Study On The Extraction, Purification And Prebiotic Activity Of Rapeseed Polysaccharides

In this paper, extraction, purification, structure and prebiotic activity of polysaccharidesfrom rapeseed were mainly studied. Firstly, single-factor experiments and response surfacemethodology were employed to optimize extraction conditions of the rapeseedpolysaccharides. Then two fractions of rapeseed polysaccharides, namely RP1and RP2,were obtained after extraction, deproteinization, decolouration, DEAE-cellulose andSephadex G-100column chromatography. The primary structures of RP1and RP2wereinvestigated. Furthermore, the anti-digestibility of RP1and RP2against artificial humangastric juice and α-amylase was estimated. Finally, the proliferative effect of the rapeseedpolysaccharides on probiotics in vitro was also studied. The main contents and results are asfollows:1. Study on the optimal extraction conditions of rapeseed polysaccharides using sodiumhydroxide (NaOH) solution as extracting agent. Firstly, the effects of several main factors(NaOH concentration, extraction temperature, extraction time and liquid-solid ratio) on theextraction yield of rapeseed polysaccharides were determined through single-factorexperiments. On the basis of that, response surface methodology along with Box-Behnkendesign was applied to optimize the extraction condition, which was NaOH concentration4.1%, extraction temperature86oC, extraction time3h and liquid-solid ratio25:1(v/w).Under the condition, the extraction yield of rapeseed polysaccharides was5.86%.2. Study on the separation and purification of single fractions of rapeseedpolysaccharides. After pretreatment of the raw material, NaOH aqueous solution was usedto extract rapeseed polysaccharides. Crude polysaccharides were obtained after theconduction of deproteinization, decolouration, precipitation, centrifugation andlyophilization successively. The crude polysaccharides were loaded on DEAE-cellulosecolumn. Then distilled water,0.05,0.10and0.20mol/L NaCl solutions were used aseluents in turn. Two main fractions eluted by distilled water and0.10mol/L NaCl solutionswere gathered and then futher purified by Sephadex G-100. Finally two fractions named asRP1and RP2were obtained. The deproteinization experiments showed that the500kDamembrane had a better result than Sevag method. In the decolouration experiment,macroporous resin AB-8showed a better effect than D101.3. Study on the chemical composition and primary structure of the rapeseedpolysaccharides. The results showed that the total sugar, protein, uronic acid and sulfatecontent of RP1were68.25%,13.26%,8.84%and9.65%, respectively. The protein portion consisted of14kinds of amino acid. And in RP2, the total sugar, protein, uronic acid andsulfate content were65.86%,19.10%,4.89%and10.15%, respectively. The protein portionconsisted of13kinds of amino acid. The HPLC analysis showed that RP1and RP2werehomogeneous polysaccharides and molecular weights were28.51kDa and6.55kDa,respectively. The GC analysis showed that both of RP1and RP2were composed ofrhamose, arabinose, xylose, mannose, glucose and galactose. The UV analysis revealed thatthey contained protein, but not contained nucleic acid. The IR spectroscopy analysisindicated that both of RP1and RP2exhibited characteristic absorption bands ofpolysaccharides. Moreover, there were absorption bands corresponding to the existence ofcarbonyl and N-H, which indicated that RP1and RP2were protein-bound polysaccharides.4. Study on the anti-digestibility of the rapeseed polysaccharides against artificial humangastric juice and α-amylase. FOS was used as positive control. The anti-digestibility of RP1and RP2were determined by measuring their hydrolysis degrees in the artificial humangastric juice and α-amylase under different pH. The results showed that the maximalhydrolysis degrees of RP1and RP2in the artificial human gastric juice were2.78%and4.02%, respectively. And they were5.07%and8.06%resoectively in the α-amylase. Itcould be seen that only small percentage (＜8.06%) of RP1and RP2were hydrolyzed,indicating their good anti-digestibility.5. Study on the proliferative effect of the rapeseed polysaccharides on probiotics.Different concentrations of crude rapeseed polysaccharides, RP1and RP2were added tothe basal media without any direct carbon source. The probiotics were inoculated and thencultivated anaerobically. After incubation for0h and48h, the bacterial number and pH ofthe media were determined. The results showed that crude rapeseed polysaccharides, RP1and RP2could stimulated the proliferation of Bifidobacterium adolescentis,Bifidobacterium infantis, Bifidobacterium bifidum and Lactobacillus acidophilussignificantly. Furthermore, the pH values of the media decreased during the cultivation. Theresults indicated that the probiotics could utilize the rapeseed polysaccharides and produceSCFA. In addition, RP1showed the best stimulatory effect on probiotics, followed by RP2and crude polysaccharides. The appropriate concentration of the rapeseed polysaccharides(RP1and RP2) for probiotics was2.0～3.0%. The growth curves of the tested strainsshowed that the probiotics reached plateau phase after24～32h cultivation whensupplemented with the rapeseed polysaccharides. The results indicated that the rapeseedpolysaccharides could stimulate the proliferation of probiotics and have potential to beexploited as novel prebiotics.