5 6 Vol. 5 No. 6 2014 6 Journal of Food Safety and Quality Jun., 2014 王廷璞 1* 1,, 赵强 2, 袁建军 (1., 741001; 2., 810001) 1 摘要 : 目的 方法,, 4,, Box-Behnken 结果 45% 85 2.5 h 1:40, 15.7 mg/g, 1.57%, 98.2%, (16.4 mg/g) 4.6% 结论 Box-Behnken,, 关键词 : ; ; Optimization of technology for extracting total flavonoids from tartary buckwheat shell by response surface methodology WANG Ting-Pu 1*, ZHAO Qiang 1, 2, YUAN Jian-Jun 1 (1. College of Life Science and Chemistry, Tianshui Normal University, Tianshui 741001, China; 2. Northwest Plateau Institute of Biology, The Chinese Academy of Sciences, Xining 810001, China) ABSTRACT: Objective To optimize the extraction technology of total flavonoids from tartary buckwheat shell. Methods The maximum extraction rate and content of total flavonoids were compared by soak extraction method, constant temperature oscillation extraction method, soxhlet extraction method, high-pressure extraction method, and ultrasonic wave extraction method. Soak extraction method was selected as the optimization extraction method. Effects of ethanol concentration, extraction temperature, extraction time and liquid to solid ratio on total flavonoids extraction rate were studied using response surface methodology, and the experiment mathematical model was established according to Box-Behnken central composite experimental design. Results The optimum extraction conditions are ethanol concentration 45%, extraction temperature85, extraction time 2.5 h, and the liquid to solid ratio 1:40. Under the optimum conditions, the flavonoids yield is 15.7 mg/g, the maximum extraction rate is 1.57%, close to predictive value16.4 mg/g. Conclusion The combination of Box-Behnken design and response surface analysis can well optimize the extraction technology of total flavonoids from tartary buckwheat shell. KEY WORDS: tartary buckwheat shell; total flavonoids; response surface methodology 基金项目 : (31360603) Fund: Supported by the National Natural Science Foundation of China (31360603) * 通讯作者 :,, E-mail: wangtp002@163.com *Corresponding author: WANG Ting-Pu, Research fellow, College of Life Science and Chemistry, TianshuiNormal University, Tianshui741001, China. E-mail: wangtp002@163.com
1630 5 1 引言 (Polygonaceae),,,,, [1] :,,, [2],, [3], ( ), [4],, (response surface methodology, RSM),,,,,, [5], [6-7], [8],,, 2 材料与方法 2.1 实验材料 (,,, ) 2.2 仪器及试剂 722 ( ); KQ-500E ( ); TDZ5-WS ( ); AUY220 ( ); DK-H24 ( ); ; (Shanghai Tauto Biotechoco Ltd. China); (AR, ); (AR, ); (AR, ); (AR, ) 2.3 实验方法 2.3.1 苦荞壳的制备, 12 h,, ;, 2.3.2 标准曲线的建立 2.3.2.1 10.6 mg, 30%, 60, 25 ml,, 0.424 mg/ml 2.3.2.2 0 0.2 0.4 0.6 0.8 1.0 1.2 ml,, 30% 1.2 ml, 5% 0.2 ml, 5 min; 10% 0.2 ml, 5 min; 1 mol/l 2 ml,, 30% 1.4 ml, 10 min 510 nm, C, A [9] 2.3.3 苦荞壳中总黄酮提取方法的选择 2.3.3.1 1.00 g, 30 ml 80%, 80 30 min, (3500 r/min)10 min, 80% 50 ml, 1 ml, OD [10] 2.3.3.2 1.00 g, 30 ml80%, 80 2 h, (3500 r/min)10 min, 80% 50 ml, 1 ml, OD [11] 2.3.3.3 1.00 g, 30 ml80%
