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热解酸化蔗糖制备低聚焦糖的研究
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摘要
蔗糖(sucrose)是世界上产量最大的高纯度碳水化合物,是食品工业中最重要的能量型甜味剂。蔗糖是α-D-葡萄糖的C1与β-D-果糖的C2通过糖苷键结合的非还原性糖,分子中含有3个伯醇羟基和5个仲醇羟基,通过羟基的醚化、酯化、取代、分解和缩聚等化学反应,可生成若干种衍生物(如:蔗糖脂肪酸酯、三氯蔗糖、蔗糖聚醚及聚氨酯树脂)。以蔗糖为原料,经过物理、化学和生物途径开发包括表面活性剂、洗涤剂、塑料、医药等多种产品,开拓蔗糖高附加值衍生物的新领域具有重要意义。
     本论文以酸化蔗糖为原料,对热化学法制备新型功能性食品添加剂—低聚焦糖的工艺及其特性进行了较为全面的研究。主要研究内容和结果如下:
     不同干法工艺和湿法工艺制备低聚焦糖,采用主成分分析、聚类分析和结构方程模型对得率、pH值、A294、A420、总糖含量及还原糖含量等指标进行统计分析。结果表明,干法和湿法工艺的得率随加热温度的增加呈现先升高后下降的趋势,常压干法和真空干法工艺的得率高于瞬时热化学法—微波干法;高压反应釜湿法工艺在较高温度时易发生树脂化和碳化,影响产物。主成分分析、聚类分析和结构方程模型显示真空干法工艺更适合制备低聚焦糖,A294与A420和还原糖含量呈显著正相关,与pH呈显著负相关;A420与总糖含量呈显著负相关,pH与总糖含量呈显著正相关;低聚焦糖样品的吸光度及总糖含量更能代表其特性。
     考察反应温度、保温时间、柠檬酸用量、氨水用量和浓度因素等对真空干法制备低聚焦糖的得率和抗氧化性的影响。单因素试验表明,反应温度、保温时间和柠檬酸用量是影响低聚焦糖得率和抗氧化性的主要因素。根据Box-Behnken design (BBD)中心组合原理,对低聚焦糖的制备工艺进行响应面优化,真空加热温度162℃,加热时间0.95h,柠檬酸用量0.94%,得率为83.91±1.82%;反应温度167℃,加热时间1.05h,柠檬酸用量1.04%,清除DPPH自由基的能力为88.61±2.13%。
     对真空干法制备的低聚焦糖的pH值稳定性、吸湿保湿性、热稳定性和抗氧化等理化特性的分析表明,在碱性环境下,低聚焦糖溶液的稳定性较差;在中性和酸性环境下,低聚焦糖溶液有较好的稳定性;在温度30℃,相对湿度95%的条件下,低聚焦糖的最大吸湿率为81.78%;相对湿度30%的条件下,最大保湿率仅为9.71%;低聚焦糖与酸化蔗糖相比,酸化蔗糖的热变性曲线中有两个较窄的峰,含有两种物质;低聚焦糖的热变性曲线中有一个较大的吸热峰,表明含有的是热变性温度相近的一类物质,其变性温度为144.53? 0.62℃;较低温度100℃制备的低聚焦糖清除DPPH自由基能力很小,可能是因于反应速率较低,生成的低聚焦糖及美拉德反应产物较少,而蔗糖含量较高;采用最优工艺条件制备的低聚焦糖具有较好的清除DPPH自由基能力,40mg/mL的低聚焦糖样液清除DPPH自由基能力达到95.96%,随后提高样液的浓度,低聚焦糖的清除DPPH自由基能力不再有明显的增强,其IC_(50)值为2.81mg/mL。参照国标考察不同反应温度和最优工艺制备低聚焦糖的色率、红色指数、黄色指数、耐盐性等色素特性,结果表明,随着反应温度的升高,色率、红色和黄色指数、浊度均呈上升趋势;旋光度呈下降趋势。最优工艺条件下制备低聚焦糖产物的色率为15000 EBC,红色指数为8.158,黄色指数为9.116,旋光度为0.717±0.120,浊度为2.180±0.160 IU。
     采用凝胶柱和活性炭-硅藻土吸附柱对低聚焦糖成分进行级分分离,通过高效液相色谱和离子色谱对低聚焦糖及其分子量分布和柱分离级分分析,活性炭-硅藻土吸附柱的分离效果优于Sephadex G-100和Bio-gel P-2凝胶柱,但活性炭-硅藻土吸附柱的回收率和级分得率较低;高效液相色谱和离子色谱法分析低聚焦糖和柱分离级分得出低聚焦糖含有葡萄糖、果糖等单糖和蔗糖、蔗果三糖及以上聚合度低聚糖,是分子量范围非常相近的一些混合物。
     采用酸水解处理低聚焦糖及其活性炭-硅藻土吸附柱分离级分,通过气质联用色谱分析,主要产物有1,6-脱水-α-D-吡喃葡萄糖、1, 6-脱水-β-D-呋喃葡萄糖、1, 6-脱水-β-D-吡喃葡萄糖;甲基化分析结果显示,大部分果糖基连接在C1和C6位上,少部分的连接在C2、C3和C4位形成分支结构。低聚焦糖在红外光谱上表现出多处吸收峰,含有糖类的特征功能团;低聚焦糖的热裂解产物主要有呋喃、酮、醛、呋喃酮、吡唑、吡咯、烯、酸酐类;酸化蔗糖含有12%~15%的水分,短时水解反应生成D-葡萄糖和果糖;受热和酸作用,进一步发生脱水、热降解和糖的复合反应,蔗糖及水解生成的单糖分子中的糖苷键断裂形成果糖正离子和葡萄糖负离子,前者与蔗糖分子中的羟基发生缩合反应,形成低聚糖,与果糖基1位的羟基结合形成1-酮糖(即蔗果三糖),与果糖基6位的羟基结合形成6-酮糖,与葡萄糖基6′位的羟基结合形成新酮糖。形成的低聚糖与果糖正离子或葡萄糖正离子进一步聚合形成分子量更大的低聚糖。
