The Preparation And Purification Of Gentiobiose

Gentiobiose is a kind of functional oligosaccharides,it promotes the proliferation of probiotic Bifidobacterium and Lactobacillus, improves food flavor, and possesses many other advantages. In this paper gentiobiose was prepared by starch hydrolysis and transglycosylation of glucose catalyzed by ?-glucosidase. Fermentation was conducted to remove monosaccharides. Gel-filtration chromatography was applied to separate gentiobiose from other sugars. Bifidobacterium proliferation was carried out to examine the ability of gentiobiose. The main findings are as follows:Wheat starch was hydrolyzed by hydrochloric acid at varied concentrations to produce gentiobiose. High concentration of hydrochloric acid showed an inhibitory effect on gentiobiose synthesis. However, low concentration of hydrochloric acid was not conducive to the hydrolysis of starch. When hydrolysis temperature was 125 ?, the yields of glucose, isomaltose, gentiobiose, cellobiose and maltose were increased with hydrolysis time. Metal ions such as K+, Ca2+, Na+, and Mg2+ showed stimulatory effects on the synthesis of gentiobiose, the strength of stimulation was K+>Ca2+>Na+>Mg2+.Gentiobiose was also prepared by transglycosylation of glucose catalyzed by ?-glucosidase. Effects of the source of ?-glucosidase, reaction temperature, p H, ?-glucosidase dose, substrate concentration, and reaction time on the synthesis of gentiobiose were investigated. The enzyme preparation of ?-glucosidase synthesized by Nanjing Forestry University was selected as the suitable enzyme source for the synthesis of gentiobiose. The optimal condition for gentiobiose synthesis was as follows. Glucose at a concentration of 900 g/L was added with ?-glucosidase at a dosage of 60 IU/g glucose. The mixture was incubated at p H5, 60? with a shaking speed of 80 r/min. After 24 h, 46.9g/L of gentiobiose was obtained. Anion exchange chromatography was used to quantitatively detection of gentiobiose. Separation and purification of the gentiobiose product was completed by ion chromatography. Gentiobiose of each reaction was separated through membrane separator. After separation, if glucose was reused in next synthesis of gentiobiose, its utilization rate reached 28.5%, 4.48 times of the rate when reuse was not conducted.Nano-filtration was applied to remove salts after hydrochloric acid hydrolysis of starch. Glucose was retained in the sugar solution and was removed by fermentation with Saccharomyces cerevisiae. The fermented solution was nano-filtrated again to separate gentiobiose from other compounds. The retention rates of gentiobiose,glycerol, and ethanol were 79.33 %, 26.53 %, and 7.21 %, respectively. The glucose in starch hydrolysate could be removed by Saccharomyces cerevisiae through fermentation. However, fermentation was not a good method for the removal of glucose presented in reaction liquid of transglycosylation, although gentiobiose is a non-fermented sugar. When the fermentation conditions were relatively suitable, in the process of ethanol production Saccharomyces cerevisiae could produce ?-glucosidase, which could hydrolyze gentiobiose into glucose.Using ?KTA Explorer chromatography system as separation platform, Bio-gel P-2 gel could separate disaccharide from glucose, improving the proportion of isomaltose and gentiobiose in the mixture. After one run elution of starch hydrolysate the content of isomaltose and gentiobiose in the mixture reached 82.1 %; the collected elution samples contained 27.3% gentiobiose. When the reaction liquid of glucose transglycosylation was treated, one run of chromatography was performed, and the portion with high gentiobiose concentration was collected, in which the concentration of gentiobiose reached 61.5%.Cation resin 99Ca/320 chromatography was used to separate gentiobiose as well. When unfermented starch hydrolysate was treated once, in the gentiobiose-rich portion the content of gentiobiose reached 50.8%. After the second run of separation, the content of gentiobiose in the gentiobiose-rich portion increased to 57.5%,with a content of isomaltose and gentiobiose up to 98.8%. When the reaction liquid of glucose transglycosylation was separated, one run treatment resulted in a gentiobiose concentration of 66.7% in the gentiobiose-rich portion of elution, whereas the second run of treatment resulted in an increase of gentiobiose concentration to 97.0%.Both glucose and gentiobiose were effective in anaerobic proliferation of Bifidobacterium adolescentis, even the proliferation effect of glucose was better than that of gentiobiose. Bifidobacterium adolescentis metabolized glucose and gentiobiose to produce lactic acid, acetic acid, and small amounts of propionic acid and butyric acid. The contents of lactic acid and acetic acid were increased with time of cultivation, while the contents of propionic acid and butyric acid were relatively stable. The balance of carbon element during the cultivation was analyzed. When glucose was used as the carbon source, the maximum difference of total carbon was 5.11% compared to the original, indication a close carbon balance during the metabolism process of glucose. Gentiobiose was also used as the carbon source for the proliferation of Bifidobacterium adolescentis. Gentiobiose containing 74.9 mmol/L carbon was metabolized for 36 h. This resulted in organic acids with 62.1 mmol/L carbon and bacterium biomass with 9.56 mmol/L carbon. The concentration of bacterium biomass increased by 3.76 times, indicating that the gentiobiose prepared in this study was an effective inducer for Bifidobacterium adolescentis which is beneficial to human health. In the process of Bifidobacterium adolescentis cultivation with gentiobiose, the maximum difference of total carbon was 11.8 % compared to the original. A close carbon balance was achieved.