6, : 1631, 80 30 min, (3500 r/min)10 min, 80% 50 ml, 1 ml, OD [12] 2.3.3.4 1.00 g, 30 ml80%, 121 30 min, (3500 r/min)10 min, 80% 50 ml, 1 ml, OD 2.3.3.5 1.00 g, 30 ml 80%, (7-8 ),, (3500 r/min)10 min, 80% 50 ml, 1 ml, OD [13] 2.3.4 苦荞壳中总黄酮的提取和含量的测定 2.3.4.1 1.0 g,,,,, 2.3.4.2 [14] 1.0 ml, 80%, OD, (mg) (mg)= CV 1 V/V 2, (%)=(CV 1 V/V 2 )/m 100% C: (mg/ml); m: (mg); V 1 : (ml); V 2 : (ml); V: (ml) 2.3.5 单因素实验,,, 2.3.5.1 15 (1.0 g), 20% 40% 60% 80% 100%, 1: 50, 3, 80, 2 h, 10 min(3500 r/min), 50 ml, OD 2.3.5.2 15 (1.0 g), 1: 10 1: 20 1: 30 1: 40 1: 50, 40%, 3, 80, 2 h, 10 min(3500 r/min), 50 ml, OD 2.3.5.3 15 (1.0g), 40%, 1: 30, 80,, 0.5 1 1.5 2.0 2.5 h, 10 min(3500 r/min), 50 ml, OD 2.3.5.4 15 (1.0g), 40%, 1: 30, 3, 50 60 70 80 90 2.5 h, 10 min(3500 r/min), 50 ml, OD 2.3.6 响应面实验设计 1,, 4 ( ),, 4 3 29 (5 ) [15] Table 1 表 1 响应面因素水平表 Factors and levels of response surface methodology Level A (h) Extraction time B ( ) Extraction temperature C (%) Ethanol concentration D (g/ml) Liquid to solid ratio +1 3.0 85 60 45 0 2.5 80 40 40-1 2.0 75 20 35 0.5 5 20 5
1632 5 2.3.7 验证性实验 Design-Expert 8.0,, 6, OD 2.3.8 精密度实验 0.424 mg/ ml 0.4 ml, 5 ml, 6, OD 2.3.9 稳定性实验 1 ml, Na 2 NO 2 -Al(NO 3 ) 3 -NaOH, 10 min, OD, 5 2.3.10 1 g, 6, 0.424 mg/ml 1 ml,, 1 ml, OD, [16] =( )/ 100% : ;,, 1 Fig. 1 Regression curve of ruti 3 结果与分析 3.1 芦丁标准曲线 C A ( 1 ), : y=9.6677x+0.0015, R 2 =0.9992, 3.2 不同方法提取苦荞壳中总黄酮的实验结果和分析 2 2,,, 1.19%, 12.9 mg/g 2 Fig. 2 Different extraction methods on effect of the extraction rate of tartary buckwheat shell total flavonoids Table 2 表 2 不同提取方法对苦荞壳中总黄酮含量提取的差异显著性比较 Comparison of significance of different extraction methods of total flavonoids in buckwheat shell extracts. Process (mg/g) Mean value 5% 5% Significant level 1% 1% Very significant level 12.9 a A 10.1 b B 7.23 c C 6.794 c C 6.59 c C
6, : 1633 3.3 单因素实验结果 3.3.1 乙醇浓度对苦荞壳中总黄酮提取率的影响 3,,, 40%, : 1.29%,,,,,, 3.3.3 提取时间对苦荞壳中总黄酮提取率的影响 5, 1~2 h, 2.5 3 h, 2.5 h, 1.44%; 3.5 h, 1.60%, 2.5 h 5 Fig. 5 Different extraction time on the effect of the extraction rate of tartary buckwheat shell total flavonoids 3 Fig. 3 Different ethanol concentrations on the effect of extraction rate of tartary buckwheat shell total flavonoids 3.3.2 料液比对苦荞壳中总黄酮提取率的影响 4,, 35,, 1: 40 3.3.4 提取温度对苦荞壳中总黄酮提取率的影响 6,, :,,,,,,,, ;, 80 4 Fig. 4 Different liquid to solid ratios on the effect of the extraction rate of tartary buckwheat shell total flavonoids 6 Fig. 6 Different extraction temperature on the effect of the extraction rate of tartary buckwheat shell total flavonoids
