基于天然低共熔溶剂的甜叶菊中甜菊糖绿色提取方法及优化

doi:10.11983/CBB21064

摘要/Abstract

摘要： 尝试利用天然低共熔溶剂(NADES)提取甜叶菊(Stevia rebaudiana)中的甜菊糖, 探索一种高效、绿色和环保的甜菊糖提取新方法。以甜叶菊干叶为原料, 对照传统提取溶剂水, 以甜菊糖中甜菊苷和莱鲍迪苷A的提取浓度作为指标, 筛选出最优的NADES提取配方, 然后通过Box-Behnken响应面法对NADES提取甜叶菊中甜菊糖的工艺条件进行筛选优化。结果表明, 提取效率最高的NADES配方为1,2-丙二醇:甘油:水=8:1:1 (v/v/v), 提取的甜菊苷浓度为2.59 mg∙mL^-1, 比水提取高16.40%, 提取的莱鲍迪苷A浓度为1.06 mg∙mL^-1, 比水提取高12.62%; 通过响应面法得到最优提取条件: 提取时间90分钟, 提取温度60°C, 超声功率为80 J∙s^-1, 预测甜菊苷提取浓度为3.49 mg∙mL^-1, 莱鲍迪苷A提取浓度为1.43 mg∙mL^-1, 与实验验证值(甜菊苷浓度为3.48 mg∙mL^-1, 莱鲍迪苷A浓度为1.42 mg∙mL^-1)接近。在最优条件下, 甜菊苷提取浓度比初始条件提高了34.36%, 莱鲍迪甘A提取浓度比初始条件提高了33.96%。NADES绿色环保, 且提取效率高于传统溶剂, 可用于甜叶菊中甜菊糖的绿色提取; 同时, 该提取方法可为后续推广至其它大宗经济植物类天然产物的绿色工业生产提供参考。

关键词: 天然低共熔溶剂, 甜叶菊, 甜菊苷, 莱鲍迪苷A, 绿色提取

Abstract: Natural deep eutectic solvent (NADES) was used to extract steviosides from Stevia rebaudiana as an efficient, green and environmentally friendly new method. Compared with traditional solvent water, the optimal NADES formula was selected to extract S. rebaudiana dry leaves by measuring the concentration of stevioside and rebaudioside A, two main components in steviosides. The extraction conditions of NADES extracting steviosides were optimized by Box-Behnken design from response surface methodology. The best NADES formula for steviosides extraction was selected: 1,2-propanediol:glycerol:water=8:1:1 (v/v/v), the extraction concentration of stevioside was 2.59 mg∙mL^-1 (16.40% higher than water), and the extraction concentration of rebaudioside A was 1.06 mg∙mL^-1(12.62% higher than water). The NADES extraction conditions were optimized by response surface methodology: extraction time was 90 min, extraction temperature was 60°C, ultrasonic power was 80 J∙s^-1, the predicted extraction concentration of stevioside was 3.49 mg∙mL^-1 and the concentration of rebaudioside A was 1.43 mg∙mL^-1, which was close to the experimental verification value (the concentration of stevioside is 3.48 mg∙mL^-1, the concentration of rebaudioside A is 1.42 mg∙mL^-1). Under the best condition, the extraction of stevioside was 34.36% higher than the initial condition, and the extraction of rebaudioside A was 33.96% higher than the initial condition. NADES is environmentally friendly and has higher extraction efficiency than traditional solvents. It can be used for the green extraction of steviosides from S. rebaudiana. At the same time, it provides a promising prospect for the green extraction of natural products from other bulk economic plants.

Key words: NADES, Stevia rebaudiana, stevioside, rebaudioside A, green extraction

缪晴, 萨比哈·帕合尔丁, 曾思瑀, 潘琪芳. 基于天然低共熔溶剂的甜叶菊中甜菊糖绿色提取方法及优化. 植物学报, 2021, 56(6): 722-731.

