【作者】 王秋霜；

【导师】 应铁进；

【作者基本信息】 浙江大学 ， 农产品加工与贮藏工程， 2008， 博士

【摘要】 近年来,随着科学技术的不断发展,低聚糖的生理功能越来越受到广大学者的关注。大豆低聚糖是大豆籽粒中含有的可溶性寡糖的总称,占大豆中总碳水化合物的7-10%。研究发现大豆低聚糖的摄入对于调整肠道菌群平衡、预防许多疾病、增强机体免疫力、延缓衰老等有着重要的作用,研究认为大豆低聚糖的主要作用在于促进双歧杆菌的增殖。有人认为,低聚糖摄入人体后会引起隔气、肠鸣、腹痛等肠胃胀气现象,然而现代安全性检验证明大豆低聚糖不是引起胀气的直接原因,食用大豆低聚糖具有良好的安全性。在豆腐皮的生产过程中,大豆经浸泡、磨浆、过滤和煮浆后,通过保温蒸发,表面结皮,每隔一定时间收获产品。随着豆腐皮的不断形成,留下粘稠状的浓浆,即甜浆。甜浆是豆腐皮生产过程的终端副产物,其中含有较高浓度的低聚糖。目前甜浆一般被用来做动物饲料;或者重新倒入煮浆池,与新鲜的豆浆一起保温揭皮,但是这样生产出来的豆腐皮质地较脆、易折断,且颜色褐黄,无光泽,膜也较厚,筋性不好。本研究的目的是系统探索从甜浆中提取大豆低聚糖的路线方案,对甜浆资源进行再利用,拓宽大豆低聚糖的生产途经,为工业上的进一步应用提供理论基础和指导。本研究采用HPLC方法对豆腐皮生产过程中大豆低聚糖的物料平衡进行了跟踪测定。结果表明,低聚糖损失于浸泡阶段、过滤阶段、成皮阶段,损失的低聚糖含量分别为1.43%、48.5%、24.18%（重量比）。浸泡对低聚糖含量的影响不大,蔗糖、棉子糖、水苏糖分别损失0.91%、0.31%和0.21%;过滤是导致低聚糖损失的重要阶段,蔗糖、棉子糖和水苏糖损失量分别占大豆中低聚糖总量的21.68%、10.76%、16.06%,成品保留有原料中14.66%的低聚糖,副产品甜浆含有原料中11.23%的低聚糖,是一种十分有潜力的大豆低聚糖提取来源。本研究系统分析了工厂采样甜浆的成分。结果显示:总糖占干重的50%以上,其中大部分物质是低聚糖,蔗糖、棉子糖、水苏糖浓度分别为51.42、6.36、34.16 mg/ml,占甜浆干重的40%左右;可溶性蛋白质占甜浆干重的近17%,功能性物质如总黄酮和皂苷含量浓度分别为0.07、0.5 mg/ml,分别占甜浆干重的0.03%、0.22%。SDS-PAGE电泳图谱显示,主要的蛋白带有9条,其分子量大小分别为:76.36、50.37、41.35、37.44、27.14、25.78、19.94、17.62、13.64 kDa。甜浆成分的分析表明,功能性低聚糖含量远远高于大豆乳清废水。从甜浆中分离提取大豆低聚糖,得率和综合能耗可能远低于以乳清废水等为原料的提取工艺。采用PES膜对大豆低聚糖粗提液进行超滤研究。要保证超滤的正常运行,甜浆溶液必须经过适当的前处理。结果表明:pH值为4.5、温度为70℃、加热5 min、CaCl₂浓度为10%是最佳的前处理方式。经过前处理的甜浆溶液中蛋白浓度降到3.99 mg/ml,蛋白质的去除率达到89%以上。超滤单因素实验采用MWCO 10000 kDa的PES超滤膜进行。结果表明,甜浆中大豆低聚糖的提取最佳方案为:压力1.75Ba,超滤温度为45℃,pH值7.0。正交试验表明,MWCO对蛋白质去除率和低聚糖保留率影响最大。对于蛋白质来说,MWCO为3000 kDa的膜蛋白质去除率最高,但是低聚糖透过率和渗透通量却处于所有实验中的最低水平;而MWCO为10000 kDa的膜低聚糖透过率最大,且渗透通量最高,蛋白质截留率偏低。50℃超滤温度对二者都比较适宜。综合渗透通量、膜衰减系数、蛋白质截留率和低聚糖透过率四方面因素考虑,认为最佳操作条件为:操作压力为1.5 Ba、温度为50℃、pH值为7.0,膜的截留分子量为10000 kDa。通过这样的操作可以截留73%的蛋白质,低聚糖透过率达到85%以上。超滤后的大豆低聚糖溶液具有很高的含盐量,必须经过脱盐处理以达到大豆低聚糖产品的相关标准。一定浓度的NaCl溶液脱盐实验证明本研究中所使用的电渗析器工作性能稳定。本实验所用的电渗析装置极限电流密度的经验模型表达式为:I_lim=4.13v^0.112c^0.033。电渗析单因素试验结果表明,大豆低聚糖溶液的最佳脱盐方案为:20 V左右的操作电压、60 L/h左右的流量、稀释15倍。正交实验确定了最佳工艺条件,即电压为20V、流量为60 L/h、稀释18倍。对低聚糖保留率影响最大的因素是电压,其次是流量,再次是稀释倍数。通过最佳的操作条件进行脱盐可以成功脱除大豆低聚糖溶液中95%以上盐类,保留90%左右的低聚糖。大豆低聚糖粗提液具有很深的颜色,本研究采用树脂脱色法对其中的色素进行脱除。以静态吸附法从10种树脂中筛选了对大豆低聚糖粗溶液脱色效果较好树脂,并探讨了吸附动力学及其机制。大孔吸附树脂DM-130和AB-8具有较高吸附量,脱色率达到70%以上,解吸率近100%。与AB-8相比,DM-130对糖的吸附较小,是脱色的较合适树脂。温度对树脂脱色效果的影响结果表明,树脂对色素的吸附更容易在低温下发生,说明吸附过程是放热的、自发的。室温20℃是脱色效果较好的温度。当树脂-糖液比例在0.13g/ml左右时可以达到较高的脱色率,超过这个比例时脱色率变化不明显。树脂对色素的吸附等温线更符合Freundlich等温线模型,说明树脂对色素的吸附是单层吸附,且树脂表面发生对多种成分的吸附。吸附动力学模型的分析表明,树脂对色素的吸附符合pseudo-second-order模型,说明吸附行为是化学吸附,也是限速步骤。粒子内扩散模型说明吸附过程是先发生了表面吸附,然后发生粒子内扩散。动态吸附实验认为最佳流速为1 ml/min,乙醇洗脱液的浓度为50%。更多还原

【Abstract】 Recently, with the development of science and technology researchers have paid considerable interest in biological functions of oligosaccharides. Soybean oligosaccharides are soluble carbohydrate in soybean seeds and account for 7-10 % of the total carbohydrates in soybean. Researches have found that the intake of soybean oligosaccharides have important functions on the balance of microflora in intestine, prevention of many illness, enhancing body immunity, prevention of aging, etc. This was attributed by some researchers to promotion of bifidobacterium proliferation by soybean oligosaccharides. Researches had tested that oligosaccharides led to flatus and bellyache. However moden safe tests have shown that soybean oligosaccharides is not the direct reason of flatus and it is safe to take soybean oligosaccharides.The production of soybean sheet from soybean follows a traditional process: After soaking milling, filtering, and boiling, soybean