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1、华中科技大学 硕士学位论文 蜂蜜及高果糖浆的鉴别检测 姓名：刘子维 申请学位级别：硕士 专业：生物化学与分子生物学 指导教师：杨祥良 20090518 华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 摘摘 要要 蜂蜜是一种传统的天然保健食品，含有糖类、蛋白质、氨基酸、维生素、微量 元素酸、色素、花粉、激素、微量活性成分等。蜜源不同，蜂蜜中的各种组分及其 含量不同，即使是同一种类的蜂蜜，由于受气候条件、土壤成分、养蜂技术等诸多 影响因素的存在，导致即使同一种蜂蜜其中的各个成分及其含量也不一样。高果糖 浆由淀粉水解产生，其中的主要的糖成分和含量与蜂蜜基本一致，因此检测蜂蜜中
2、 掺伪高果糖浆一直是研究的难点，但是在蜂蜜中的主要成分的的含量比例是一定的， 相对与人为将淀粉水解后的高果糖浆而言也是有一定的区别。本文通过糖类的薄层 色谱法和淀粉酶的同工酶电泳的方法快速表征蜂蜜中的糖和淀粉酶。以便和高果糖 浆加以区别，我们在蜂蜜的检测中做了以下几个方面的工作 (1) 对蜂蜜中的常规指标诸如水分，灰分，和酸度做了检测，水分和还原糖的含 量共同影响着蜂蜜的粘度，灰分在一定的程度上可以反应蜂蜜中的蛋白含量，酸度 则可以表示蜂蜜中的单糖的含量，蜂蜜的水分，灰分，酸度的数值都在规定的范围 之内，而且可以看到二号糖浆的以上 3 个值更加的接近于蜂蜜。但是从这些指标的 检测结果中很难看出
3、蜂蜜和糖浆的实质区别，只能作为一个一般的质量测定的指标 存在。 (2) 我们采用薄层色谱的方法对蜂蜜中的糖类进行了鉴别， 首先通过化学滴定法 测定了蜂蜜和糖浆的的葡萄糖，蔗糖，果糖，以及还原糖的含量，对蜂蜜和糖浆中 的糖的主要组分有一个定量的判断，然后采用薄层色谱法进行蜂蜜中糖类成分的分 析，通过对普通硅胶G板，高效硅胶G板和手工自制版 3 种固定相的筛选，我们最后 采用普通硅胶G板作为我们薄层展层的固定相。 在流动相的筛选中我们采用的 3 个不 同体系的流动相作为展开剂，最后确定了甲醇-乙酸乙酯-水-氨水体系为最佳的展开 系统，对甲醇-乙酸乙酯-水-氨水各个比例的微调确定了最佳比例为 9：5
4、：1：1.5， 在这个比例下蜂蜜和糖浆的各个糖的组分都分离明显，相对于国家标准而言有很大 的改进，薄层图片上可以分辨的斑点从 3 个提高为 6 个，且分离效果提升，最上端 I 华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 和最下端斑点的Rf值的差从 0.18 提升至 0.31，从薄层色谱图上可以看到糖浆相对于 蜂蜜而言在图的最下方多出 2 个斑点。 (3) 蜂蜜中含有 - 淀粉酶及少量的自身转化酶，我们对蜂蜜中的淀粉酶进行酶 活测定。采用了硫酸铵抽提蜂蜜中的淀粉酶，对抽提蛋白所使用的硫酸铵的饱和度 从抽提后的蛋白含量以及抽提后蛋白的淀粉酶活性 2 方面进行了评价，最后
5、确定了 40%饱和度硫酸铵为最佳的饱和度， 在这个饱和度下蛋白含量和蛋白中淀粉酶的活性 都得到了最佳保持。最后采用了抽提后的蛋白进行了同工酶电泳检测。 关键词关键词：蜂蜜 糖类 淀粉酶 薄层色谱 II 华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 Abstract Honey is a traditional health food which contains carbohydrates, proteins, amino acids, vitamins, trace elements in acid, pigment, bee pollen, hormones, a
6、ctive ingredients and so on. The various components and their content are quietly different due to various sources of honey, Even the same types of honey, many influencing factors such as Climatic conditions, soil composition, bee-keeping technology will cause the fails of analysis results which sho
7、uld be totally the same composition and the similar content. High Fructose Corn Syrup are hydrolysis from starch, the main composition and content is similar to the honey. So detection of complex of honey and High Fructose Corn Syrup isnt a easy work .But compared to High Fructose Corn Syrup come fr
8、om starch which is hydrolysis by people , the main composition of honey and the ratio of those composition is consistent, the honey and High Fructose Corn Syrup will show some difference both in chemical and physical respects. Our aim is adopting Thin-layer chromatography(TLC) and Isozyme electropho
9、resis to identify Carbohydrate and Amylase in a convenient way. So we can tell difference between honey and High Fructose Corn Syrup. We take hand in The following aspects in honey detection. (1) Several conventional indicators, such as moisture, ash, and acidity are detected in our honey specimen.
10、The content of aqua and reducing sugar take a combined effect on the viscosity of honey. Ash respect the content of protein in honey in some ways. Acidity is related to proportion monosaccharide. The mark of moisture, ash, and acidity are within the scope of the provisions, and we can perceive that
11、the second syrup is more similar to the honey. But the result of those indicators show little substantive distinction between honey and High Fructose Corn Syrup, exist just as a normal indicator of quality determination. (2) We adopt Thin-layer chromatography(TLC) to distinguish carbohydrate of hone
12、y. above all we evaluate the content of glucose, sucrose, fructose and reducing sugar content in both honey and High Fructose Corn Syrup by Chemical titration. Then we got a quantitative assessment to the main composition of honey in both honey and High Fructose Corn Syrup. After that we analysis th
13、e carbohydrate by TLC, We compare III 华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 ordinary silica gel G plates, high-performance silica gel G plate and hand-made plates, finally we select ordinary silica gel G plates as our stationary phase. In the selecting of mobile phase, We use three different systems a
14、s our show layer agents. Finally we adopt ethyl-acetate methanol-water-ammonia as the best show layer system. We regulate proportion of ethyl: acetate methanol: water: ammonia, and then we got the best ratio is 9:5:1:1.5. Under the condition, there is a conspicuity improvement on separation both in
15、the honey and High Fructose Corn Syrup, and got a better effect compare to national standard. The spot showed on TLC increase from 3 to 6, and the effect of separation became better. The disparity of Rf between spot in the top and bottom increase from 0.18 to 0.31. we can easily perceive two extra s
16、pot on the TLC of High Fructose Corn Syrup . (3) Honey contain some Amylase and some self-converting enzyme, we test the activity of Amylase in honey. The way of ammonium persulfate extraction protein is adopted in our experiment. We appraise the consequent of protein extraction using distinctive saturation of ammonium persulfate refering to two aspect: Protein content and activity of Amylase, after that we got the best saturation of ammonium persulfate is forty percentage. Under that
