| Lotus seed oligosaccharides(LOS)are natural carbohydrate with low degree of polymerization isolated from lotus(Nelumbo nucifera Gaertn.)seeds.In our previous study,3 kinds of oligosaccharides(LOS3-1,LOS3-2 and LOS4)were separated by medium-pressure liquid chromatography(MPLC)via the conditional optimization and structural characterization.The in vitro probiotics test results have shown that LOS exerted potential prebiotic properties due to their proliferation promotion in Bifidobacterium and Lactobacillus and increased the production of short-chain fatty acids.In order to further research the prebiotic effects and intestinal regulation of LOS,the effects of LOS on the intestinal flora structure and metabolites in mice during the growth process and its mechanism were investigated.The theoretical basis for the application of LOS and the lotus functional research fields were provided in this work.(1)Comparative study on the fermentation characteristics of LOS by the intestinal flora in vitroThe effects of LOS on the number of specific flora and metabolites in fermentation broth were compared by using in vitro fermentation method,and the prebiotic effects of LOS were further evaluated by probiotics index.The results showed that the carbohydrate-protein fermentation balance was affected by the supplementation of LOS,which resulted in increasing on the production of short-chain fatty acids(acetic acid and butyric acid)and decreasing in the amount of ammonia content.Both of them also contributed to a significantly lower the pH of fermentation broth.The PI value of each carbon source decreased with the extension of fermentation time.However,the number of Bifidobacterium and Lactobacillus in the fermentation broth with LOS3-2 and LOS4 could more significantly increased at 12 h(p<0.05).The number of Bacteroides and Escherichia coli in broth decreased at 24 h.The PI value of the carbon source with LOS3-2 and LOS4 was significantly higher than glucose(Glc)group at 12 h(p<0.05).The probiotic effect of different structures of oligosaccharides showed a certain difference such as LOS3-2 and LOS4 were effective at increasing prebiotic index and conducive to carbohydrate-protein fermentation balance.(2)Effect of LOS on nutritional status and metabolites in miceThe effects of LOS3-1,LOS3-2 and LOS4 on the nutritional status and the carbohydrate-protein fermentation balance of Balb/C mice were studied.The results showed that the daily food intake,body weight gain and feed efficiency could significantly increase by LOS4 treated.There was no significant effect of oligosaccharides on intestinal tissue weight except LOS4 could increase the cecum wall weight(p<0.05).The content weight of caecum and colon was significantly increased in LOS4 group(p<0.05).The content of acete,propionic,butyrate in the feces of the mice during the intragastric administration could be increased by feeding LOS,resulting in an improvement of mineral apparent absorption and water holding capacity of the feces;while the isobutyric acid,isovaleric acid and ammonia content were relatively decreased,which indicated that LOS exert favorable impact on the carbohydrate-protein fermentation balance.Meanwhile,LOS4 showed stronger acid production and inhibition of protein fermentation.(3)Effect of LOS on intestinal morphology and gut satiety hormone secretion in miceTo illustrate the relationship between feeding,weight gain,mineral absorption and various LOS,some appetite-related hormones,including serum insulin,petide-YY(PYY)and glucagon-like peptide-1(GLP-1)under fasting state or acute treatment were measured,the glucose level and intestinal morphology evaluated as well.The change in serum glucose following 21 days ingestion was not statistically significant between groups(p>0.05).Compared with control group,LOS3-2 and LOS4 significantly decreased the level of serum PYY and decreased the level of insulin and GLP-1 in some extent.After acute treatment at 30 min.LOS4 significantly lower PYY and GLP-1 level,with 25.9%and 25.3%reducing above basal levels,respectively.The acute feeding results further confirmed that LOS had lowing effects on PYY and GLP-1 levels.The intestinal morphology results showed that dietary supplementation of LOS could decrease the myenteron thickness and crypt depth of ileum,while increasing the villus height and V/C value(p<0.05).Those suggested that dietary supplementation with LOS could improve the morphological structure of intestinal mucosa and enhance the ability of absorbing nutrients.LOS4 could increase the myenteron thickness of cecum and colon,which caused an increasing in weight of the intestinal wall.Meanwhile,the cecal and colonic crypt depth were greater in LOS group may be due to the increase of short-chain fatty acid generation and stimulation of cell proliferation.(4)Metagenomic insights into the regulatory effects of LOS on the composition of fecal microbiota in miceThe high throughput 16S rDNA metagenomic sequencing techniques were used to investigate the effects of LOS on the composition of fecal flora in male Balb/C mice.The results showed that LOS exerted regulatory effect with a certain structure-activity relationship between the different monomer structures.At the phylum level,both LOS3-1 and LOS4 significantly the elevated abundance of Bacteroidetes and Actinobacteria,decreased the abundance of Firmicutes and F/B value.At the genus level,LOS3-1 significantly increased the relative abundance of Alloprevotella,Barnesiella and Bifidobacterium and Lactobacillus.LOS3-2 promoted the abundance of Allobaculum,Alloprevotella,Butyrivibrio and Natronoflexus.LOS4 significantly raised the abundance of Bifidobacterium,Butyrivibrio,Lactobacillus and Natronoflexus.All kinds of LOS showed obvious inhibitory effect on Eisenbergiella,Oscillibacter and Peptococcus.The regulation effect of different LOS on the microbiota suggested that a single oligosaccharide composition can be utilized by multiple bacteria groups and promoted its growth.However,other flora may be inhibited by the mechanism of competition or metabolite antagonism. |
