and found that E-cadherin is the core protein of intestinal adhesion.In order to further investigate the effect of D.rugosa SD-17 on adherin junction of mouse colon,colon tissue and Caco-2 cells were stimulated by D.rugosa SD-17,and the results showed the gene expression level of E-cadherin was significantly increased.In addition,the expression levels of other related proteins(β-catenin,ZO-1,etc.)attached to adherin junction were up-regulated to varying degrees.These results show that,D.rugosa SD-17 increased the gene expression level of adherin junction-associated proteins in the colon of mice,and promoted the maintenance of intestinal structure.In conclusion,a beneficial strain D.rugosa SD-17 with strong colonizing ability was successfully obtained in this study.This strain has obvious strain specificity,has good probiotics to the chick,and can optimize the intestinal flora and maintain the intestinal structure.This study laid a foundation for further study on the mechanism of intestinal probiotics,and provided guidance for the development and application of intestinal probiotics in livestock and poultry. |
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