Qing Miao, Pa-he-er-ding Sabiha, Siyu Zeng, Qifang Pan. Green Extraction Method and Optimization of Steviosides from Stevia rebaudiana by Natural Deep Eutectic Solvent. Chinese Bulletin of Botany, 2021, 56(6): 722-731.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21064

https://www.chinbullbotany.com/CN/Y2021/V56/I6/722

图/表 14

表1 不同组合的天然低共熔溶剂

Table 1 Different combinations of natural deep eutectic solvents

No.	Component 1	Component 2	Component 3	Mole ratio (v/v)
NADES-1	1,2-propanediol	Glycerol	Water	8:1:1
NADES-2	1,2-propanediol	Glycerol	Water	1:1:1
NADES-3	1,2-propanediol	Glucose	Lactic acid	3:1:5
NADES-4	1,2-propanediol	Water		2:1
NADES-5	1,2-propanediol	Glycerol		1:2
NADES-6	Glycerol	Water		9:1

表2 标准品溶液浓度

Table 2 Standard solution concentration

No.	1	2	3	4	5	6
Stevioside (mg∙mL^-1)	4	2	1	0.5	0.25	0.125
Rebaudioside A (mg∙mL^-1)	3	1.5	0.75	0.375	0.1875	0.09375

表3 响应面试验的因素与水平

Table 3 Factors and levels of response surface methodology

Level	Factor
Level	A: Temperature (°C)	B: Power (J∙s^-1)	C: Time (min)
-1	60	50	30
0	70	65	60
1	80	80	90

表4 梯度洗脱程序

Table 4 Gradient elution procedure

Time (min)	Mobile phase C (%)	Mobile phase B (%)
0	80	20
3	80	20
15	30	70
20	30	70
20.1	80	20
27	80	20

图1 用不同溶剂提取的甜菊苷浓度 * P<0.05; NADES-1-6见表1。

Figure 1 Extraction concentration of stevioside in different solvents * P<0.05; NADES-1-6 are shown in Table 1.

图2 用不同溶剂提取的莱鲍迪苷A浓度 * P<0.05; NADES-1-6见表1。

Figure 2 Extraction concentration of rebaudioside A in dif- ferent solvents * P<0.05; NADES-1-6 are shown in Table 1.

图3 超声功率对甜菊苷(A)和莱鲍迪苷A (B)浓度的影响不同小写字母表示差异显著(P<0.05)。

Figure 3 The effects of ultrasonic power on the concentration of stevioside (A) and rebaudioside A (B) Different lowercase letters indicate significant differences (P< 0.05).

图4 提取温度对甜菊苷(A)和莱鲍迪苷A (B)浓度的影响不同小写字母表示差异显著(P<0.05)。

Figure 4 The effects of extraction temperature on the concentration of stevioside (A) and rebaudioside A (B) Different lowercase letters indicate significant differences (P< 0.05).

图5 提取时间对甜菊苷(A)和莱鲍迪苷A浓度(B)的影响不同小写字母表示差异显著(P<0.05)。

Figure 5 The effects of extraction time on the concentration of stevioside (A) and rebaudioside A (B) Different lowercase letters indicate significant differences (P< 0.05).

表5 响应面实验结果

Table 5 Response surface experimental results

Run units	A: Tempe- rature (°C)	B: Power (J∙s^-1)	C: Time (min)	Stevioside concentration (mg∙mL^-1)	Rebaudioside A concentration (mg∙mL^-1)
1	70	50	30	2.30	1.11
2	80	65	90	2.08	0.90
3	70	80	90	2.77	1.03
4	70	50	90	0.11	0.09
5	70	80	30	1.71	0.70
6	60	65	90	2.57	1.18
7	60	65	30	2.59	1.06
8	80	80	60	2.00	0.83
9	60	80	60	2.52	1.02
10	70	65	60	1.52	0.64
11	70	65	60	1.52	0.64
12	70	65	60	1.52	0.64
13	80	50	60	0.06	0.02
14	70	65	60	1.52	0.64
15	70	65	60	1.52	0.64
16	80	65	30	1.31	0.65
17	60	50	60	0.49	0.35

