【作者】 张帆；

【导师】 宋宏新； 李敏康；

【作者基本信息】 陕西科技大学 ， 食品科学， 2013， 硕士

【摘要】 低聚木糖是一种功能性低聚糖，具有很强的促进双歧杆菌增殖能力，常作为重要的益生因子应用于保健食品中。小麦麸皮富含半纤维素，是制备低聚木糖的良好原料。本文以小麦麸皮为原料，采用两种生物酶法制备低聚木糖。首先，探讨了以小麦麸皮为固态发酵基质培养产木聚糖酶的微生物，在其生长期诱导产木聚糖酶降解麦麸以制备低聚木糖的可行性，为麸皮综合利用及低聚木糖制备提供新途径；其次，以商品木聚糖酶加入粗提麦麸木聚糖中制备低聚木糖；最后，对两种生物酶法从小麦麸皮制备低聚木糖的工艺效果进行了比较。首先通过透明圈法及固态发酵预实验对实验菌种进行选择。三种产酶微生物（木霉Trichoderma sp.、黑曲霉Aspergillus niger、球毛壳Chaetomiumglobosum）接种于以粗提木聚糖为唯一碳源的透明圈测定培养基中，培养72h后三者均产生水解圈，说明三个菌种均能产生木聚糖酶。木霉通过固态发酵所得可溶性总糖含量较黑曲霉和球毛壳高，且平均聚合度适中，适合固态发酵制备低聚木糖；而黑曲霉和球毛壳经固态发酵所得低聚木糖的平均聚合度偏低，不适合通过固态发酵的方法制备低聚木糖。以木霉为实验菌株进行固态发酵实验，考察了起始pH、起始含水量、接种量、表面活性剂用量、发酵时间、低聚木糖添加量、温度对固态发酵的影响，通过测定固态发酵提取液中还原糖、可溶性总糖的浓度、平均聚合度确定最佳单因素条件为：（以底物10g麦麸为基准）起始pH4.0、起始含水量10mL、接种量1%、表面活性剂用量0.05%、发酵时间84h、低聚木糖的添加量0.25%、发酵温度为28℃培养60h后转入37℃培养24h。在单因素实验基础上，选定发酵温度、发酵时间、低聚木糖添加量后设计四因素三水平的正交实验，确定各因素的主次顺序为：起始含水量>起始pH>表面活性剂用量>接种量，确定最佳固态发酵工艺为：（以底物10g麦麸为基准）起始含水量10mL、表面活性剂用量0.05%、起始pH5.0、木霉接种量1.5%。在正交实验确定的最优条件下进行验证性实验，固态发酵提取液中可溶性总糖含量为198.62mg·g^-1，还原糖含量为49.32mg·g^-1，低聚木糖含量为149.3mg·g^-1，平均聚合度为4.027，麦麸木聚糖降解率为66.21%。对最佳条件组合下的固态发酵提取液经硅胶薄层层析，分析结果显示主要成分为木三糖、木四糖。以碱法粗提麦麸木聚糖为底物，以酶解液中的还原糖浓度、可溶性总糖浓度、低聚木糖平均聚合度为衡量指标，考察了酶解时间、酶质量分数、底物质量分数、酶解温度、pH五个单因素对木聚糖酶酶解麦麸木聚糖效果的影响，确定了最佳单因素条件为：酶解时间4h，酶质量分数15g·L^-1，底物质量分数50g·L^-1，酶解温度40℃，pH5.5。在单因素基础上，选定底物质量分数50g·L^-1及酶解时间4h，选取酶质量分数、酶解温度、pH为考察因素的三因素三水平的Box-Behnken实验，得可溶性总糖含量Y对酶质量分数X¹、酶解温度X²、pH X3的多元二次回归方程为：Y=9.54＋0.37X¹－0.56X²＋0.55X³＋0.006X¹X²－0.86X¹X³－0.32X²X³－0.28X₁²－0.58X₂²－0.38X₃²。模型能够较好的对实验进行分析和预测，通过响应面分析，确定因素间的主次顺序：酶解温度>pH>酶质量分数，进一步确定最佳酶解工艺为：酶质量分数10.7g·L^-1，酶解温度36℃，pH6.0。在最佳酶解条件下，该法所得可溶性总糖含量为200.9mg·g^-1，还原糖含量为93.52mg·g^-1，低聚木糖含量107.38mg·g^-1，平均聚合度为2.148，麦麸木聚糖酶降解率66.97%。通过薄层层析结果显示，酶解液的主要成分为木二糖、木三糖。比较了两种制备低聚木糖的生物酶法方案和工艺的差异，重点比较了两类生物酶法对木聚糖的降解情况。以微生物固态发酵麦麸制备低聚木糖虽然没有商品木聚糖酶所制备的低聚木糖的有效成分多，但由微生物经诱导产生的木聚糖酶对木聚糖具有一定酶解效果，能够实现微生物以麦麸为固态发酵基质生长，并在生长期诱导所产木聚糖酶降解麦麸同步进行，简化低聚木糖制备工艺，方法具有可行性，为制备低聚木糖提供了新途径。更多还原

