Optimization of Ultrasound-assisted and Pectinase-added Extraction for Uronic Acid from Auricularia auricula (L. ex Hook) Underw
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摘要:
目的 优化黑木耳糖醛酸提取工艺，提高黑木耳资源的开发、应用价值。 方法 通过比较热水、超声波、微波、光波、中性蛋白酶、纤维素酶、果胶酶等方法的黑木耳糖醛酸提取率，得到最佳提取方法；通过单因素试验比较黑木耳粒径、果胶酶添加量、液料比、提取温度、提取时间、pH值、超声功率、超声时间等对黑木耳糖醛酸提取率的影响，得到关键影响因素；在单因素实验基础上，以液料比、提取温度、超声功率和pH值为自变量，利用4因素3水平响应面法优化黑木耳糖醛酸提取工艺。 结果 超声波、微波、光波等3种物理破壁方法和中性蛋白酶、纤维素酶、果胶酶等3种生物酶法中分别以超声波法和果胶酶法最佳；单因素提取黑木耳糖醛酸的最佳条件分别为：黑木耳粒径58 μm、果胶酶0.25%、液料比100 mL·g-1、提取温度50℃、提取时间2 h、pH值5.50、超声功率540 W、超声时间30 min；确定响应面最优工艺参数为：液料比110 mL·g-1、pH5.60、超声功率540 W、提取温度49℃。本研究在超声波协同果胶酶提取黑木耳糖醛酸的最佳条件下，糖醛酸提取率达7.95‰，比传统热水法提高了201%。 结论 通过超声波与果胶酶协同提取超微粉碎的黑木耳粉的糖醛酸工艺，确定响应面最优工艺参数为：液料比110 mL·g-1、pH5.60、超声功率540 W、提取温度49℃。 Abstract:Objective To improve the development and application value of resourcos of Auricularia auricula (L. ex Hook) Underw, extraction process of uronic acid from Auricularia auricula (L. ex Hook) Underw was optimized. Method By comparing the uronic acid yield, applications of ultrasound, microwave, light, neutral protease, cellulase, and/or pectinase were incorporated in the extraction to maximize the production. Critical processing conditions including substrate particle size, pectinase dosage, solvent to substrate ratio, temperature, time, pH, ultrasonic power, and ultrasound application time were evaluated in a single factor test. The ultrasound-assisted process with added pectinase was selected for the process optimization using the response surface method with 4 factors and 3 levels. Result The optium method for extracting uronic acid from A.auricula were ultrasound and pectinase. The optimum extraction conditions were determined to include the substrate particle size of 58 μm, pectinase addition at 0.25%, solvent to substrate ratio at 100 mL·g-1, temperature at 50℃, 2 h process duration, pH at 5.50, and 540 W ultrasonic treatment for 30 min. The response surface experiment indicated that a solvent to substrate ratio of 110 mL·g-1, pH at 5.60, ultrasonic power of 540 W, and temperature at 49℃ would maximize the uronic acid yield at 7.95‰, which was 201% of what a traditional hot water method delivered. Conclusion The newly established highly efficient process to extract uronic acid from ultra-fine A. auricula powder applied 540 W ultrasound, added pectinase, used 110 mL per gram of substrate, adjusted pH to 5.60, and maintained a temperature at 49℃ throughout the process, which optimized the extration process of uronic acid of A.auricula, improved the economic value and provided reference for exploiting resources of A.auricula. -
Key words:
- Auricularia auricular /
- uronic acid /
- ultrasound /
- pectinase /
- process optimization
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图 1 不同粒径对糖醛酸提取率的影响
Figure 1. Effect of substrate particle size on uronic acid extraction rate
图 2 不同果胶酶添加量对糖醛酸提取率的影响
Figure 2. Effect of pectinase dosage on uronic acid extraction rate
图 3 不同液料比对糖醛酸提取率的影响
