The Fermentation Product Of Ganoderma Lucidum By Probiotics Improved The Intestinal Barrier Function And Ameliorated Immunosuppression In Mice

ObjectiveIn this study,a probiotic fermentation system of ganoderma lucidum fruiting body was established,and the prevention and therapeutic effect and mechanism of Ganoderma lucidum fruiting body fermentation liquid in animal models with intestinal barrier damage and animal models with immune suppression were studied,expecting to become a new type of drug and food homologous preparation for the treatment of clinical diseases.The 1st part:Ganoderma lucidum fruiting body-probiotics fermentation is developed under laboratory conditions.1.Materials and methodsFirstly,the probiotics that can grow normally in the liquid medium of Ganoderma lucidum were screened out from six kinds of probiotics,including Bacillus subtilis,Lactobacillus acidophilus,Bifidobacterium brevis,Lactobacillus brevis,Bifidobacterium pseudospectorum and Lactobacillus garsseri by the method of viable bacteria count.The fruiting body concentration of low,medium and high Ganoderma lucidum was set as 7.5mg/m L,15mg/m L and 30mg/m L,and then the optimum fruiting body concentration of Ganoderma lucidum was determined by detecting the OD value and the total sugar content of the probiotics fermentation broth.The optimized probiotics strains were co-fermented with the Ganoderma lucidum medium under the appropriate concentration,and the changes of physicochemical properties of the fermentation broth were analyzed by HPLC-MS and p H test.2.ResultsIn terms of the selection of probiotics,Bacillus subtilis,Lactobacillus acidophilus and Bifidobacterium brevis can grow normally in MRS liquid medium containing Ganoderma fruiting body.We found that when the Ganoderma lucidum fruiting body medium concentration is 30mg/m L,Bacillus subtilis,Lactobacillus acidophilus,Lactobacillus acidophilus and Bifidobacterium breve OD value of the mixed bacteria fermented liquid is higher,and the total sugar content is higher,so using Bacillus subtilis,Lactobacillus acidophilus and Bifidobacterium breve mixed strains of probiotics as probiotics,Ganoderma lucidum fruiting body culture medium concentration of 30mg/m L to make the Ganoderma lucidum fruiting body probiotics fermentation broth for subsequent experiments.The composition changes of Bacillus subtilis-Ganoderma lucidum fruiting broth,Lactobacillus acidophilus and Bifidobacterium breve mixed probiotics-Ganoderma fruiting broth before and after fermentation were quantitatively analyzed by HPLC-MS,And after fermentation,various Ganoderma acid components decreased and p H value decreased.and it had the function of transforming Ganoderma acid A into Ganoderma acid C2 for mixed probiotics.3.ConclusionBacillus subtilis,Lactobacillus acidophilus and Bifidobacterium breve can decompose and use the constituents of Ganoderma acid,and have the function of biotransformation to the constituents of Ganoderma acid.The 2st part:Treatment of intestinal barrier damage model with Bacillus subtilis-Ganoderma lucidum fruiting body fermentation liquid1.Materials and methodsFor Bacillus subtilis-Ganoderma lucidum fermentation broth to evaluate the treatment the intestinal barrier function is impaired in mice model,40 healthy male SPF BALB/c mice(20+2 g)were randomly divided into five experimental group,respectively is healthy controls group(CON),intestinal barrier damage model group group(CS),Bacillus subtilis-Ganoderma lucidum fermentation liquid treatment group(FT),Bacillus subtilis-Ganoderma lucidum fermentation liquid to prevent group group(FP),Ganoderma lucidum medium group(GL).Except for CON group and FP group,the other 3 groups were given 400mg/m L ceftriaxone sodium 0.2 m L,twice a day for7 days,to establish the intestinal barrier damage model.On the 8th day,MRS medium of CS group was given 0.2m L;FT group Bacillus subtilis-Ganoderma lucidum fermentation liquid 0.2 m L;Ganoderma lucidum solution in GL group was 0.2 m L for7 consecutive days.FP group was given Bacillus subtilis-Ganoderma lucidum fermentation solution for prevention on the basis of intragastric administration of ceftriaxone sodium on the first 7 days,and then given 0.2 m L Bacillus subtilis-Ganoderma lucidum fermentation solution on the second 7 days.The CON group was intragastrically administrated with normal saline as control on the first 7days,and intragastrically administrated with 0.2m L MRS medium on the second 7days.On the 15th day,mice in each group were sacrificed.Calculating cecal index;Hematoxylin-eosin staining(H&E)was used to examine the level of colonic tissue damage.ELISA was used to detect serum LPS,IL-10,TNF-α,IL-6 levels.The proportion of immune cell subsets in spleen of each group was compared by flow cytometry.High throughput 16S r RNA analysis of intestinal microecological differences.2.ResultsCeftriaxone