Grading Alcohol Precipitation Of Resistant Starch Derived From Lotus Seed, And Its Probiotics Effects

Resistant starch (RS) which escapes digestion in the small intestine could be completely or partially fermented in the colon, and has many beneficial effects for human health. It was proved that eating a diet rich in RS may help people lose weight and solve the problem of obesity. Food containing RS could lower serum glucose and insulin levels, and might play a role in controlling type Ⅱ diabetes. The metabolic product of RS may also inhibit cholesterin synthesis, affect lipid metabolism, and help people prevent intestinal diseases by promoting the growth of beneficial microorganisms. Our previous study showed that the molar masses of Lotus Seed Resistant Starch(GP-LRSS) which prepared by autoclaving method were distributed mainly in the range of 1.0×104-1.0×107 g/mol. GP-LRS3 had the rough surface structure, B-type crystal structure, and high degree of double helix structure. GP-LRS3 could promote Bifidobacterium proliferation and could be fermented by Bifidobacterium to produce higher content of butyric which would be beneficial to intestinal environment. While the in vivo experiment is needed to understand the prebiotics effects of GP-LRS3, and the research on prebiotics effects of different molar masses of Lotus Seed Resistant Starch is required. Therefore BALB/c mice were fed diet containing GP-LRS3 in this research. The in vivo benefits of GP-LRS3 were assessed from the content of short-chain fatty acid in the colon and the growth of flora fecal, in order to verify the conclusion of our prior study. Ethanol was used to treat GP-LRS3 by Stepwise Ethanol Precipitation to get different molecular weight distribution of GP-LRS3 fractions. The optimal GP-LRS3 fraction which could promote beneficial bacterium proliferation was obtained by in vitro experiment.High amylose maize starch (HAMS) was added as the positive control. The effects of GP-LRS3 on the intestinal flora of mice and the mechanism of this action were studied. The results showed that GP-LRS3 displayed no abnormal effects on the growth of mice. Middle and high dose of GP-LRS3 could control the weight of mice, lower the feed convertion efficiency, and enhance the apparent absorption of minerals. GP-LRS3 could effectively alter the constitution of microbial colonies in intestinal of mice, reduce the number of the pathogenic bacterium such as Porphyromonadaceae Helicobacter、Rikenellaceae and Clostridium, and increase the number of bacterium such as Butyrivibrio、Stomaiobaculum which will produce short-chain fatty acids. Middle and high dose of GP-LRS3 could significantly increase the content of acetic acid, propionic acid, butyric acid, isobutyric acid and lactic acid. The number of murine fecal bacteria indicated that GP-LRS3 could obviously promote the proliferation of Lactobacillus and Bifidobacterium than HAMS. This study verified our prior research results, and illustrated that GP-LRS3 could promote the proliferation of the beneficial bacterium and enhance the content of short-chain fatty acids, especially butyric acid, which could improve the body health.The conditions for Stepwise Ethanol Precipitation of GP-LRS3 were as follows:3% concentrations of lotus seed starch,100℃ of autoclaving temperature,15 minutes of autoclaving time,20% and 30% of ethanol concentrations. Under these conditions two fractions of GP-LRS3 were separated including GP-LRS3-1 and GP-LRS3-2. The molecular characteristics of GP-LRS3-1 and GP-LRS3-2 showed that the Mw values of GP-LRS3-1 and GP-LRS3-2 were 8.49 X 104 and 2.23 X 104 g/mol, and the Mw/Mn values of GP-LRS3-1 and GP-LRS3-2 were 2.28 and 1.56. Over 50% molar masses of GP-LRS3-1 were distributed mainly in the range of 1.0×04-2.0×104, and the molar masses of GP-LRS3-2 were distributed mainly in the range of 2.0×104×-4.0×104 g/mol, accounted for 73.10% of total GP-LRS3. GP-LRS3-1 and GP-LRS3-2 displayed a highly branched and uniform spherical conformation in DMSO. TEM results demonstrated that GP-LRS3-1 and GP-LRS3-2 had rough surface structure.According to the results of animal experiments, GP-LRS3 increased the number of Bifidobacterium and Lactobacillus, therefore the proliferation effects in vitro of each fraction of GP-LRS3 were studied. Glucose (GLU) and high amylose maize starch (HAMS) were added as blank control and positive control, respectively. The effects of GP-LRS3, GP-LRS3-1 and GP-LRS3-2 in the context of different Bifidobacterium were investigated. The results showed that the stimulative effect of GP-LRS3 and GP-LRS3-1 on Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium adolescenti were superior to that of 30% GP-LRS3, GLU and HAMS. Lotus Seed Resistant Starch could be well used by Bifidobacterium to produce large amount of acetic acid and butyric acid after metabolism. Furthermore GP-LRS3 could enhance the tolerance of Bifidobacterium to the lower pH and to the high concentration of bile salt environment. TEM results indicated that the surface structure of GP-LRS3-1 and GP-LRS3-2 were changed a lot after being fermented. GP-LRS3-1 and GP-LRS3-2 were observed with rougher surface and platy structure.Glucose (GLU) and high amylose maize starch (HAMS) were added as blank control and positive control, the role of GP-LRS3, GP-LRS3-1 and GP-LRS3-2 in the context of different Lactobacillus was investigated. The results showed that the stimulative effect of GP-LRS3 on Lactobacillus acidophilus and Lactobacillus delbrueckii subsp.bulgaricuswas superior to that of other groups. Lotus Seed Resistant Starch could be used by Bifidobacterium to produce large amount of acetic acid and lactic acid metabolism. Under pH 1.5, Lactobacillus acidophilus could well survive in GP-LRS3, GP-LRS3-1, HAMS and GP-LRS3-2 medium, but Lactobacillus delbrueckii subsp.bulgaricus only survived in GP-LRS3 medium. GP-LRS3 could enhance the tolerance of Lactobacillus to the high concentration of bile salt environment. TEM results demonstrated that the surface structure of GP-LRS3-1 and GP-LRS3-2 after being fermented by Lactobacillus had rougher surface and platy structure, similar with the structure of GP-LRS3-1 and GP-LRS3-2 after being fermented by Bifidobacterium.