     采用抗生素微生物检定法评价低聚焦糖的抑菌能力和抗生素效价,低聚焦糖的抑菌效果顺序:金黄色葡萄球菌>大肠杆菌>枯草芽孢杆菌>藤黄微球菌;对黑曲霉和黑根霉等霉菌则无抑菌效果;分析低聚焦糖样品对人体肠道总厌氧菌、有益菌(双歧杆菌和乳酸菌)、肠道内最大量菌群(拟杆菌)及条件致病菌(肠杆菌)的影响,分别以低聚焦糖或葡萄糖为唯一碳源进行发酵,接种肠道菌群24h后发酵液的吸光度均增大,反应温度140~160℃制备的低聚焦糖更适合肠道微生物的生长;低聚焦糖对肠道益生菌——双歧杆菌和乳酸杆菌的生长具有增殖作用,对拟杆菌和肠杆菌具有一定的抑制作用。
Sucrose is high purity carbohydrates and important energy sweeteners in food industry, and is the world's largest production in sugar industry. Sucrose is non-reducing sugar linked by the glycosidic bond ofα-D-glucose C1 andβ-D-fructose C2, containing three primary alcohol hydroxyl and five secondary alcohol hydroxyl groups. Various derivatives (i.e: sucrose fatty acid ester, sucralose, sucrose polyether and polyurethane resin) can be obtained through the chemical reaction of the hydroxyl group with the etherification, esterification, substitution, decomposition and condensation polymerization. Sucrose could be exploited as the surface active agent, detergent, plastic, medicine, new additives and various products by physical, chemical, and biological way. Thus, exploring new territory of high value added products of sucrose has the vital significance.
     In this paper, the new functional additives STOC, prepared from acidified sucrose by the thermochemical method, were comprehensive researched, including the preparation process and characteristics. The main contents and results were as follows:
     The STOC was prepared by different dry and wet process, and the principal component analysis and structural equation model were used to statistically analyze every index. These results showed traditional and vacuum dry process were superior to microwave dry process, and high pressure reactor process was superior to pulse electric field process in yield, and the yield of dry and wet process increased within the 100-160 oC with a subsequent decrease. However, the resinification and carbonation reaction occurred under more than 160 oC in high pressure reactor process. The principal component analysis and structural equation model showed the vacuum dry process was more suitable for the preparation of STOC. The absorption value at 420nm significantly positive correlated with reducing sugar and the absorption value at 294nm, and significantly negative correlated with pH value. The absorption value at 420nm significantly negative correlated with total sugar content, and significantly positive correlated with pH value. Absorbance and total sugar content were the more representatives of its characteristics.
