Study On The Extraction, Enzymatic Synthesis Technology Ofα-galactooligosaccharides And Its Prebiotic Effect

Oligoaccharides with a-galactosidic linkages, a-linked galactooligosacchrides (a-GOS), belonging to low molecular weight, of non-reducing sugars, are widely distributed in the plant kingdom. Due to the lack of a-galactosidase in the upper intestinal tract of human, a-GOS could avoid digestion and reach the colon as substrates for luminal fermentation by the microbial flora, it could stimulate the growth of Bifidobacterium bifidum. a-GOS have many other physiological fructions, such as dominating pathogen organisms, preventing constipation and diarrhea, protecting liver, decreasing blood pressure, reducing blood lipid, enhancing immune, promoting the absorption of nutrition and Ca+, prevention and treatment of lactose dyspepsia, improving lipid metabolism and anticancer activities. Therefore, it has been paid much attention to the production, especially its enzymatic synthesis methods. a-GOS will be widely used in food, pharmaceutical, feed and brewing industries. In this article, we studied the production of a-GOS by extraction from legumes and synthesized from galactose by reverse reaction of a-galactosidase DS from Aspergillus niger.Firstly, the compositions of free sugars in ten kinds of common legume seeds were investigated through high performance liquid chromatography. A Sugar-D column was used with acetonitrile-water solution (75:25, v/v) as the mobile phase at a flow rate of1mL/min, and a refractive index detector was used for the detection of sugars. Under such conditions, it ensured good separation for the sugars with stable baseline. The results showed that oligosaccharides in all10legume seeds belonged to a-GOS, consisting of sucrose, maltose, raffinose, ciceritol, stachyose and an unknown pentasaccharides, which was the main sugar in faba beans and mung beans, was isolated and purified by column chromatography, and identified as verbascose by means of1H-NMR and ESI-TOF-MS. Discriminant analysis was applied in order to differentiate samples with different amounts of sugars. The results showed that there were considerable variations in the levels of sucrose between cultivars, which ranged from0.24%～8.03%. Substantial differences in a-GOS contents were also detected, which ranged from4.01%～9.81%. The contents of raffinose, stachyose and verbascose were within the range of0.41～2.18%,0.25～4.67%, and0.38～3.32%, respectively. Ciceritol could only be detected in chickpeas and was the main sugar in the chickpea sample, which accounted about51%of the total a-GOS. Verbascose could be detected in almost all the legumes except for white beans and black beans by HPLC.With high amount of a-GOS and lower amount of sucrose, chickpea could be a better choice for obtaining a-GOS for use as prebiotics in functional foods.Secondly, we sysnthesized a-linked galactooligosacchrides (a-GOS) from galactose by reverse reaction of a-galactosidase DS from Aspergillus niger. The a-galactosidase used could effectively catalyze the reverse reaction and4products were detected in the reaction mixture by HPLC. Based on the retention times of standard sugars, there are2kinds of α-galactobiose(a-Gal2), which could be positional isomers, and2kinds of α-galactotriose(α-Gal3). Two of the products, α-Gal2and α-Gal3were isolated and purified by column chromatography separately, and identified as α-(1â†'6)Gal2and α-(1â†'6)Gal3by means of’H-NMR,13C-NMR and ESI-TOF-MS. Then we also investigated the effects of different factors (substrate concentration, enzyme concentration, reaction pHs and temperatures) on the formation of α-GOS. The result showed that the most important factor in increasing the yield of reverse reaction products is the substrate concentration.α-galactosidase DS was not inhibited even in high substrate concentration, and effectively catalyzed the reverse reaction. The yield of α-GOS increased greatly as the initial concentration of galactose increased to90%(w/v). Under the optimum conditions, the yield of α-GOS reached about30%, including79%α-Gal2and21%α-Gal3.Thirdly, florescence in situ hybridization(FISH) was used to analyze the amount and spatial distribution of bacteria in the colon,16S rRNA specific probe used with DAPI total cell staining and dilute technology, FISH can in situ detect the amount and spatial distribution of bacteria in the colon efficiently. The substrates were incubated in24h batch culture fermentations of human faecal bacteria. FISH was used to determine changes in populations of bifidobacteria, lactobacilli, clostridia, bacteroides and total bacteria. Short-chain fatty acids (SCFAs) production were also measured by HPLC. a-GOS gave a significant increases in bifidobacterium, lactobacillus and total bacterial numbers, a limited number of Clostridium histolyticum and bacteroides during the incubations. However, galactose appeared to be a more selective prebiotic as it did not significantly stimulate growth of bacterial groups which were not probiotic in nature. a-GOS produced the highest levels of SCFAs. Thus, a-GOS might be useful as a new physiologically functional food material. At the same time, FISH as a new technology, has been widely used in the study of determine the short and the amount of bacterium.