sodium induced intestinal flora dysregulation and intestinal mucosal barrier function in mice,and the body weight of mice in CS group was reduced.Cecum swelling;Increased intestinal permeability and inflammatory infiltration;Mature T cells,CD4+T cells and CD8+T cells were overregulated;the abundance and diversity of intestinal flora decreased,and the abundance of Enterococcus increased significantly,while the abundance of Lactobacillus decreased.Under the intervention of Bacillus subtilis-Ganoderma lucidum fermentation broth,the body weight of FT group and FP group returned to normal level.The infiltration of inflammatory cells in colon tissue was improved.Decreased intestinal permeability;Inflammatory cytokine content and lymphocyte ratio tended to normal level;The relative abundance of Lactobacillus,bacteroidetes,Blautia in the intestinal microbiota were increased,while the relative abundance of Mycoplasma,Anaeroplasma and Enterococcus were decreased.3.ConclusionThe results showed that Bacillus subtilis-Ganoderma lucidum fermentation broth could improve the impaired intestinal barrier function by reducing intestinal permeability,regulating the proportion of lymphocytes,and adjusting the structure of intestinalmicrobiota.The 3st part:Treatment of immunodeficiency model with mix probiotics(Lactoba cillus acidophilus,Bifidobacterium breve)-Ganoderma fermentation broth1.Materials and methodsFor Lactobacillus acidophilus,Bifidobacterium breve-Ganoderma lucidum fermentation broth to evaluate the treatment the low immunity mice model of fermented liquid test,32 male BALB/c mice(6 to 8 weeks,weight 18 to 22 g)were randomly divided into 4 groups,including normal control group(CON group)and low immunity model group(DEX),Lactobacillus acidophilus,Bifidobacterium breve-Ganoderma lucidum fermentation broth(FT)and Ganoderma lucidum fruiting body medium(GL).Mice in the other three groups were injected intraperitoneally with 20mg/(kg.d)dexamethasone every day for consecutive 7 days,except for CON group,to establish the hypoimmune model.MRS medium 0.2 m L in the CON group,0.2 m L in the DEX group,0.2 m L in the FT group,0.2 m L in the Ganoderma lucidum medium 0.2 m L in the GL group,and were fed continuously for 14 days from day 8.On the 22nd day,mice in each group were euthanized and sacrificed.Weight curve drawing,determination of weighing index,spleen H&E staining microscopic evaluation of colonic pathological tissue damage,ELISA method to detect the mice serum IL-17,TNF-αlevel,through the flow cytometry comparison between groups of mice spleen immune cell subgroup percentage,A sword bean element(Con A)stimulus spleen lymphocytes in vitro comparison between groups of T lymphocyte proliferation response,16 s r RNA high-throughput sequencing analysis structure of intestinal microbiota;Ganoderic acid The metabolitesas that fermented by Lactobacillus acidophilus and Bifidobacterium brevis to stimulate Peyer`s patches(PPs)cells in vitro,and then the proportion of lymphocytes was detected by flow cytometry.2.ResultsMice in the DEX group showed immunosuppression.Compared with the CON group,the H&E staining of colon tissue of mice in the DEX group showed serious pathological damage;Serum TNF-αlevel increased;The proportion of CD4~+T,CD8~+T and CD3~+T lymphocytes in spleen decreased significantly.Splenic lymphocyte transformation function decreased significantly;The abundance of intestinal microbiota decreased and the relative abundance of Lactobacillus significantly decreased,while the relative abundance of Sphingomonas and Enterococcus increased.Histological damage and inflammatory infiltration in FT group were alleviated by Lactobacillus acidophilus,Bifidobacterium breve-Ganoderma lucidum fruitbody fermentation broth intervention.;weight recovery;spleen index increased;serum IL-17level decreased;the proportion of CD4~+T,CD8~+T,CD3~+T lymphocytes in spleen increased.The function of T lymphocyte transformation in spleen of mice was significantly improved.The fermentation broth of Lactobacillus acidophilus,Bifidobacterium breve-Ganoderma lucidum also increased the richness of intestinal microbiota in FT and restored the composition of intestinal microbiota,especially decreased the abundance of Sphingomonas and Enterococcus in mice with low immunity,and increased the abundance of high-quality intestinal micribiota-Lactobacillus.Metabolites of Ganoderic Acid A fermented by Lactobacillus acidophilus and Bifidobacterium breve can stimulate the activation of mice CD4~+T cells of PPs in vitro.3.ConclusionThe results showed that Lactobacillus acidophilus,Bifidobacterium breve-Ganoderma lucidum fruitbody fermentation broth could improve the immune suppression of mice in terms of inhibiting the secretion of inflammatory cytokines,enhancing the function of T lymphocytes,and regulating intestinal microbiota.