Study On The Preparation And Regulatory Mechanisms Of Lotus Seed Oligosaccharides To Probiotics And Pathogens In The Intestinal Tract

In the private sector, lotus seeds are used in the diet. Medical applications include treatment of diarrhea, inflammation, arrhythmia, cancer, and skin diseases; however, they can also be used as an antidote and hepato-protective supplement. In recent years, the potential nutritional and effective characteristics of lotus seeds have gradually been determined, which have also shown to exhibit utility in the context of functional foods, cosmetics, pharmaceuticals, and in other commercial applications.Currently, the prebiotic potential of oligosaccharides on intestinal microbial ecology is focused on several common oligosaccharides, and studies that have attempted to explore the effects of lotus seed oligosaccharides (LOS) on the gut microbiota remain largely unknown and important gaps in current knowledge need to be explored. Our prior studies have shown that oligosaccharides, polysaccharides and starch in the lotus seed have highly effective proliferative effects on bifidobacteria. Therefore, this study has used LOS as a general focus of research activity. Prompt extraction, separation processes, the specific structure of the monomer, and the regulatory mechanisms that modulate intestinal flora in mice on exposure to LOS are comprehensively studied. Growth and inhibitory effects of LOS on probiotics and adhesion of Enterohemorrhage Escherichia coli (EHEC) to the colorectal cell-line Caco-2, were investigated by in vitro experimental models, respectively. Improved ingredients of LOS are further studied for their specific targeted sites of action for both Bifidobacterium and Enterohemorrhage Escherichia coli (EHEC), respectively.Ultrasonic microwave-assisted extraction technology of LOS was optimized by response surface methodology. The results showed that the conditions for optimal ultrasonic microwave-assisted extraction of LOS were:extraction time 325 s, liquid ratio 10 mL/g, ultrasonic power 300 W, and a microwave power of 250 W. Under these conditions, the theoretical yield of LOS, trisaccharides and tetrasaccharides was 10.998%、 3.953% and 4.891%, respectively, which was consistent without any significant differences seen in the context of theoretical predictions.Compared with the traditional hot water extraction method, the yield was increased by 76.59%,17.47% and 27.21% respectively, and the time was shortened by 12.18 times,8.92 times, and 1.16 times according to measurements taken by an ultrasonic and a microwave-assisted extraction method.The optimal conditions for the preparation and separation of LOS by preparative chromatography were as follows:a flow rate of 30 mL/min, an injection volume concentration of 0.2 g/mL, and an injection volume of 1mL. Under these conditions, four types of LOS monomer were separated. The composition ratio for the optimal conditions of the mobile phase of LOS, as determined by thin layer chromatographic development layers were:ethyl acetate/ethanol/water/ammonia (volumetric ratio of 9:11:8:0.9); HPLC analysis results showed that the four prepared LOS monomers had a high degree of purity. Purities were 98.07%,98.18%,99.74% and 92.67%, respectively.In addition, infrared spectroscopic results showed that compound two from LOS contained the β-glycoside bond. Moreover, compounds 3-1,3-2, and compound 4, contained characteristic molecular absorption peaks of mannose, the a-glycoside bond type furanose, and a-D-glucopyranose. The degree of compounds that can be derived from LOS can be determined by MS, and the molecular weight of the lotus oligosaccharides was 342, 504,504 and 666, respectively. Monosaccharide compositional analysis and NMR showed that the a-type glycosidic bond, and the pyran ring were the preferred conformation of LOS. In addition, four monomers from LOS were separated, one of which was lactose, and the remaining chemical structure for compound 3-1 was Man-α-1�'6-Glu-α-1�'2-α-Fru, and the structure for the compound 3-2 was Man-α-1�'6-Man-α-1�'6-Glu-α/β, and for compound 4, the structure was Man-α-1�'6-Man-α-1�'6-Glu-α-1�'2-α-Fru. In addition, mannitol was also included in the LOS extraction.Prebiotic fructo-oligosaccharides are added as a positive control. Regulation of the intestinal flora by LOS was studied. The results showed that weight gain was promoted in mice by LOS. Simultaneously, LOS displayed no abnormal effects on any of