Enzymatic Synthesis Non-Digestive Oligosaccharides And Evaluation Its Physiological Function

Non-digestive oligosaccharides are different with ordinary oligosaccharides, which belong to prebiotic as a class of function components. Discovery of novel non-digestive oligosaccharides is interesting issue in the world of prebiotic. With the development of enzyme technology, this question would be resolved gradually. Because structure of non-digestive oligosaccharides is special and its isolation and purification is very difficulty. In the past, prebiotic properties were evaluated with commercial oligosaccharide products or the products with simple isolation. It can not confirm which one definite oligosaccharide in the mixture plays the most important in the anerobe fermention. Also it can not confirm structure of oligosaccharides for prebiotic properties and inhibition ability to cancer cells. In previous research, it has been observed that chemical structures of oligosaccharides (the number or type of saccharidemoieties and the position and conformation of links between the saccharides) are important in determining the prebiotic properties of oligosaccharides, especially the selectivity of fermentation and digestibility in the colon. Therefore, there is the important for understanding of specific structure-function relationship of prebiotics. This paper include five chapter, emphasizing on enzymatic synthesis non-digestive oligosaccharides withβ-D-galactosidases from different origin; isolation, purification and structure characterization of pure components non-digestive oligosaccharides; optimization of synthetic conditions of non-digestive oligosaccharides; study on prebiotic properties with different structure of pure components non-digestive oligosaccharides; study on inhibition ability to human gastric cancer BGC-823 cells with different structure of pure components non-digestive oligosaccharides, respectively. Main results are listed as follows:1. Enzymatic synthesis non-digestive oligosaccharides withβ-D-galactosidases from different originIn the present study,β-D-galactosidase from Bacillus circulans, Penicillum multicolor and Aspergillus oryzae were used to biocatalyst with lactose, lactose and sucrose, lactose and N-acetylglucosamine as substrates, respectively. The reaction mixture was followed by high performance liquid chromatography focus on many kinds of transfer products at different reaction condition with different substrates. Transfer products was isolated using high pressure-, medium pressure- and normal-pressure liquid chromatography combined with charcoal and Celite column and gel exclusion column. Then nine kinds of oligosaccharides were obtained. This oligosaccharides and a kind of commercial FOS mixture named QHT-90S were identified by by UV, ESI-TOF-MS,1H-NMR and 13C-NMR.β-D-Galactosidase from B. circulans was proved to be suitable biocatalyst for major oligosaccharides withβ-(1â†'4) linkage and minor products withβ-(1â†'3) orβ-(1â†'6) linkages. Degree of polymerization (DP) of the transfer products was 2 to 4. Three oligosaccharides ofβ-D-Galp-(1â†'4)-β-D-Galp-(1â†'4)-α-D-Glcp-(1â†'2)-β-D-Fruf,β-D-Galp-(1â†'4)-β-D-Galp-(1â†'4)-β-D-Glcp-(1â†'2)-β-DD-GlcpNAc andβ-D-Galp-(1â†'4)-β-D-Galp-(1â†'4)-D-GlcpNAc was not reported in the past and named firstly.β-D-Galactosidase from P. multicolor was proved to be suitable biocatalyst for major oligosaccharides withβ-(1â†'6) linkage and minor products withβ-(1â†'4). DP of the transfer products was 3 to 4.2. Optimization of enzymatic synthetic conditions of non-digestive oligosaccharidesIn this paper, the effects of synthetic conditions including reaction temperature, concentration of substrate, molar ratio of donor/acceptor and enzyme concentration on the formation of different pure components of GOS, lactosucrose and its analogues, N-acetyllactosamine and its derivates were examined. We found that the major reaction conditions were temperature, concentration of substrate, molar ratio of donor/acceptor, whereas enzyme concentration was secondary condition.The optimal reaction conditions for all kinds of pure components was listed as fllowed:(1) 4’-GOS3:40℃, pH 6.0,60% lactose concentration,1.0 U/mL enzyme concentration (derived from B. circulans) after 6 h; (2) 4’-GOS4:40℃, pH 6.0,60% lactose concentration,1.0 U/mL enzyme concentration (derived from B. circulans) after 8 h; (3) 6’-GOS3:50℃, pH 5.0,70% lactose concentration,0.3 U/mL enzyme concentration (derived from P. multicolor) after 6 h; (4) 6’-GOS4:50℃, pH 5.0,70% lactose concentration,0.3 U/mL enzyme concentration (derived from P. multicolor) after 8 h; (5) lactosucrose and 4’-galactosyl-lactosucrose:40℃, pH 6.0,60% total carbohydrate concentration (lactose,30%; sucrose,30%); 1.0 U/mL enzyme concentration (derived from B. circulans) after 6 h; (6) allo-lactosucrose:50℃, pH 6.0,60% total carbohydrate concentration (lactose,30%; sucrose,30%); 1.0 U/mL enzyme concentration (derived from B. circulans) after 12 h; (7) LacNAc, 4’-galactosyl-LacNAc and 4’-digalactosyl-LacNAc:40℃, pH 6.0,1.0 M total carbohydrate concentration (lactose,0.5 M; GlcNAc,30%); 1.0 U/mL enzyme concentration (derived from B. circulans) after 4,5 and 6 h of reaction