表6 甜菊苷浓度的回归模型方差分析

Table 6 Analysis of the model variances of stevioside concentration

Source	Sum of squares	Degree of freedom	Mean square	F value	P-value
Model	9.98	9	1.11	8.00	0.0060**
A: Temperature	0.9351	1	0.9351	6.75	0.0355*
B: Power	4.58	1	4.58	33.08	0.0007**
C: Time	0.0188	1	0.0188	0.1355	0.7237
AB	0.0018	1	0.0018	0.0128	0.9130
AC	0.1520	1	0.1520	1.10	0.3297
BC	2.64	1	2.64	19.09	0.0033**
A²	0.0265	1	0.0265	0.1910	0.6752
B²	0.4768	1	0.4768	3.44	0.1059
C²	1.21	1	1.21	8.71	0.0214*
Residual	0.9697	7	0.1385
Lack of fit	0.9697	3	0.3232
Pure error	0.0000	4	0.0000
Cor total	10.95	16
R²	0.9114
R_adj²	0.7975
R_pred²	-0.4173
Adeq precision	10.9996

表7 莱鲍迪苷A浓度的回归模型方差分析

Table 7 Analysis of the model variances of rebaudioside A concentration

Source	Sum of squares	Degree of freedom	Mean square	F value	P-value
Model	1.51	9	0.1682	4.53	0.0294*
A: Temperature	0.1834	1	0.1834	4.94	0.0616
B: Power	0.4988	1	0.4988	13.45	0.0080**
C: Time	0.0126	1	0.0126	0.3396	0.5783
AB	0.0044	1	0.0044	0.1185	0.7408
AC	0.0040	1	0.0040	0.1083	0.7517
BC	0.4567	1	0.4567	12.31	0.0099**
A²	0.0173	1	0.0173	0.4660	0.5168
B²	0.0951	1	0.0951	2.56	0.1534
C²	0.2538	1	0.2538	6.84	0.0346*
Residual	0.2597	7	0.0371
Lack of fit	0.2597	3	0.0866
Pure error	0.0000	4	0.0000
Cor total	1.77	16
R²	0.8536
R_adj²	0.6653
R_pred²	-1.3429
Adeq precision	7.9551

图6 提取温度、超声功率和提取时间对甜菊苷和莱鲍迪苷A的响应面分析 (A) 温度与功率的交互作用对甜菊苷提取影响的响应面; (B) 温度与时间的交互作用对甜菊苷提取影响的响应面; (C) 功率与时间的交互作用对甜菊苷提取影响的响应面; (D) 温度与功率的交互作用对莱鲍迪苷A提取影响的响应面; (E) 温度与时间的交互作用对莱鲍迪苷A提取影响的响应面; (F) 功率与时间的交互作用对莱鲍迪苷A提取影响的响应面

Figure 6 Response surface analysis of extraction temperature, ultrasonic power and extraction time for stevioside and rebaudioside A (A) Response surface (3D) showing effect of temperature and power on stevioside; (B) Response surface (3D) showing effect of temperature and time on stevioside; (C) Response surface (3D) showing effect of power and time on stevioside; (D) Response surface (3D) showing effect of temperature and power on rebaudioside A; (E) Response surface (3D) showing effect of temperature and time on rebaudioside A; (F) Response surface (3D) showing effect of power and time on rebaudioside A

表8 不同提取条件对甜菊苷和莱鲍迪苷A提取浓度比较

Table 8 Extraction concentration of stevioside and rebaudioside A by different extraction conditions

	Temperature (°C)	Power (J∙s^-1)	Time (min)	Stevioside concentration (mg∙mL^-1)	Rebaudioside A concentration (mg∙mL^-1)
Initial condition	60	65	30	2.59±0.04	1.06±0.08
Optimal condition	60	80	90	3.48±0.36*	1.42±0.18*

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