1634 5 3.4 响应面实验结果 Design-Expert 8.0 ( 3), (Y),, : Y=14.50+1.26A+0.39B-0.084C+1.93D+0.058AB+ 0.29AC+0.61AD+0.55BC+0.23BD-0.32CD-3.07A 2-1.5 4B 2-0.58C 2-0.39D 2 R 2 =0.8473, 84.73%, CV,, CV 10.93%,, [17] 4, Model (Prob F) 0.01( ),,, ;, 4, (A 2 ) P 0.01, ; (B 2 ) 0.05 P 0.01, ; P 0.05, Table 3 表 3 响应面实验数据 Data of response surface methodology No. A (%) Ethanol concentration B (g/ml) Liquid to solid ratio C (h) Extraction time D ( ) Extraction temperature Y (mg/g) Total flavonoid content 1 20.00 40.00 2.00 80.00 10.098 2 40.00 40.00 3.00 75.00 11.081 3 40.00 45.00 2.00 80.00 11.235 4 40.00 40.00 2.50 80.00 14.468 5 40.00 35.00 2.50 75.00 9.865 6 40.00 40.00 2.00 75.00 12.425 7 40.00 40.00 2.50 80.00 12.606 8 40.00 40.00 2.50 80.00 14.804 9 40.00 40.00 2.50 80.00 14.106 10 40.00 40.00 3.00 85.00 15.114 11 40.00 40.00 2.50 80.00 16.537 12 60.00 40.00 2.50 75.00 10.434 13 20.00 45.00 2.50 80.00 9.218 14 40.00 35.00 2.00 80.00 10.977 15 40.00 45.00 2.50 85.00 15.528 16 40.00 35.00 3.00 80.00 11.546 17 20.00 35.00 2.50 80.00 9.089 18 60.00 35.00 2.50 80.00 11.546 19 20.00 40.00 3.00 80.00 9.322 20 20.00 40.00 2.50 75.00 8.728 21 60.00 40.00 3.00 80.00 11.986 22 20.00 40.00 2.50 85.00 9.529 23 40.00 45.00 2.50 75.00 10.176 24 40.00 40.00 2.00 85.00 17.726 25 60.00 40.00 2.00 80.00 11.598 26 60.00 45.00 2.50 80.00 11.908 27 60.00 40.00 2.50 85.00 13.667 28 40.00 45.00 3.00 80.00 14.002 29 40.00 35.00 2.50 85.00 14.313
6, : 1635 Table 4 表 4 方差分析 Analysis of mean square Source Sum of Squares df Mean Square F F Value Prob F Model 137.85 14 9.85 5.55 0.0014 A 19.14 1 19.14 10.78 0.0054 B 1.87 1 1.87 1.05 0.3227 C 0.085 1 0.085 0.048 0.8303 D 44.73 1 44.73 25.20 0.0002 AB 0.014 1 0.014 7.646E-003 0.9316 AC 0.34 1 0.34 0.19 0.6689 AD 1.48 1 1.48 0.83 0.3769 BC 1.21 1 1.21 0.68 0.4233 BD 0.20 1 0.20 0.12 0.7395 CD 0.40 1 0.40 0.23 0.6415 A 2 61.32 1 61.32 34.54 < 0.0001 B 2 15.38 1 15.38 8.66 0.0107 C 2 2.15 1 2.15 1.21 0.2892 D 2 1.00 1 1.00 0.56 0.4663 Residual 24.85 14 1.78 Lack of Fit 16.87 10 1.69 0.84 0.6247 Pure Error 7.99 4 2.00 Cor total 162.71 28 mean 12.19 R-Squared 0.8473 CV % 10.93 P, : D( ) A( ) B( ) C( ) 3.5 苦荞壳中总黄酮提取的响应面法分析与优化 7,, 1: 40~1: 43,,,, 8,, 38%~52%,,, 7 Fig. 7 Interactive influence of both extraction time and liquid to solid ratio on the content of total flavonoids
