| β-lactoglobulin(β-Lg) allergy often leads to digestive allergies or allergic gastroenteritis,damages intestinal micro ecological balance, and impact on infant growth and health of body and mind seriously.This thesis takes the L.acidophilus obtained by previous research which can effectively alleviate the β-Lg allergy as research object.PCR-DGGE method was applied to monitor mice colonic fecal flora diversity of the normal group,the allergy group and different dose of L.acidophilus group.Changes of mice intestinal metabolite in each group was measured by high performance liquid/gas chromatography and automatic amino acid analyzer.ELISA method is adopted to determinate the content of total Ig E in serum and observe the number of eosinophils and allergy symptoms.Analysis of the relationship between probiotics regulating intestinal flora changes, metabolic activity and host health provides new theoretical basis for revealing the mechanism of probiotics to alleviate allergies.1.Establishment and validation of animal modelFemale BALB/c mice were divided randomly into normal saline control group, β-Lg sensitized group and different concentration of L.acidophilus gavage group(n=15). Mice in L.acidophilus group were administrated with different concentration of bacterial suspension 3times a week for three weeks since the first day(low concentration of 10μg/m L, middle concentration of 50μg/m L,high concentration of 250μg/m L). β-Lg allergen sensitized group and L.acidophilus group were given intraperitoneal injection with 0.01 m L/g allergen(1 m L Freund’s adjuvant +1 m L 2mg/m L β-Lg) in seventh, fourteenth, twenty-first days.Control group were intraperitoneal with saline. In the twenty-third day of the experiment, oral β-Lg provoked allergic(1mg/g) 2 times(interval 30min).After the assessment of allergy symptoms the mice were killed and the index was detected. Experimental results show that, compared with the normal mice, β-Lg sensitized group of mice allergic symptoms are significant, and the content of serum total Ig E and eosinophil numbers increased significantly(P < 0.05), suggesting that the animal model was established successfully.And L.acidophilus can effectively relieve allergy symptoms and reduce the level of antibodies in the serum of allergic mice,and there is dependence between quantity and effect.2.Effect of L. acidophilus on mice intestinal flora diversity effectsUsing PCR-DGGE to analyze the diversity of the intestinal flora,it can find that The dominant bacteria in control group is Bacteroides,uncultured Bacteroidetes,Firmicutes,Bacteroides door. Compared with the control group,the number of L.actobacillus and Bifidobacterium were significantly reduced and the number of clostridium increased significantly in the sensitized group; and the L. acidophilus could significantly regulate the balance of intestinal flora(P<0.05), and it was proportional to the gavage dose. Using Quantity One software to analyze PCR-DGGE graph spectra, and the richness of sensitized group colonic microflora of mice DGGE graph spectra was 30±2, which is significantly lower than that of the control group mice35.67±1.15(P<0.05). At the same time, sensitized group colon flora of mice Shannon-Wiener index and maximum Shannon-Wiener index were significantly lower than the control group(P <0.05).L.acidophilus intervention can significantly improve the intestinal flora of the indexes of diversity,there is no significant difference among middle bacteria,high bacteria group and control group(P > 0.05).3.The effect of L. acidophilus on mouse intestinal metabolite of β-Lg allergyApplication of high performance liquid/gas chromatography and automatic amino acid analyzer to analyze changes of each group mouse intestinal metabolite. The experimental results show that, compared with the control group, the content of lactic acid in colon feces of sensitized group decreased significantly(P < 0.05); There was a remarkable rise of acetic acid and short chain fatty acid and a remarkable decrease of propionic acid, butyric acid, isobutyrate, isovalerate was significantly lower(P < 0.05).And L.acidophilus intervention can improve allergic intestinal lactic acid and short chain fatty acid metabolism. In addition, the method of principal component analysis of aminos acid content in mice colon feces showed that the score of the control group is significantly higher than that of sensitized group, L.acidophilus intervention can improve the comprehensive score, and the high and middle dose group of L. acidophilus was more significant in promoting intestinal amino acid metabolism than the low dose group.The experiment indicates that, L.acidophilus intervention can alleviate effect on β-Lg allergy,the immune mechanism may be closely related to L.acidophilus regulating intestinal flora quantity and variety, improving intestinal lactic acid, short chain fatty acid,amino acid metabolism, and then inhibiting inflammation. |
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