| Probiotics are the general name of live microorganisms that are colonized in human intestinal tract and r EProductive system,can improve the balance of microbiota and play a beneficial role to the host health.Pediococcus pentosaceus,Gram-positive,belonging to the genus Pediococcus of Str EPtococcus,has been approved as an edible species which can be used as food supplementation.P.pentosaceus has been widely applied in animal production,food fermentation industry,highly valued by researchers,and considered as a potential probiotic strain.Many studies have proved that P.pentosaceus exerted antioxidant,antibacterial,anti-inflammatory and immunoregulatory activities.Tuberculosis is a major zoonotic infectious disease caused by Mycobacterium tuberculosis infection,which affects human health and the development of animal husbandry and has a great impact on the national economy and people’s livelihood around the world.The global emergence,spread and prevalence of drug-resistant tuberculosis make the prevention,control and treatment of tuberculosis more difficult.It is particularly important to introduce new strategies to control tuberculosis and improve the treatment outcome.The probiotic potential of P.pentosaceus provides a new idea for the prevention and treatment of tuberculosis.Because its anti-mycobacterium tuberculosis activity and its role in regulating the immune response in the process of infection with Mycobacterium tuberculosis are still unclear,nine strains of P.pentosaceus isolated previously in our laboratory were used to evaluate the anti-M.smegmatis activity of P.pentosaceus in vitro.The zebrafish model was applied to analyze the regulatory effect of P.pentosaceus on lipopolysaccharide(LPS)-induced inflammation,and to clarify the role of P.pentosaceus in regulating immune response and phagocytosis of macrophages in the process of M.smegmatis infection,so as to provide scientific theoretical basis for elucidating the beneficial bioactivity of P.pentosaceus and promoting its application in prevention and treatment of tuberculosis.In the first part,the anti-M.smegmatis activity of P.pentosaceus was evaluated in vitro.The fermentation supernatants of nine strains of P.pentosaceus were collected respectively.After freeze-drying,the antimicrobial activity of P.pentosaceus metabolites against M.smegmatis was evaluated by agar diffusion method by measuring the diameter of inhibitory zones.By adjusting the p H value of fermentation supernatant,whether its antibacterial activity was related to organic acids was analyzed,and the metabolites of P.pentosaceus with antibacterial activity were further determined by enzyme treatment.The experimental results showed that the metabolites of LY-8 and LY-9 strains showed the strongest antibacterial effect at the concentration of 120 mg/m L,and the metabolites of LY-8 strain had the strongest antibacterial effect at the concentration of 240 mg/m L.Short chain fatty acids and bacteriocin may be the bacteriostatic substances in the supernatant of LY-6 and LY-7 strains,and bacteriocin may be the bacteriostatic substances in the supernatant of other strains.In the second part,the zebrafish model was used to explore the regulatory effect of P.pentosaceus on LPS-induced inflammatory response.Zebrafish embryos were treated with different concentrations of P.pentosaceus metabolites.According to the survival curve of zebrafish,the proper concentrations of the metabolites were 1.5 μg/L,1 μg/L and 0.5 μg/L.The inflammatory model of zebrafish was induced by LPS,and the expression of inflammatory factor mRNA was detected by Quantitative Real-time PCR to explore the anti-inflammatory activity of P.pentosaceus.The results showed that the metabolites of nine strains of P.pentosaceus could significantly reduce the expression of pro-inflammatory factors il6 and il1β,and increase the expression of anti-inflammatory factor il10.The anti-inflammatory activity of LY-8 strain was negatively correlated with the dose,and its anti-inflammatory effect was stronger than other strains.In the third part,the in vitro cell model was used to clarify the anti-inflammatory activity of P.pentosaceus and its role in regulating immune response and phagocytosis of macrophages during M.smegmatis infection.Cell Counting Kit-8(cck-8)and trypan blue counting method were used to determine the Experimental concentrations of nine strains of P.pentosaceus.According to the results,concentrations of 25 μg/m L,12.5 μg/mL and6.25 μg/mL were used.The inflammatory model of mouse monocyte macrophage Raw264.7 was induced by LPS in vitro,and the macrophage model infected by M.smegmatis was established.The expression of inflammatory factor m RNA was detected by q RT-PCR.According to the results,nine P.pentosaceus strains significantly down-regulated the expression of pro-inflammatory factors IL-6 and IL-1β,and up-regulated the expression of anti-inflammatory factor IL-10.During the infection of M.smegmatis,nine P.pentosaceus strains played a regulatory role in cellular immune response,and LY-8 strain had the strongest anti-inflammatory effect,and its anti-inflammatory activity was negatively correlated with dose,and its ability to regulate immune response was the strongest.In the fourth part,in order to explore the effect of P.pentosaceus on the phagocytosis of macrophages,macrophages were infected by M.smegmatis,the alterations of the cytoskeleton was determined at the protein level,and the fluorescence intensity of bacteria in macrophages was observed by laser confocal microscope.The results showed that nine P.pentosaceus strains could promote the remodeling of cytoskeleton and significantly enhance the fluorescence intensity of bacteria in macrophages.P.pentosaceus LY-8exerted the strongest ability to promote phagocytosis of M.smegmatis by macrophages.The above Experimental results show that P.pentosaceus has anti-M.smegmatis activity and anti-inflammatory activity,can regulate the immune response in the process of M.smegmatis infection,and improve the phagocytic ability of macrophages by affecting cytoskeleton.Among the nine strains of P.pentosaceus,LY-8 strain has better antibacterial,anti-inflammatory and immune response regulating ability than other strains,but the specific mechanism needs to be further explored. |
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