time, respectively; (8) allo-LacNAc:60℃, pH 6.0,1.0 M total carbohydrate concentration (lactose,0.5 M; GlcNAc,30%); 1.0 U/mL enzyme concentration (derived from B. circulans) after 12 h.3. Study on prebiotic properties with different structure of pure components non-digestive oligosaccharidesIn this paper, the different pure components of GOS, FOS, lactosucrose and its analogues with different glycosidic bonds and DP was studied by anaerobe faecal fermentation in vitro. Total bacterial population, beneficial bacterium and harmful bacterium were enumerated by fluorescent in situ hybridization (FISH). Prebiotic properties were evaluated by prebiotic index (PI). In addition, their effects on production of SCFA and lactic acid were also investigated aiming to understanding of specific structure-prebiotic properties relationship of these oligosaccharides.With series of GOS, FOS and two ordinary oligosaccharides (lactose and sucrose) as substrates in vitro anaerobe fermentation, it was found that (galactosyl)n-glucose (GOS, n=1-3) presented higher PI than glucose-(fructosyl)n. (FOS, n=1-3). For Bifidobacterium spp., series of GOS would appear to be more selective than others, corresponding to high amounts of acetic and lactic acids and PI, A comparison among linkages showed higher PI values for GOS withβ-(1â†'6) linkages (6’-GOS3) than forβ-(1â†'4) linkages. For Lactobacillus/Enterococcus spp., series of FOS would appear to be more selective than others. Butyric acid production from series of GOS fermentation were also higher than FOS, butyric acid was also confirmed ability to reduce the risk of developing gastrointestinal cancer. PI was influenced by DP of substrates. For trisaccharides, the PI values obtained at 12 h of incubation were higher than those with tetra-, and penta-oligosaccharides at 24 h. The DP of oligosaccharides was high, the prebiotic properties maintained more time. Apart from two digestible disaccharides (lactose and sucrose), which showed significant change in Bacteroides-Prevotella group and Clostridium histolyticum group (clustersâ… andâ…¡) population, the rest of the oligosaccharides did not present significant differences during the whole process compared with the no treatment control.With a kind of GOS (4’-GOS3), raffinose and lactosucrose and its analogues as substrates in vitro anaerobe fermentation, it was found that trisaccharide with structure formula of (galactosyl)2-glucose (4’-GOS3) presented higher PI than lactosucrose with structure formula of galactosyl-glucosyl-fructose. Meanwhile, the increase of Bifidobacterium spp. and Lactobacillus/Enterococcus spp. as well as the population fractions of these genera compared to the total bacteria counted in overall fermentation time. However, in oligosaccharide with structure of (galactosyl)2-glucose, ifβ-(1â†'4) linkages between two galactosyl residue was changed toβ-(1â†'3) linkages, PI of oligosaccharide will changed notable. In overall fermentation time, the PI of allo-lactosucrose was higher than PI of 4’-GOS3, corresponding to the highest amount of total acids. Raffinose withα-glycosidic bond would raise Clostridium histolyticum group (clustersâ… andâ…¡) population, this is disadvantage of PI elevation. Also the lowest levels of butyric accumulated in the presence of raffinose. As mentioned above, PI was influenced by DP of substrates.4. Study on inhibition ability to human gastric cancer BGC-823 cells with different structure of pure components non-digestive oligosaccharidesIn this paper, proliferation inhibition of human gastric cancer BGC-823 cells (tumor inhibition rate,%) was studied in vitro with the different pure components of GOS, FOS, lactosucrose and its analogues with different glycosidic bonds and DP. This study will provide a basis for the study on the inhibition effects of different pure oligosaccharides for BGC-823 cells. In some extents, this information will explain oligosaccharides’ structure in influence of biologic activity gastric cancer BGC-823 cells.Results demonstrate all the non-digestive oligosaccharides at certain concentration have the inhibition to growth of BGC-823, with time and dose dependent. With increasing of concentration, tumor inhibition rate (%) was increasing. At same concentration, with prolongation of reaction time, tumor inhibition rate (%) was increased. Comparison with tumor inhibition rate (%) of oligosaccharides at the lowest concentrate (10 mg/mL), results indicate tumor inhibition rate (%) of allo-lactosucrose withβ-(1â†'3) linkage higher than lactosucrose,4’-GOS3 and 4’-GOS4 withβ-(1â†'3) linkages. Series of FOS withβ-(1â†'2) linkage obtained higher tumor inhibition rate (%); however, two kinds of GOS (6’-GOS3 and 6’-GOS4) withβ-(1â†'6) linkage obtained lower tumor inhibition rate (%) in all experiments. There is no significant difference between the trisaccharide and tetrasaccharide of same kind of oligosaccharides. In conclusion, tumor inhibition rate (%) is significant difference among different kinds of oligosaccharides. Oligosaccharides structure is crucial reason for tumor inhibition rate, including kinds of glycosidic bonds, saccharides composition etc., however, DP of oligosaccharide was no significant difference for tumor inhibition rate (%).