论文总字数：17268字

摘 要

黑蒜是因具有高抗氧化性，抗癌，抗衰老的功能而备受关注的新养生食材，但是在制备黑蒜时，无法避免的美拉德反应也会产生某些有害的产物如丙烯酰胺，5-羟甲基糠醛，呋喃酮等。本实验采用高效液相色谱技术，建立黑蒜中5-羟甲基糠醛的含量测定方法，并对样品前处理提取步骤进行优化，以提高5-羟甲基糠醛的提取率，提高实验的准确性。最后检测不同产地黑蒜5-羟甲基糠醛的含量，并进行比较。结果显示，使用色谱柱Sepax HP-C₁₈(4.6×250 mm, 5 μm)，以0.1%甲酸甲醇-0.1%甲酸水（5:95）为流动相，流速为1.0 mL/min,进样量为20 μL，在284 nm检测波长下，2.00-64.04 μg/mL (r=0.99996)内呈良好的线性关系；且在使用LC-C18小柱，超声20 min，提取3次的条件下，回收率最高。云南独头黑蒜，云南多瓣黑蒜，山东多瓣黑蒜中5-羟甲基含量分别为259.21，1777.49，644.5 mg/Kg，不同产地与品种的黑蒜5-羟甲基糠醛含量有明显差别，其中以云南多瓣黑蒜中5-羟甲基糠醛的含量最高。

关键词：黑蒜 5-羟甲基糠醛 高效液相色谱法

Determination of 5-Hydroxymethyl Furfural Content in Black Garlic by HPLC

Abstract

Black garlic, as a new garlic product attracts attention for its beneficial functions, including antibacrerial, antioxidant and anticancer activities,also be given some harmful products through Maillard reaction, such as acrylamide, 5-Hydroxymethyl Furfural (5-HMF), furosine, etc. This experiment adopts HPLC to the establish a method of determining 5 - HMF content in the black garlic. In order to improve the accuracy of the experiment, the experiment also optimizes the sample pretreatment step and improve the extraction rate of 5 - HMF. Finally, the content of 5-hydroxymethyl furfural in garlic in different areas of productions was tested and compared. The result shows when used Sepax HP-C₁₈(4.6×250 mm, 5 μm), serving 0.1% formic acid methanol solution-0.1% formic acid solution(5:95) as a mobile phase at a flow rate of 1.0 mL/min and sample loading was 20 μL. While the detection wavelength was 284 nm, the liner range of 5-HMF was 2.00-64.04 μg/mL (r=0.99996). In addition, using LC-C18 SPE, ultrasonic time was 20 min, the recovery rate was the highest with the extraction of 3 times.The content of 5-HMF in Yunnan single black garlic, Yunnan multi-pedaled black garlic, Shandong multi-pedaled black garlic is 259.21，1777.49 and 644.5 mg/Kg respectively.It is clear that the contents of 5-HMF in different breeds and places of production are significantly different, in which one from Yunnan multi-pedaled black garlic is the highest.

Key words: black garlic; 5-Hydroxymethyl Furfural (5-HMF); HPLC

目 录

摘 要......................................................................................................Ⅰ

Abstract...................................................................................................Ⅱ

第一章 文献综述......................................................................................1

1.1 黑蒜概述.............................................................................................................1

1.2 5-羟甲基糠醛概述...............................................................................................1

1.3 测定5-羟甲基糠醛技术.....................................................................................2

1.3.1 紫外分光光度法（UV）................................................................................2

1.3.2 高效液相色谱法（HPLC）............................................................................2

1.3.3 液相色谱质谱法（LC-MS）..........................................................................3

1.3.4 气相色谱质谱法（GC-MS）.........................................................................3

1.4 高效液相色谱技术（HPLC）............................................................................3

1.4.1 HPLC原理....................................................................................................3

1.4.2 HPLC优点....................................................................................................3

1.4.3 使用HPLC注意事项...................................................................................4

1.5 固相萃取技术.....................................................................................................4

1.5.1 固相萃取技术概述......................................................................................4

1.5.2 固相萃取技术原理......................................................................................5

1.6 本课题研究的目的及意义.................................................................................5

1.7 本课题的研究内容.............................................................................................5

第二章 实验材料与方法..........................................................................6

2.1 实验仪器与设备.................................................................................................6

2.2 实验试剂.............................................................................................................7

2.3 实验样品.............................................................................................................7

2.4 HPLC条件确定...................................................................................................7

2.4.1 5-羟甲基糠醛最大吸收波长的测定............................................................7

2.4.2 5-羟甲基糠醛出峰时间测定........................................................................8

2.4.3 色谱条件的测定..........................................................................................8

2.5 黑蒜中5-HMF含量测定...............................................................................................8

2.5.1 样品制备方法..............................................................................................8

2.5.2 供试液制备方法..........................................................................................8

2.5.3 标准曲线及相关参数的测定......................................................................8

2.5.4 方法的检出限和定量限测定......................................................................8

2.5.5 精密度试验..................................................................................................9

2.5.6 回收率考察..................................................................................................9

2.5.7 提取条件优化..............................................................................................9

2.5.8 不同产地黑蒜中5-HMF含量测定............................................................9

第三章 实验结果与讨论.........................................................................10

3.1 HPLC参数确定.................................................................................................10

3.2 标准曲线及相关系数.......................................................................................11

3.3 方法的检出限和定量限...................................................................................12

3.4 精密度...............................................................................................................12

3.5 回收率考察.......................................................................................................12

3.6 提取条件优化结果...........................................................................................13

3.6.1 SPE小柱优化..............................................................................................13

3.6.2 超声时间优化............................................................................................13

3.6.3 提取次数优化............................................................................................14

3.6.4 优化总结....................................................................................................15

3.7 不同产地黑蒜中5-HMF含量..........................................................................15

第四章 结论与展望.................................................................................17

4.1 结论...................................................................................................................17

4.2 展望...................................................................................................................17

参考文献...................................................................................................18

致谢...........................................................................................................21

1. 文献综述

1.1 黑蒜概述

大蒜在我国是有着悠久种植历史的药食同源的农作物，黑蒜（black garlic）的原料是大蒜，再经过生物发酵加工技术加工大蒜，将其制得名为黑蒜的新型食材。黑蒜具很高的营养价值，主要营养成分有异亮氨酸，赖氨酸等多种氨基酸，蛋白质，脂肪，B族维生素，膳食纤维，糖等[^[1]]，脂肪和水分含量降低，易于储存和运输，微量元素如钙，铁，锌等显著提高，综合营养价值明显高与普通的新鲜大蒜[^[2]]。它还具有良好的药用价值，从2006年黑蒜被研究出有强大的抗肿瘤能力后，人们又相继发现它的其他作用，如强力调节血糖水平，延缓糖尿病发展[^[3]]，显著抑制胰腺癌Panc-1细胞的生长能力[^[4]]，提高人体免疫系统[^[5]]，降低胆固醇水平，有效防止肥胖等，进而黑蒜受到越来越多中老年人的青睐与喜爱[^[6]]。

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