Figure 3. Effect of solvent-to-substrate ratio on uronic acid extraction rate
图 7 不同超声功率对糖醛酸提取率的影响
Figure 7. Effect of ultrasonic power on uronic acid extraction rate
图 8 不同超声时间对糖醛酸提取率的影响
Figure 8. Effect of ultrasonic application time on uronic acid extraction rate
图 9 响应面法优化不同因素对糖醛酸提取率的影响
Figure 9. Effect of various factors on uronic acid extraction rate based on response surface methodology
表 1 黑木耳糖醛酸提取条件
Table 1. Various methods to extract uronic acid from A. auricula
方法
Method提取条件
Extraction conditions热水Hot water 液料比50 mL·g-1, 温度80℃, 时间2.5 h 超声波Ultrasonic 液料比50 mL·g-1, 超声功率480 W, 超声时间20 min, 温度60℃ 微波Microwave 液料比50 mL·g-1, 微波功率600 W, 提取时间1.5 min/次，共提取3次(＜40℃) 光波Light wave 液料比50 mL·g-1, 提取功率400 W, 提取时间1.0 min/次，共提取3次(＜40℃) 中性蛋白酶Neutral protease 液料比50 mL·g-1, 酶用量120 U·g-1木耳(1.25%), 提取温度45℃, pH 7.0, 时间2.5 h 纤维素酶Cellulase 液料比50 mL·g-1, 酶用量100 U·g-1木耳(0.2%), 提取温度50℃, pH5.0, 时间2.5 h 果胶酶Pectinase 液料比50 mL·g-1, 酶用量100 U·g-1木耳(0.2%), 提取温度55℃, pH5.0, 时间2.5 h 
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表 2 黑木耳糖醛酸的单因素提取条件
Table 2. Conditions for uronic acid extraction from A. auricula by single-factor experiment
提取因素
Extraction factors因素水平
Factor level其他提取条件
Other extraction conditions粒径Particle size /μm 880, 600, 270, 212, 150, 150, 106, 75, 58 液料比50 mL·g-1, 超声功率480 W, 超声时间20 min, 酶用量0.25%, 提取温度55℃, pH 5.0, 提取时间2 h 果胶酶Pectinase /% 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00 粒径58 μm, 液料比50 mL·g-1, 超声功率480 W, 超声时间20 min, 提取温度55℃, pH 5.0, 提取时间2 h 液料比Liquid material ratio/(mL·g-1) 20, 40, 50, 60, 80, 100, 110, 120 粒径58 μm, 超声功率480 W, 超声时间20 min, 酶用量0.25%, 提取温度55℃, pH 5.0, 提取时间2 h 温度Temperature /℃ 40, 45, 50, 55, 60, 65 粒径58 μm, 液料比60 mL·g-1, 超声功率480 W, 超声时间20 min, 酶用量0.25%, pH 5.0, 提取时间2 h 提取时间Extraction time/h 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 粒径58 μm, 液料比60 mL·g-1, 超声功率480 W, 超声时间20 min, 酶用量0.25%, 提取温度50℃, pH 5.0 pH值pH value 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 粒径58 μm, 液料比60 mL·g-1, 超声功率480 W, 超声时间20 min, 酶用量0.25%, 提取温度50℃, 提取时间2 h 超声功率Ultrasound power/W 240, 360, 420, 480, 540, 600 粒径58μm, 液料比60 mL·g-1, 超声时间20 min, 酶用量0.25%, 提取温度50℃, pH 5.0, 提取时间2 h 超声时间Ultrasound time/min 10, 20, 30, 40, 50, 60 粒径58 μm, 液料比60 mL·g-1, 超声功率480 W, 酶用量0.25%, 提取温度50℃, pH 5.0, 提取时间2 h
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表 3 试验因素水平
Table 3. Codes for factors and levels of experiment
水平
Level液料比
Liquid material ratio/(mL·g-1)提取温度
Extract temperature/℃超声功率
Ultrasonic power/WpH值
pH value-1 90 45 480 5.0 0 100 50 540 5.5 1 110 55 600 6.0
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表 4 不同提取方法对黑木耳糖醛酸提取率影响
Table 4. Effect of method on uronic acid extraction rate
项目
Items热水
Hot water超声波
Ultrasonic微波
Microwave光波
Light wave中性蛋白酶
Neutral protease纤维素酶
Cellulase果胶酶
Pectinase糖醛酸
uronic acid/‰2.64±0.03e 3.94±0.12ab 3.44±0.06d 3.44±0.25cd 3.72±0.16bc 3.79±0.14b 3.96±0.02a 注：同行数据后不同小写字母表示差异显著(P＜0.05)。
Note：Different lowercase letters means significant differences in the same line (P＜0.05).
