| Natto is a traditional Japanese fermented food,made of soybean fermented by Bacillus natto.It keeps the nutritional value of soybean,and it was richs in active ingredients such as natto kinase and glucopeptide.The nutritional and functional components of soybean is popular in the research of soybean.There are many domestic and foreign researches on natto,however,natto products and the relationship between the immune and intestinal flora research reported were less.The Bacillus natto preparation?BNPr?and natto lyophilized powder?NLP?were made from Bacillus natto liquid or solid state fermentation,and made a components compare analysis,the normal mice and intestinal flora imbalance in mice being used as model,the immune and the effect on the regulation of intestinal flora in mice was studied using in vivo experimental,and ultimately to find the correlation between intestinal flora and immune regulation.The main research contents and results in this thesis are as follows:1?The main nutrients were measured to study the main component of the fermented natto and compare the nutrition components of NLP and BNPr with the soybean,the changes of main components were explored during the fermentation process.The results showed that compared with soybean powder,ash and amino acid nitrogen content in NLP were increased significantly.The crude fiber,crude fat,and moisture content in NLP were decreased significantly.Compared with liquid culture product of Bacillus natto,the crude protein content in BNPr was increased extremely significantly,while the ash and amino acid nitrogen content were increased significantly.The moisture content was decreased extremely significantly and the crude fiber content was decreased significantly.In addition,after fermentation,the natto kinase content in BNPr and NLP increased,and the soybean isoflavone and phytic acid content in NLP were decreased extremely significantly,and vitamin K2content decreased significantly.2?The BNPr to antibiotics mediated immunomodulatory effects and the influence of the secretion of cytokines in SPF mice were studied in this paper.Results show that,compared with model group,the spleen and thymus index was extremely significant increased.The production of serum IL-2,IL-10,TNF-alpha,IFN-gamma were increased significantly in BNPr regulation group.The iNOS activity and the albumin,globulin and globulin ratio in mice serum and the phagocytizing effect of the chicken red blood cell by peritoneal macrophage?PM?in experimental group was increased significantly.The mice serum hemolysin level in experimental group was significantly higher than control group.The spleen lymphocyte transformation factor levels in experimental groups were increased significantly compared with the model group.Compared with control group,the spleen index of BNPr preventional group close to normal level.The percentage and index of phagocytosis were raised with the increase of the concentration of the preparation.The release of IL-2 was significantly increased in high dose BNPr regulational group,the level of TNF-alpha was adjusted to normal levels in middle dose BNPr regulational group.The secretion of IL-10 was significantly reduced in middle dose BNPr prevention group.The content of albumin and globulin in middle dose BNPr prevention group were higher than control group.3?The NLP to antibiotics mediated immunomodulatory effects and the influence of the secretion of cytokines in SPF mice were studied in this paper.Results show that,compared with model group,low dose of NLP of spleen and thymus index was extremely significant higher than model group.The production of serum IL-2,IL-10,TNF-alpha,IFN-gamma were increase extremely significantly in NLP regulational group compared with model group.The iNOS activity and the albumin,globulin and globulin ratio in mice serum and the phagocytizing effect of the chicken red blood cell by peritoneal macrophage?PM?in experimental group was extremely significant higher than model group.The NO level in high and low doses of NLP regulational group was extremely significant higher than model group.The mice serum hemolysin level in control group and regulational group and preventional group were extremely significant higher than control group.The spleen lymphocyte transformation factor levels in experimental groups were increased significantly compared with the model group.Compared with the blank control group,the half hemolysis value of NLP regulational group was increased significantly.The phagocytosis function of macrophage was extremely significant enhanced and the phagocytosis percent of macrophages and red blood cell count of chicken was extremely significant increased.The iNOS activity and the content of albumin were extremely significant increased in high dose of NLP regulational group,the albumin and globulin content in middle dose of NLP preventional group was increased.4?Miseq method was used to analyze the BNPr to study the regulation of the intestinal flora in mice.The antibiotics was used to make a intestinal microflora-imbalance model,and the BNPr was lavaged to regulate intestinal flora,the diversity of the intestinal flora was analyzed,and the clustering analysis was made to analyze the change of intestinal flora.The results showed that the OTU coefficient was decreased with the increase of the BNPr dose.The chao's coefficient,Shannon coefficient,and the ACE coefficient were significantly reduced compared with control group,and it was the highest in middle-dose regulational group.Simpson coefficient was up to 0.2633 in low-dose regulational group.Good coverage coefficient in control group were significantly lower than that of other groups,and it's highest in middle-dose group?0.9868?.Dilution curve shows that the complex degree of the community was increased firstly and then decreased,finally into balance.The main phylum before modeling were bacteroidetes,actinomyces and the proteabacteria,and the firmicutes were increased while the bacteroidetes were decreased during the prophage and the anaphage of the lavigation,and the ratio of the firmicutes and the bacteroidetes was decreased since the lavigation stopping.As the results of the clustering analysis,the similarity at the OTU,phylum and genus level were the highest before the modeling,and extremely significant decreased after modeling,and significantly increased during the prophage and the anaphage of the lavigation,and it was closed to the normal level after stopping the lavigation.Probiotics were highest before modeling,and it were reduced significantly after modeling,and it were returned to 82.6%of normal levels in the anaphage of the lavigation,15 d after the stop lavigation were back to 86%.The change of pathogenic bacteria was on the contrary,it were lowest before modeling and it were 18.73 times of pre-modeling,and it were extremely significant decreased in anaphage,and it returned to normal levels at last.5 Miseq methods were used to analyze the NLP to study the regulation of the intestinal flora in mice.The antibiotics was used to make a intestinal microflora-imbalance model,and the NLP was lavaged to regulate intestinal flora,the diversity of the intestinal flora was analyzed,and the clustering analysis was made to analyze the change of intestinal flora.The results showed that the DNA extraction was high quality and it can be used in subsequent experiments,stripe numbers were extremely significant decreased and then returned to normal levels.With the increasing of the NLP dose,the diversity indexes varied.The Chao's coefficient were increased,the Shannon coefficient and ACE coefficient were increased firstly and then decreased,and the Simpson coefficient and Good coverage coefficient were decreased firstly and then increased.The diversity index of middle-dose regulational group was higher than others.The main phylum before modeling were Bacteroidetes and Firmicutes,and the Firmicutes were decreased while the Bacteroidetes were increased during the prophage,it appeared the tendericutes which was never appear,more than 92%of the phylum in model group was Proteobacteria.The Firmicutes was continue increased during the anaphage of the lavigation,and the ratio of the proteobactera was decreased while Firmicutes was extremely significant increased in model group.The proportion of Firmicutes was increased,and the Proteobactera was decreased while the Firmicutes was increased since the lavigation stopping.From the genus levels,the lactobacillus was the highest before modeling,and it extremely significant decreased in the prophage of lavigation,and it significant increased in the anaphage and the stopping of lavigation.As the results of the clustering analysis,the similarity at the OTU,phylum and genus level were the highest before the modeling,and extremely significant decreased after modeling,and significantly increased during the prophage and the anaphage of the lavigation,and it was closed to the normal level after stopping the lavigation.The bacillus natto was 0.27%in the prophage of lavigation,and it reached to 0.32%in the anaphage of lavigation,when stopped lavigation,the proportion of the Bacillus natto was 0.21%.The levels of the probiotics,and the proportion of probiotics and pathogenic bacteria were the highest before modeling,and it extremely significant decreased after modeling.When lavigated the NLP,the levels were increased and almost reached to normal when stopped lavigation,it showed an curve of V,and the variation of the pathogenic bacteria was the opposite. |
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