| Xanthan gum is a kind of complex exopolysaccharide produced by Xanthomonas campestris.Since it acquired’generally recognized as safe’（GRAS）approval in 1968,xanthan gum has become widely used as a food additive due to its unique rheological properties and excellent stabilizing and thickening effects.Xanthan oligosaccharides（XGOS）could be prepared by hydrolyzing xanthan gum.With special biological activities such as high antioxidant,anti-apoptotic and prebiotic activity,XGOS have high added value and broad application prospects.Enzymatic degradation of xanthan gum has the advantages of mild reaction conditions and controllable process,but specific enzymes are difficult to obtain,and the macromolecular properties of xanthan gum lead to low enzymatic hydrolysis efficiency.To solve the problems,an endo-β-1,4-glucanase（Persi Cel4）derived from camel rumen metagenome was expressed in Pichia pastoris GS115.XGOS were directly prepared by coupled fermentation system of P.pastoris and X.campestris CCTCC M2015714.Finally,XGOS were isolated and extracted,and the structure and bioactivity of them were studied.The main research results are as follows:（1）The recombinant P.pastoris GS115-p PIC9K-persi Cel4 was constructed by heterologous expression of Persi Cel4 derived from camel rumen metagenome.The fermentation conditions were optimized and the enzymatic properties of Persi Cel4 were studied.The results showed that the optimal fermentation conditions were p H 6.0,28℃,liquid content of 20%and methanol content of1.5%.The activity of Persi Cel4 in 7 L fermenter reached 17.97 U·m L-1.The optimal catalytic conditions of Persi Cel4 were p H 7.0 and 75℃,and can directly hydrolyze commercial xanthan gum to obtain XGOS-1 with an average molecular weight of 1536 Da,which accounting for 48.17%of the hydrolyzed products.In addition,a new production strategy of XGOS was established by adding enzyme solution during the fermentation process of X.campestris.XGOS-2 with an average molecular weight of 1808 Da was obtained,accounting for 51.50%of the hydrolyzed products.（2）The coupled fermentation system was established with recombinant P.pastoris GS115-p PIC9K-persi Cel4 and X.campestris.Persi Cel4 expressed by P.pastoris could directly hydrolyze xanthan gum produced by X.campestris to prepare XGOS.The optimal fermentation conditions were as follows:P.pastoris was inoculated with 4 times the inoculated amount(v·v-1)of X.campestris at36 h,and the p H of coupled fermentation stage was controlled at 7.0.Methanol（0.75%of the fermentation liquid volume）was added every 24 h until the end of fermentation.At the shaking flask level,the maximum total sugar content of the coupled fermentation system was 11.40 g·L-1,and the average molecular weight of the product（XGOS-3）was 2500 Da which accounting for 93.79%.The maximum total sugar content in the coupled fermentation system was 17.85 g·L-1 in the 7 L fermenter,where the low molecular weight xanthan gum（52 030 Da）accounted for 96.64%.（3）XGOS-1,XGOS-2 and XGOS-3 prepared by the above three methods（conventional hydrolysis,enzyme solution feeding and coupled fermentation）were extracted and purified,and then the structures were analyzed by monosaccharide composition analysis,MALDI-TOF MS,FT-IR spectrometry and NMR spectrometry.The degrees of polymerization of XGOS-1 were 8 and 9,the degrees of polymerization of XGOS-2 were 5 and 10,and the degree of polymerization of XGOS-3was 14.All the three XGOS had high antioxidant activity.When the concentration of XGOS-1 was 5g·L-1,the scavenging rate of DPPH radical reached 76.47%.When the concentration of XGOS-3 was5 g·L-1,the scavenging rate of hydroxyl radical reached 89.74%.In vitro probiotic fermentation experiments showed that three XGOS can effectively promote the growth of some human intestinal probiotics,and produce short chain fatty acids and lactic acid which are beneficial to human intestinal health. |
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