【Abstract】 Xylooligosaccharides（XOs）, a sort of functional oligosaccharides，were ableto promote the proliferation of bifidobacterium, which were usually applied inhealth food as a sort of significant prebiotic. Wheat bran was rich ofhemicellulose so it was a good raw material for preparation of XOs. Two kindsof biological enzymatic methods were studied to produce XOs taking wheat branas raw material. Firstly, the preparing XOs by solid-state fermentation withmicroorganisms was discussed using wheat bran as a sole substrate, whichprovided a new idea of wheat bran comprehensive utilization and XOspreparation. Secondly, the preparing XOs by adding commercial xylanase intocrude extracts of xylan were studied. Finally, the processing efficiency of twokinds of biological enzymatic methods of preparing XOs from wheat bran wascompared.The experimental strain was selected through the transparent circle methodand preliminary solid-state fermentation experiments. Three microorganismsproducing xylanase （Trichoderma sp., Aspergillus niger, Chaetomium globosum）were inoculated in transparent circle assay medium with crude extracts of xylanas sole carbon source. Hydrolysis circles appeared by72h. It was indicated thateach of the strains can produce xylanase. Total soluble sugar content bysolid-state fermentation with Trichoderma sp. was higher than that of Aspergillusniger and Chaetomium globosum with a moderate average degree ofpolymerization. It was suitable to prepare XOs by solid-state fermentation. Thesolid-state fermentation with Aspergillus niger and Chaetomium globosum werenot fit to prepare XOs because of lower average degree of polymerization.Then the optimal conditions of XOs producing by solid state fermentationwith Trichoderma sp. were researched. The factors influenced on solid-statefermentation including initial pH, initial moisture content, and inoculation volume, dosage of surfactant, fermentation time, XOs concentration andtemperature were investigated. The results were determined by total solublesugar concentration, reducing sugar concentration, the average degree ofpolymerization. The optimal conditions of single-factor experiments were asfollows:（on the basis of10g wheat bran as substrate） the initial pH was4.0andthe initial moisture content was10mL inoculated by1%and adding0.05%ofsurfactant, then fermented for84h. The XOs addition was0.25%and fermentedat28℃for60h then transferred to37℃for24h. The orthogonal experiment offour factors and three levels was designed on the basis of single factorexperiment results and the influencing of factors was confirmed as follows:initial moisture content> initial pH>dosage of surfactant>inoculation volume.The optimum conditions were as follows:（on the basis of10g wheat bran assubstrate） initial pH was5.0and initial moisture content was10mL inoculated by1.5%and adding0.05%of surfactant. The total soluble sugar content was198.62mg·g^-1, and reducing sugar content was49.32mg·g^-1, and the XOs content was149.3mg·g^-1with the average degree of polymerization of4.027and xylandegradation rate of66.21%under the optimal conditions. Fermentation brothunder the optimal conditions was analyzed by thin layer chromatography and theresults showed that the main ingredients were xylotriose and xyloteraose.The enzymolysis technology was investigated by determining total solublesugar concentration, reducing sugar concentration and the average degree ofpolymerization with alkali crude extracts of xylan as substrate. The single factorof enzymatic time, enzyme concentration, substrate concentration, reactiontemperature, pH was studied and the optimal conditions were as follows: theenzyme concentration was15g·L^-1, the substrate concentration was50g·L^-1, thepH was5.5, enzymatic hydrolyzed at40°C for4h. Selected the substrateconcentration of50g·L^-1hydrolyzed for4h, the Box-Behnken experiment ofthree factors（enzyme concentration X¹, reaction temperature X², pH X3） and threelevels which was designed on the basis of single factor experiment results.Multiple quadratic regression equation was obtained by determining the totalsoluble sugar content as follows: Y=9.54＋0.37X¹－0.56X²＋0.55X3＋0.006X¹X²－0.86X¹X3－0.32X²X3－0.28X²1－0.58X²2－0.38X²3. This model was suitablefor experimental analysis and forecast. Response surface analysis confirmed that the influencing sequences of factors: reaction temperature>pH>enzymeconcentrate. The optimum conditions were as follows: enzyme concentration was10.7g·L^-1incubating at36°C, pH6.0. The total soluble sugar content was200.9mg·g^-1, and reducing sugar content93.52mg·g^-1, and XOs content107.38mg·g^-1with the average degree of polymerization of2.148and xylan degradation rate of66.97%under the optimal conditions. Enzymatic hydrolysate under the optimalconditions was also analyzed by thin layer chromatography and the resultsshowed that the main ingredients were xylobiose and xylotriose.The technological and processing differences between two kinds ofbiological enzymatic methods for preparing XOs were compared withdegradation of xylan. It indicated that the effective components by addingcommercial xylanase into crude extracts of xylan were superior to preparing XOsby solid-state fermentation using wheat bran as a sole substrate withmicroorganism. In spite of that, the latter method showed that somemicroorganisms were capable of inducing xylanase and hydrolyzing of xylan atthe same time of growth and simplified the technological process. Thetechnology was feasible to prepare XOs from wheat bran by solid statefermentation with microorganism and provide a new way for XOs preparation.更多还原