Agaro Oligosaccharides Prebiotics Effect Andthe Research Of Its Effect On Intestinal Flora

Agar is a kind of water-soluble polysaccharide,which is widely used in food,medicine and daily chemical industries.In food,agar can be used as food gelling agent,stabilizer andthickener,and thus increase the viscosity of food and make it tough and smoothness.In medicine,agar is mainly used as microbial culture medium.Agaro oligosaccharides,as a product of agar,have excellent water solubility and antioxidant activity which were beneficial to the absorption and immunity by human body.In this dissertation,we mainly focus on the research of agaro oligosaccharides and explored its effect on prebiotics as well as intestinal flora.Agaro oligosaccharides in different polymerization degree which prepared through agar enzyme enzymolysis and hydrochloric acidhydrolysis were used to explore their difference in antioxidant and prebiotics effects.Then,the oligosaccharide with best probiotic effect was seleted to do feeding tests and vitro fermentation experiments.The first step was to distinguish different bacterial flora through plate colony-counting methods.Then the agaro oligosaccharides were added to screen those bacteria which can grow well.Through 16s rRNA analysis,strain could be identified by constructing a phylogeny tree.First of all,different degree of polymerization of agaro oligosaccharides were prepared by controlling the enzyme hydrolysis time or hydrochloric acid hydrolysis time.And sugar substructure of the enzyme and acid prepared oligosaccharides were tested by mass spectrometry.Based on the different structures,antioxidant capacity of the oligosaccharides was investigated.The results showed that enzyme oligosaccharides and acid oligosaccharides had certain antioxidant capacity.Among them,the best reducing ability was DP5 enzymatic oligosaccharide and DP3 acid oligosaccharide.The best scavenging ability of DPPH free radical was DP5oligosaccharide.The best scavenging ability of ABTS⁺free radical was DP3 oligosaccharide,and the clearance rate was above 99%in the concentration range of 15?35 mg/mL.The scavenging capacity of DP8 acid oligosaccharide on·OH and·O₂^-were higher than that of the other oligosaccharides,and the clearance rate were 38.79%and 44.3%at the concentration of 35mg/m L,respectively.Secondly,on the basis of agaro oligosaccharide biological activity determination,the effect of different DP of acid and enzymatic agaro oligosaccharides on the growth of Lactobacillus,Streptococcus,Bacillus subtilis,and E.coli were studied.The study found that the enzymatic agaro oligosaccharides could promote the growth of Lactobacillus and Streptococcus but acid agaro oligosaccharides could promote the growth of bacillus subtilis.DP3 enzymatic agaro oligosaccharides had the best growth probiotic effect,and thus they were used in the feeding experiment.Compared with the group indexes of body length,body weight growth rate,feed conversion rate,specific growth rate,DP3 oligosaccharides had a greater promotion effect on dragon crayfish growth than the control group without sugar additon.The gas production,pH value and NH₃-N concentration were measured by fermentation in vitro.Finally,the prawn intestinal contents was inoculated on the selection medium.The total number of aerobic bacteria mainly including Escherichia coli,Vibrio parahaemolyticus,and a few opportunistic pathogen Staphylococcus aureus.Anaerobic bacteria were mainly Lactobacillus,Bifidobacterium,and a small amount of Clostridium perfringens.Then,agaro oligosaccharides were added into selective culture-medium.Compared with original culture medium,it was found that there were significant changes in the number and morphology of the colonies on the agar medium.The growth promoting strain 11,31,41,61 were selected from the plates for 16s rRNA gene sequence analysis and phylogenetic tree was constructed.The preliminary determination of strain 11 was Vibrio parahaemolyticus,strain 31 Vibrio alginolyticus,strain 41 Vibrio alginolyticus and strain 61 Vibrio parahaemolyticus.After that,the effect of agaro oligosaccharide on the growth of bacteria were deteced.Under the condition of oligosaccharide addition,OD₆₀₀ of strain 11 and strain 31 increased 15.2%and 19.5%respectively comparing to control group.The results indicated that agaro oligosaccharides had significant growth-promoting effect on strain 11 and strain 31.