Study On The Production Of α-Galactooligosaccharides

α-Galactooligosacchrides(α-GOS),belonging to low molecular weight,of non-reducing sugars,are widely distributed in the plant kingdom.An especially large amount of these saccharides occurs in generative parts of plants,where they perform protective physiological functions.Due to the lack ofα-galactosidase in the upper intestinal tract of human,sugars withα-galactosidic linkages could avoid digestion,and reach the colon as substrates for luminal fermentation by the microbial flora.As a result,α-galactosides could promote the growth of Bifidobacteria.These characteristics allowα-GOS to be considered as prebiotics.Consequently,it has aroused interests in usingα-GOS as ingredients in functional foods in order to adjust the composition of colonic microflora,contributing to human health in many ways.In this article,we studied the production ofα-GOS by extraction from legumes and synthesized from galactose by reverse reaction ofα-galactosidase DS from Aspergillus niger.Firstly,the compositions of free sugars in soybean seeds and chickpea 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 of 1 mL/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.Through the analysis,we found that soybean seeds contained fructose,sucrose,melibiose,raffinose,stachyose,and two unknown substances,while chickpea seeds contained ribose,fructose,sucrose,maltose, raffinose,stachyose,verbasecose,and two unknown substances.An unknown trisaccharide, which was the main sugar in chickpea seeds,was isolated and purified by column chromatography,and identified as ciceritol by means of ¹H-NMR and ESI-TOF-MS.With high amount ofα-GOS and lower amount of sucrose,chickpea could be a better choice for obtainingα-GOS for use as prebiotics in functional foods.Secondly,the extraction procedure of oligosaccharides from chickpea seeds was optimized and sufficient extraction was achieved with 50%of ethanol-water in a ratio of 10:1 for solvent to defatted chickpea meal at 50℃for 30 min.Using the optimal extraction conditions,the contents ofα-GOS and sucrose in 19 cultivars of chickpea seeds were determined with the aim of selecting those with high amounts ofα-GOS and low contents of sucrose for use as ingredients in functional foods.Statistic analysis showed that there were significant variations in the levels of sucrose between species,ranging from 1.80%-5.22%.Substantial differences inα-GOS contents were also detected,especially in the verbascose content which could only be detected in 7 samples.The contents of raffinose, ciceritol,stachyose and verbascose ranged from 0.46%-0.92%,3.04%-5.06%, 1.64%-3.09%,and 0.27%-0.70%,respectively.With the highest amount ofα-GOS and a low amount of sucrose,the 171 chickpea seeds was the best choice for obtainingα-GOS as prebiotics in functional foods.Thirdly,we sysnthesizedα-linked galactooligosacchrides(α-GOS) from galactose by reverse reaction ofα-galactosidase DS from Aspergillus niger.Theα-galactosidase used could effectively catalyze the reverse reaction and 4 products were detected in the reaction mixture by HPLC.Based on the retention times of standard sugars,there are 2 kinds ofα-galactobiose(α-Gal2),which could be positional isomers,and 2 kinds ofα-galactotriose(α-Gal3).One of the main products,α-Gal2 was isolated and purified by column chromatography,and identified asα-(1→6)Gal2 by means of ¹H-NMR,¹³C-NMP and ESI-TOF-MS.Then we also investigated the effects of different factors(substrate concentration,enzyme concentration,reaction pHs and temperatures) on the formation ofα-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 ofα-GOS increased greatly as the initial concentration of galactose increased to 90%(w/v). Under the optimum conditions,the yield ofα-GOS reached about 30%,including 79%α-Gal2 and 21%α-Gal3.