the blood indices in mice. Moreover, droppings and intestinal contents of the mice at different feeding periods were collected and analyzed by DGGE with the specific aim of determining the advantage that colonies display when constituting the intestinal and fecal flora. Middle dose of LOS can effectively alter the constitution of microbial colonies in both intestinal and fecal flora of mice. Additionally, the high dose of LOS displayed similar effects to that seen with fructooligosaccharides. The altered relative number of murine fecal bacteria was determined by quantitative PCR, which showed that the number of microorganisms of the genus Bifidobacterium, Lactobacillus, Bacteroides, and Butyrivibrio were significantly improved in murine feces. By contrast, the colonization by Clostridium and Enterobacteriaceae were reduced in the intestine. Acetic acid, propionic acid, butyric acid, and isovaleric acid were significantly increased in the feces and intestinal contents of the mouse (p<0.05).According to the test results completed in mice, and due to the good growth results between LOS and bifidobacteria or lactobacilli, each monomer of LOS and probiotic strain were fermented in vitro to investigate the role of the monomer of LOS in the context of probiotic strains. Carbon was not added as a control or added with FOS as a positive control. Results for each monomer that was fermented showed that Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, and B. bifidum all effectively utilized Glc as a carbon source, from which, acetic acid, and lactic acid were produced after metabolism. Moreover, Lactobacillus acidophilus effectively used LOS3-1 to proliferate, and large amounts of acetic and lactic acid were produced after metabolism. Both Bifidobacterium adolescentis, and Bifidobacterium longum were effectively used by LOS4 to proliferate, following which, acetic, lactic, and butyric acid were significantly higher than other carbon sources after metabolism (p<0.05).The hydroxy labeling method was used by FITC, which was a successfully formed fluorescent oligosaccharide product as shown by LOS3-1-FITC, LOS3-2-FITC and LOS4-FITC. Results of UV-visible absorption spectra scans showed that the maximum absorption wavelength of LOS3-1-FITC, LOS3-2-FITC and LOS4-FITC was 455 nm. Excitation and emission spectra of fluorescence showed that the solution fluorescence effect was good with a concentration of more than 0.012 μmol/mL for LOS3-1-FITC, LOS3-2-FITC and LOS4-FITC where the optimum excitation wavelength was 490 nm and the fluorescence maximum emission wavelength was 518 nm. LOS4 and Bifidobacterium adolescentis are used as test subjects based on results of p between LOS4 and Bifidobacterium adolescentis were located both outside and inside of bacterial cells. Single bacteria of Bifidobacterium adolescentis fermented LOS4 in one cycle and did so within one hour.robiotics fermentation characteristics in vitro, which are further studied on sites of action between LOS4 and Bifidobacterium adolescentis. Laser confocal microscopy indicated that the action siteEHEC was labeled with the fluorescent dye FITC. Caco-2 cells were cultured in differentiation side brush border microvilli, which also displayed the cell polarity associated with intestinal epithelial cells that simulated intestinal epithelial cell adhesion state. Apoptosis of normal Caco-2 cells occurred after infection by EHEC in the absence of an exogenous interference. Effects of interference suppression under exogenous carbohydrate on adherence of EHEC to Caco-2 cells were LOS4>MOS>Man>LOS3-2> LOS3-1. Carbohydrate also somewhat influenced the bacterial cell membrane after adhesion was evident between carbohydrate and the cell membrane.LOS4 significantly reduced r, which is membrane fluorescence anisotropy value, which indicated that LOS4 may display greater binding sites with the surface receptor of EHEC. LOS4 can also significantly inhibit mRNA expression of Lrp and Fim as cell surface receptors of EHEC. However, these did not display any significant difference with MOS (p>0.05).The results of laser confocal microscopy showed that sites between LOS4 and EHEC were located to the bacterial cell surface. Many surface adhesion sites had not shown accelerated accumulation until 12 h. Binding sites were located at the adhesion surface side of EHEC. In addition, binding sites were located to the EHEC adhesion surface side. Finally, LOS4 did not penetrate the interior of EHEC.