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表 5 响应面法试验方案及结果
Table 5. Response surface design and results on uronic acid extraction
序号
No.液料比
Liquid-material ratio/(mL·g-1)提取温度
Extract temperature/℃超声功率
Ultrasonic power/WpH值
pH value糖醛酸提取率
Uronic acid extraction rate/‰1 90 45 540 5.5 6.38±0.28 2 110 45 540 5.5 7.36±0.07 3 90 55 540 5.5 5.81±0.60 4 110 55 540 5.5 6.78±0.41 5 100 50 480 5.0 5.58±0.10 6 100 50 600 5.0 5.96±0.49 7 100 50 480 6.0 6.02±0.10 8 100 50 600 6.0 6.38±0.28 9 90 50 540 5.0 5.83±0.13 10 110 50 540 5.0 5.84±0.10 11 90 50 540 6.0 5.37±0.22 12 110 50 540 6.0 6.60±0.21 13 100 45 480 5.5 6.69±0.42 14 100 55 480 5.5 5.74±0.34 15 100 45 600 5.5 6.51±0.08 16 100 55 600 5.5 6.37±0.09 17 90 50 480 5.5 5.16±0.27 18 110 50 480 5.5 6.65±0.14 19 90 50 600 5.5 5.27±0.35 20 110 50 600 5.5 6.10±0.52 21 100 45 540 5.0 6.92±0.23 22 100 55 540 5.0 5.91±0.23 23 100 45 540 6.0 6.49±0.42 24 100 55 540 6.0 6.85±0.46 25 100 50 540 5.5 7.70±0.19 26 100 50 540 5.5 7.65±0.23 27 100 50 540 5.5 7.75±0.48 28 100 50 540 5.5 7.93±0.53 29 100 50 540 5.5 7.84±0.28
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表 6 回归模型方程方差分析
Table 6. Variance analysis of regression equation
方差来源
Source平方和
Sum of squares自由度
Degrees of freedom均方
Mean squareF值
F valueP值
Pvalue显著性
Significance模型Model 17.18 14 1.23 29.53 ＜0.0001 ** A 2.53 1 2.53 60.90 ＜0.0001 ** B 0.6960 1 0.6960 16.75 0.0011 ** C 0.0469 1 0.0469 1.13 0.3061 D 0.2324 1 0.2324 5.59 0.0330 * AB 0.0000 1 0.0000 0.0006 0.9808 AC 0.1089 1 0.1089 2.62 0.1277 AD 0.3721 1 0.3721 8.96 0.0097 ** BC 0.1640 1 0.1640 3.95 0.0668 BD 0.4692 1 0.4692 11.29 0.0047 ** CD 0.0001 1 0.0001 0.0024 0.9616 A2 5.65 1 5.65 136.11 ＜0.0001 ** B2 0.7999 1 0.7999 19.25 0.0006 ** C2 6.80 1 6.80 163.62 ＜0.0001 ** D2 4.78 1 4.78 115.12 ＜0.0001 ** 残差Residual 0.5816 14 0.0415 － － － 失拟项Lack of fit 0.5315 10 0.0531 4.24 0.0882 － 纯误差Pure error 0.0501 4 0.0125 － － － 总误差Cor total 17.76 28 － － － － 标准偏差Standard deviation 0.2038 － R2 0.9672 － － 平均值Average value 6.46 － RAdj2 0.9345 － － 变异系数Coefficient of variation/% 3.15 － RPred2 0.8232 － － 注：“－”表示无。*、**分别表示在0.05、0.01水平差异显著。
Note：‘－’means no. *，** means significant difference at 0. 05 and 0. 01 level，respectively.
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