ABSTRACT
Objective To investigate the effects of Lactobacillus rhamnosus GG (LGG) colonization in early life on intestinal barrier and intestinal development in offspring mice and its possible mechanism.Methods Six C57BL/6 pregnant mice with the same conception time of 6 weeks were selected and randomly divided into experiment group given 108 cfu/ml LGG live bacteria and control group given LGG inactivated bacteria by gavage from the 18th day of pregnancy until natural birth.The progeny mice in the two groups were continued to be gavaged with 107 cfu/ml of LGG live bacteria or LGG inactivated bacteria on days 1-5 of birth.The body weight changes of 3 week'progeny mice were recorded.The colonization of LGG bacteria in offspring mice was detected at 2nd and 3rd weeks.The mRNA of intestinal proinflammatory cytokines and tight junction molecules were evaluated by real-time PCR method.HE,immunohistochemistry,immunofluorescence staining and enzyme-linked immunosorbent assay were used to evaluate the intestinal barrier of 3-week old off spring mice.Results Compared with the control group,the progeny mice of the experiment group showed no significant difference in body weight at the first week,and the body weight increased at the second week and the third week [2ndweek:(3.790±0.240) g vs.(4.326±0.140) g,t=3.707,P=0.006;3rd week:(7.295±0.326) g vs.(8.040±0.370) g,t=3.130,P=0.011].LGG colonization can be detected only in the feces of progeny mice in the experiment group.Intestinal colonization can promote the growth of small intestine villi and colon crypt depth [jejunum:(320.000±22.514) μm vs.(265.100±15.611) μm,t=8.258,P＜0.001;ileum:(150.500±13.099) μm vs.(111.000±11.308) μm,t=9.958,P＜0.001;colon:(295.000±15.209) μm vs.(233.100±6.678) μm,t=9.129,P＜0.001].Compared with the control group,the number of goblet cells in the colonic crypt of the experiment group increased (11.62 ± 0.780 vs.35.24 ±1.370,t=15.000,P＜0.001),and the relative mRNA expression levels of pro-inflammatory factors as IFN-γ (1.280±0.232 vs.0.512±0.206,t=4.970,P=0.001),IL-6 (1.364±0.271 vs.0.941±0.215,t=2.452,P=0.040),IL-10 (1.341±0.320vs.0.744±0.294,t=2.762,P=0.025)andTNF-α (3.702±0.150 vs.2.581±0.500,t=2.553,P=0.034) in the experiment group decreased;the expression levels of the intimate tight junction molecules (Claudin3) (1.283±0.152 vs.1.881±0.172,t=4.932,P=0.001) and the atresia protein molecule (Occludin) (1.164±0.342 vs.0.812±0.224,t=3.67,P=0.016) significantly increased.Conclusion Early life LGG colonization protects the intestinal barrier by inhibiting lowgrade intestinal inflammation.This study will lay the experimental foundation for the supplementation of probiotics in early life so as to prevent intestinal diseases.
ABSTRACT
Fish oil is an important nutrient component in rainbow trout bone, and the optimization of extraction by enzymatic hydrolytic method is of great significance.This study selected the alkaline protease as the hydrolytic enzyme, and optimized the process conditions of enzymatic hydrolysis of rainbow trout fish using single factor analysis method.Effects of several factors on the extraction of fish oil were studied, including the ratio of material to liquid, pH, enzymatic hydrolysis time, enzymatic hydrolysis temperature and amount of enzyme.Fatty acids were identified by gas chromatography-mass spectrometry (GC-MS).Results showed that the optimum extraction parameters of enzymatic hydrolysis were as follows: 2000 U/g alkaline protease, ratio of material to liquid of 1∶1 (w/w), pH 7.5, and extraction at 55℃ for 3h.It was found that the main composition of rainbow trout bone oil was unsaturated fatty acid with the content of 80.4% (w/w).The relative content of monounsaturated fatty acid and polyunsaturated fatty acid was about 76.9% (w/w) and 23.1% (w/w), respectively.The total content of EPA and DHA was 3.4% (w/w).This study optimized the extraction method of rainbow trout fish oil, analyzed and identified the main volatile compounds, and identified the main substances contributing to fish oil flavor.The method thus was of significance for the analysis and identification of fish oil products.