sheet is formed in the milk surface kept at certain temperature, and could be harvested at some time interval. With the harvest of soybean sheet thick and yellow slurry was left, namely the sweet slurry. Sweet slurry contains a considerable amount of soybean oligosaccharides. Normally it is used as feedstuff for animals or fed back to the soymilk pool to produce next batch of soybean sheet. However soybean sheet produced by this method was not in good merchant quality, for it was thick, easily broken, dark color with less luster. The aim of this research was to explore the method for separation and purification of soybean oligosaccharides from sweet slurry, thus to tap the potential of the sweet slurry resource and to provide alternatives for the production of soybean oligosaccharides. The research results may provide theory basis and guidence for value-added industry utilization of sweet slurry.HPLC method was used in this research to trace the flow of soybean oligosaccharides during the production of soybean sheet. The results showed oligosaccharides losses were lost in soaking, blanding and filtrating, sheet forming stages, representing 1.43 %、48.5 %、24.18 % （on weight）. Soaking did not show much influence on oligosaccharides content. Surcose, raffinose and stachyose losses were 0.91 %、0.31 % and 0.21 % respectively. Blanding and filtrating was the most important step for oligosaccharides losses compared with other steps. Surcose, raffinose and stachyose losses were 21.68 %、10.76 %、16.06 % relative to the total oligosaccharides in soybean seeds. The final product soybeansheet contains 14.66 % of the total oligosaccharides. Still 11.23 % of oligosaccharides were in the sweet slurry.The component of the sweet slurry sampled from factory was analysed in this research. Results indicated that the total carbohydrate accounted for 50 % of the dry matter in sweet slurry and most of them were oligosaccharides, which represented 40 % of the total dry weight. Soluble protein accounted for 17 % of the total dry weight. Functional substance such as toal flavone and saponins concentration were 0.07 and 0.5 mg/ml respectively, representing 0.03 %、0.22 % （dry weight）. SDS-PAGE result showed nine protein bands in the electrophoresis graph, and the molecule weight was 76.36、50.37s 41.35、37.44、27.14、25.78、19.94、17.62、13.64 kDa respectively. The analysis of the sweet slurry showed functional oligosaccharides content was much higher than that in soybean whey water. And the recovery percentage and total energy consumption were far lower than from the whey water.UF experiment was conducted with PES membrane. The sweet slurry must be pretreated in order to guarantee the proper function of UF. The pretreatment experiment showed that the best pretreatment condition was pH 4.5, 70℃, heating 5 min and 10 % CaCl₂ concentration. After the pretreatment the protein concentration was dropped to 3.99 mg/ml and 89 % of protein was removed. Single factor trials used MWCO 10000 kDa PES membrane. The results showed that the optimum conditions for extracting oligosaccharides from sweet slurry were: 1.75 Ba TMP, 45℃temperature and pH 7.0. Orthogonal intersection trials revealed that MWCO became the major affecting factor on oligosaccharides retention and protein removal. Membrane with MWCO 3000 kDa was best for protein removal while oligosaccharides retention