摘要
绿色小麦是我国特有的保健小麦品种,它富含硒、锌、铁、钙等20多种微量元素,且含量高于普通小麦。为提取天然绿色色素和开发绿色小麦有色食品,本论文依次以中性乙醇、酸性乙醇为溶剂,采用渗漉法提取绿麦麸色素,并用大孔吸附树脂、薄层层析等方法对其进行分离纯化,测定其紫外和红外吸收光谱,且研究其稳定性,所获主要结果是：
1、获得了提取绿色素粗提物的适宜工艺条件。通过单因素和正交试验的比较分析得到最佳提取工艺条件：pH为1.0,乙醇浓度为70%,料液比为1：10,提取温度是60℃,提取时间是1h,提取2次,色素的提取率接近83%。
2、从绿色色素粗提液中获得棕色沉淀物。该沉淀具有加碱溶解、加酸性乙醇溶解以及加酸沉淀的特性。采用正相硅胶板,以氯仿-甲酸-乙酸乙酯(16：3：1)为展开剂展开进行薄层层析,并用三氯化铁试剂进行酚类特征显色反应测试,得到其Rf=0.39。有酚类特征显色反应,圆点呈绿或蓝色,因此推测该棕色沉淀含有酚类物质。
3、未被AB-8大孔树脂吸附的绿色溶液经浓缩过滤后在4℃冷存时获得白色沉淀。用酸性乙醇溶液溶解该白色沉淀,采用反相硅胶板,以15%乙腈、2滴冰醋酸为展开剂进行层析,得到Rf=0.3,但拖尾相当严重且点不圆,加入有机碱后效果亦不好,在254nm紫外灯下观察有吸收。用Molish试剂测试无糖的特征反应。
4、测得该白色沉淀的一些其它特征为：酸性乙醇溶液中呈现绿色,难溶于中性乙醇,其色调随pH不同而改变,在中性、近中性条件下接近无色,碱性条件下呈棕红色且有沉淀产生。该物质最大吸收峰与次吸收峰的吸光度比值随pH变化,可推断其分子结构也随pH值而变化。该绿色物质有生物碱试剂反应。经红外光谱和生物碱试剂反应可初步推断,该色素可能含有酰胺,即该绿色物质不属于花青素类色素,可能为生物碱类色素。
5、用AB-8大孔树脂对其它色素物质的最佳吸附条件为：温度为室温；色素适宜的吸附pH值为1.0；色素浓度(吸光度值)为0.612×100,这时树脂吸附量最大；吸附时间为24h；吸附流速为1.5mL／min。解析时依次选择浓度为10%、30%、40%的乙醇溶液进行梯度洗脱。
6、绿麦麸色素中绿色物质的稳定性研究结果表明：(1)室内自然光和室内避光条件下稳定性较好；(2)对高温的耐受性不好,在20℃～80℃时稳定性较好,温度达到100℃时,吸光度值增加明显；(3)在酸性强(pH为1.0～4.0)时较稳定；(4)耐还原剂的能力较强；(5)耐氧化剂的能力较强；(6)耐糖性能一般；(7)盐浓度小于10%时,稳定性较好,盐浓度大于15%时,耐受力差；(8)柠檬酸对其稳定性影响不大；(9)K+、Ca2+、Zn2+、Mg2+对其稳定性影响不大,Cu2+、Fe3+、Fe2+对其有破坏作用。
Green wheat is an unique species bred in China. Compared to ordinary wheat species, green wheat is rich more than 20 types of trace elements, like selenium, zinc, iron, and calcium. In this thesis, the natural green pigments was extracted by percolation method from green wheat bran and fractionated by neutral ethanol, acidic ethanol solution successively. The resulting rough extract was separated by macro-porous resin and thin-layer chromatography (TLC). The stability of the final extract was subsequently analyzed by ultraviolet and visible spectrometry (UV) and infrared spectrometry. The results are as follows:
1. Rough pigment extract was obtained. An optimized extraction parameters achieved by means of single-factor and orthogonal experiments were pH 1.0,70% ethanol solution, dried bran/solvent (1/10),60℃,1 hr, for two times. Extraction yield was 83%.
2. Brown precipitate was obtained from ethonal extract. The precipitate was soluble in alkaline water or acidic ethanol solution, but was insoluble in acidic water. Its Rf value was 0.39 determined by normal phase silica gel TLC, with chloroform:methane acid:ethyl acetate (16:3:1) as mobile phase. It was visualized by ferric chloride. This result was speculated that the brown precipitate were likely to contain phenol compounds.
3. Green solution that was not absorbed by AB-8 macro-porous resin was condensed and filtered, placed in a 4℃refrigerator while a white precipitate appeared. The white precipitate was dissolved using acidic ethanol, and subjected to reversed-phase silica gel thin layer chromatography with 15% acetonitrile and 2 drops of glacial acetic acid as mobile phase. Rfwas determined to be 0.3, though trailing phenomenon was severe and speckles are not strictly round. Absorption was observed under 254nm UV light. The reaction of Molish reagent indicated that it may not contain carbohydrate.
4. Other characteristics of white precipitate were as follows:The hue of the green pigment which exists in the acidic extraction part changed with different pH values. The pigment was green in the acidic pH, nearly colorless in the neutral pH; brown-red with precipitate in the alkaline pH. The ratio of the absorbance of the maximum and secondary absorption peak of the pigment changed with different pH values, which showed that the molecular structure of the pigment was changed with different pH values. The infrared spectrogram of the pigment and color reaction of alkaloid reagent indicated that it may contain amides structure. As a result, green pigment of green wheat was not anthocyanidin but alkaloids.
5. Optimum purification condition for rough pigment extract by AB-8 macro-porous resin was:ambient temperature, pH 1.0, absorption:0.612×100. Absorption period 24 hours, absorption rate 1.5 mL/min. Desorption was conducted using ethanol solvent in concentration order of 10%、30%、40%.
6. Study on stability of green pigment showed that:(1)more stable in in-door natural lighted or dark conditions; (2)susceptible to high temperature, more stable at 20-80℃; at 100℃, dramatic increase in absorption was observed; (3)stable at high acidic condition (pH 1.0-4.0); (4)stable to reducing agents; (5)stable to oxidizing agents; (6)some degree of sugar resistant; (7)more stable when salinity was below 10%, unstable when above 15%; (8)citric acid had negligible effect on its stability; (9)K+、Ca2+、Zn2+、Mg2+ had slight effect on its stability, Cu2+、Fe3+、Fe2+ had destructive effect on its stability.
引文
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