食品安全质量检测学报 1636 第5卷 量的最大值 图 8 提取温度和乙醇浓度对总黄酮含量的交互影响 Fig. 8 Interactive influence of both extraction temperatures and ethanol concentration on the content of total flavonoids 由图 9 可见, 总黄酮含量随提取时间的延长和温 度的提高快速增加, 在达到峰值以后, 开始快速下 图 10 提取温度和料液比对总黄酮含量的交互影响 Fig. 10 Interactive influence of both extraction temperatures and liquid to solid ratio on the content of total flavonoids 降 根据浸提动力学理论, 时间的延长有助于总黄酮 的充分扩散溶出, 同时温度的升高使得分子解附和 扩散运动速度加快, 从而提高了总黄酮的浸出速率 和得率 因此, 适当延长时间和提高温度有助于提高 总黄酮的提取率和浸出量 由图 11 可见, 响应面开口向下, 随着每个因素 的增大, 响应值总黄酮含量也增大, 当总黄酮含量 增大到极值后, 随着乙醇浓度和料液比的增大, 总 黄酮含量逐渐减小 该模型有稳定点, 且稳定点是 最大值 图 9 提取时间和提取温度对总黄酮含量的交互影响 Fig. 9 Interactive influence of both extraction time and extraction temperatures on the content of total flavonoids 图 11 乙醇浓度和料液比对总黄酮含量的交互影响 Fig. 11 Interactive influence of both ethanol concentration and ratio of material to solvent on the content of total flavonoids 由图 10 可见, 总黄酮含量随提取温度的升高 由图 12 可见, 乙醇浓度一定时, 随着提取时间 呈直线上升趋势, 随料液比的增大呈先升高后下 的增加, 总黄酮含量发生很小的变化; 提取时间一定, 降的趋势 在提取温度最高时, 可以取得总黄酮含 总黄酮含量随着乙醇浓度的升高而逐渐变大, 到达
6, : 1637,,,,, 12 Fig. 12 Interactive influence of both extraction time and ethanol concentration on the content of total flavonoids 7~12,, ;,, [18], ;, : AD BC CD AC BD AB 3.6 苦荞壳中总黄酮提取条件的确定 : 46%, 85, 2.4 h, 1: 41,, 16.4 mg/g 3.7 验证性实验 Design-Expert 8.0,,, : 45%, 85, 1: 40, 2.5 h,, 15.7 mg/g, 4.3%,, 3.8 精密度实验 5, 4.81 mg/g, RSD=4.35%,, 3.9 稳定性实验 6, 13.82 mg/g, RSD=8.50%, 10 min, 3.10 回收率实验 7,, 98.16%, RSD 1.59%,,, Table 5 表 5 精密度实验结果 Precision of the experimental results No. (ml) Volume of Standard liquid OD (mg/g) Total flavonoid content (mg/g) Mean value RSD(%) 1 0.4 0.190 4.874 2 0.4 0.198 5.081 3 0.4 0.185 4.745 4 0.4 0.179 4.590 4.81 4.35 5 0.4 0.198 5.081 6 0.4 0.175 4.486
1638 5 Table 6 表 6 稳定性实验结果 Stability of the experimental results (min) Time (ml) Volume OD (mg/g) Total flavonoid content (mg/g) Mean value RSD(%) 0 1 0.601 15.502 10 1 0.552 14.235 20 1 0.543 14.002 13.83 8.50 30 1 0.500 12.890 40 1 0.486 12.529 表 7 回收率实验结果 Table 7 The recovery test results (g) Buckwheat shell quality (mg/g) Total flavonoid content (mg) Rutin content (mg/g) Estimated value (%) Recovery (%) Average recovery RSD(%) 1.0004 14.985 0.424 15.399 97.5 1.0001 14.675 0.424 15.089 97.6 1.0002 14.823 0.424 15.244 99.2 98.16 1.59 1.0002 14.801 0.424 15.218 98.4 4 结论, :,,, ;, [17],,,,,,,,,,,,,,,,,, [6-8, 19] 4, Box-Behnken,,, : 45% 85 2.5 h 1: 40, 15.7 mg/g, 参考文献 [1],,,. [J].
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1640 5 [J]., 2013, 06: 83 89. 作者简介 Zhao HF, Wei YM, Wang TP, et al. Study on optimization of extraction technology and detectionfor rutin from stigma maydis by response surface analysis [J]. Pratacultural Sci, 2013, 06: 83 89. ( 责任编辑 : 杨翠娜 ),, E-mail: wangtp002@163.com 酶技术在食品工程中的应用 专题征稿 2014 8 2014 7 30 E-mail 投稿方式 : www.chinafoodj.com Email tougao@chinafoodj.com 食品安全质量检测学报 编辑部