and permeate flux were the lowest in all trials. The oligosaccharides retention and permeate flux attained the highest level with MWCO 10000 kDa membrane, while the protein removal was relatively low. Temperature of 50℃was good for both protein removal and oligosaccharides retention. Considering the efficiency of protein removal, oligosaccharides retention, permeate flux and membrane decline, the optimum conditions for UF were 1.5 Ba pressure, 50℃temperature, pH 7.0, MWCO 10000 kDa. After this treatment 73 % of protein was removed and more than 85 % of oligosaccharides were retained.The permeate fluid after UF contains large quantities of salts and must be removed to meet the oligosaccharides product standard. Electrodialysis （ED） was employed for the desalination process. NaCl solution was used first to test the stability of the ED apparatus. NaCl desalination trial showed that the ED setup was stable. The experience model for limited current density （LCD） of the ED apparatus employed in this experiment was I_lim=4.13 v^0.112c^0.033. Single factor trial of ED showed that the optimum conditions for desalination was 20 V operating voltage, 60 L/h flow rate and diluting 15 times. Orthogonal intersection trials revealed that 20 V operating voltage, 60 L/h flow rate and diluting 18 times were the best condition for desalination and oligosaccharides retention. Operating voltage was the major affecting factor of oligosaccharides retention, then the flow rate and finally the diluting times. After ED more than 95 % salt in the crude soybean oligosaccharides solution was removed and 90 % of oligosaccharides was retained.The crude soybean oligosaccharides solution was in dark color. Resins were used in the experiment to remove color. Static adsorption method was used to screen the best resin for decolorization from 10 resins and adsorption kinetic of coloured impurities onto resins was also studied. The results showed that more than 70 % of the pigments were removed in the final product by the macroporous adsorption resin DM-130 and AB-8 and the desorption was almost completely. Comparaed with AB-8, resin DM-130 had relatively less adsorption of sugar solution and it was the best resin for decoloration. The adsorption process was favored at lower temperature, indicating the process was exothermic and spontaneous. And room temperaure of about 20℃was better for decolorization. When the ratio of resin mass/oligosaccharides solution volumn was approximately 0.13 g/ml higher decolorization efficiency could be obtained in our experiment. Decolorization efficiency did not change much when resin mass-oligosaccharides solution volumn ratio was higher than 0.13 g/ml. The adsorption isotherm better agreed with the Freundlich model, indicating the monolayer sorption of coloured impurities onto macroporous resins and that the impurities were of multi-component nature rather than single component. Adsorption kinetic of coloured impurities onto resins fitted the pseudo-second-order kinetic model, this indicated that the adsorption process was chemical adsorption which was the speed limiting step. The intraparticle diffusion model showed that the adsorption process occurred by both surface sorption and intraparticle diffusion. The kinetic data showed the best flow speed was 1 ml/min, and the concentration of ethanol washing solution was 50 %.更多还原