     Using the the heating temperature, holding time, the citric acid concentration and ammonia water amount and concentration as the index, the yield and DPPH scavenging ability of STOC was studied and optimized. The single factor experiments showed the heating temperature, holding time and citric acid concentration were key factors.The Box-Behnken design (BBD) was used to optimize the above critical factors, the optimal technological parameters were obtained as follows: the heating temperature 162 oC, the holding time 0.95h, the citric acid concentration 0.94%. The experimental values (83.91±1.82%) of yield was coincidental with those predicted. The heating temperature 167 oC, the holding time 1.05h, the citric acid concentration 1.04%, the DPPH scavenging ability was 88.61±2.13%.
     The pH value stability, moisture absorption and retentation, heat stability and DPPH scavenging ability of STOC were studied to analyze the physicochemical characteristics. The results showed the stability of STOC was poor in alkaline environment, and better in neutral and acidic conditions. The biggest moisture absorption was 81.78% under temperature 30 oC, relative humidity 95%, and the biggest moisture retentation was 9.71% under relative humidity 30%. DSC analysis illustrated that the acidified sucrose thermal denaturation curve had two narrow peak, contained two substances; however, the STOC thermal denaturation curve had a bigger absorption peaks, including plenty of similar substances having the thermal denaturation temperature at 144.53 oC. The DPPH scavenging ability of STOC prepared at 100 oC was very lower due to the lower reaction rate and content of STOC. But, the DPPH scavenging ability of STOC prepared at the optimum process was 95.96% when the concentration was 40mg/mL, and the IC50 was 2.81 mg/mL. With the reaction temperature increasing, the color rate, red and yellow index, and turbidity were on the rise, and the optical rotations to drop. The color rate was 15000 EBC, red index was 8.158, yellow index was 9.116, the optical rotation was 0.717±0.120, and turbidity was 2.180±0.160 IU under the optimal technological parameters.
     The STOC ingredients were separated by the gel column and active-carbon adsorption column, using HPLC and ion chromatography to monitor the carbohydrate content and molecular weight of STOC or separation. The results showed active-carbon adsorption column was superior to Sephadex G-100 and Bio-gel P-2 column in separation of STOC, but the recovery and yield of STOC was low. HPLC and ion charmatography analysis showed STOC contained monosaccharide (glucose and fructose), sucrose, 1-kestose and more polymerization degree oligosaccharides.
     The hydrolysis and GC-MS were used to analyze the STOC and the ingredients of active-carbon adsorption column separation. Results showed 1,6-dehydration-α-D-pyranoglucose, 1,6-dehydration-β-D-furanglucose and 1,6-dehydration-β-D-pyrano- glucose were the main ingredinets.The methylation analysis indicated that most of fructosyl were incorporated into C1 and C6, and a few part incorporated into C2, C3 and C4 to form branched structure. The infrared spectrum showed the STOC contained functional group characteristics of the sugar. The pyrolysis products of STOC prepared from different temperature was furan, ketones, aldehyde, furan ketone, pyrazole, and anhydride. Acidified sucrose contained 12%-15% water, and D-glucose and fructose was produced after the short-term hydrolysis reaction. And with the heating and acid effect, the reaction of dehydration, thermal degradation and the sugar complex reaction were happened, the fructosyl cation and glucosyl anion were formed by the scission of glycosidic bond. And then the fructosyl cation reacted with the hydroxyl to form oligosaccharide, linking with one location hydroxyl of fructose formed 1-ketose, six location hydroxyl of fructose formed 6-ketose, 6′location hydroxyl of glucose formed neoketose. Thus, reaction could occur with sucrose, glucose (also formed by the scission of sucrose), and to form more molecular weight oligosaccharides by polymerization.
     Antibacterial ability and antibiotics titer evaluation of STOC were appreciated by microbiological assay of antibiotics. Moreover, it could conclude that the orders of the antibacterial ability were as follows: Staphylococcus aureus > Escherichia coli > Bacillus subtilis > Micrococcus luteus, and no antibacterial effect to Aspergillus niger and Rhizopus nigricans. The effect of STOC on total anaerobes, beneficial bacterium (bifidobacterium and lactobacillus), major flora (bacteroides) and pathogen (enterobacter) of human intestinal was evaluated in batch cluture using STOC or glucose as the only carbon source. The 24 h in vitro fermentation experiments revealed that STOC affected the growth of gut bacteria during mixed culture growth and increased the absorbance of culture. The products prepared from 140-160 oC were more suitable for intestinal microbial growth; Lactobacilli and bifidobacteria were increased in the anaerobic bacteria enumerated by the STOC, and bacillus and enterobacter were certainly inhibited.
引文
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