超声耦合双水相体系提取茶皂素的工艺优化与机制探讨

doi:10.3864/j.issn.0578-1752.2022.01.014

Abstract

Abstract:

【Objective】 Tea saponin is a naturally active substance with wide application prospective, and its purity is the key factor limiting its application and value. In this study, the optimal conditions for ultrasonic-assisted two-phase extraction (UAATPE) of tea saponin from camellia meal were explored, and the possible extraction mechanism was discussed in order to develop an efficient, high-purity extraction technology, so as to provide a technical guidance for high-value utilization of camellia meal. 【Method】 On the basis of the single factor experimental results, the key factors affecting the yield of tea saponin were screened by Plackett Burman design, and the extraction process was optimized by Box-Behnken design. The extraction efficiency of tea saponin using the UAATPE method was evaluated by comparing the extraction yield and purity to the traditional ethanol extraction method and water extraction method. Scanning electron microscopy (SEM) was employed to analyze the microstructure of camellia meal, and the possible mechanism of extraction by the UAATPE method was discussed. 【Result】 The key factors affecting the yield of tea saponin were mass fraction of ethanol, mass fraction of ammonium sulfate, and ultrasonic time. The process parameters optimized by Box-Behnken design were as follows: an ethanol content of 27.50% (W/W), an ammonium sulfate content of 19.60% (W/W), a liquid-solid ratio of 50﹕1, an ultrasonic time of 32 min, an ultrasonic power of 300 W and an extraction temperature of 60℃, and the yield of tea saponin at (26.21±0.54)% was achieved under the optimized conditions. Compared with the traditional method of ethanol extraction, the purity of tea saponin extracted by the UAATPE method increased by 6.57% (P<0.05), although the difference of the yield of tea saponin extracted by these two methods was not significant. Compared with the traditional method of water extraction, the yield and purity of tea saponin extracted using the UAATPE method were increased by 17.74% and 40.23%, respectively (P<0.01). The microstructure showed that owing to the effect of the camellia meal treated by the UAATPE method, the ultrasonic cavitation accelerated the tissue damage of the camellia meal, producing a large number of voids and strong surface shrinkage, which effectively promoted the release of tea saponin in the camellia meal. In the process of the UAATPE extraction, the tea saponin first entered the bottom phase with high electrical conductivity through solid-liquid extraction in the two-phase water system, and then moved to the top phase with high polarity through liquid-liquid extraction, achieving primary purification and improving the purity of tea saponin. 【Conclusion】 The UAATPE method could significantly improve the yield and purity of tea saponin, thus providing a new novel method for the efficient utilization of camellia meal.

Key words: camellia meal, tea saponin, ultrasound, aqueous two-phase extraction, Plackett-Burman, response surface design

DU JinTing,ZHANG Yan,LI Yan,WANG JiaJia,LIAO Na,ZHONG LiHuang,LUO BiQun,LIN Jiang. Optimization and Mechanism of Ultrasonic-Assisted Two-Phase Extraction of Tea Saponin[J].Scientia Agricultura Sinica, 2022, 55(1): 167-183.

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References 41

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水平Level	A 乙醇质量分数 Concentration of ethanol (%)	B 硫酸铵质量分数 Concentration of ammonium sulphate (%)	C 液固比 Solvent-to-material ratio	D 提取温度 Extraction temperature (℃)	E 功率 Ultrasonic power (W)	F 超声时间 Ultrasonic time (min)
-1	26	18	40﹕1	50	250	20
+1	30	22	60﹕1	70	350	40

试验顺序 Test no.	A	B	C	D	E	F	茶皂素得率 Tea saponin yield (%)
1	+1	-1	+1	+1	+1	-1	22.58
2	-1	-1	-1	-1	-1	-1	22.88
3	+1	+1	-1	+1	+1	+1	18.97
4	-1	+1	+1	+1	-1	-1	22.10
5	+1	+1	+1	-1	-1	-1	19.74
6	-1	+1	+1	-1	+1	+1	19.51
7	-1	-1	+1	-1	+1	+1	22.76
8	-1	+1	-1	+1	+1	-1	22.54
9	+1	-1	-1	-1	+1	-1	21.34
10	-1	-1	-1	+1	-1	+1	21.26
11	+1	+1	-1	-1	-1	+1	18.57
12	+1	-1	+1	+1	-1	+1	19.98

方差来源 Sources of variation	平方和 Quadratic sum	自由度 DOF	均方 Mean square	F值 F value	P值 P value
模型 Model	25.50	6	4.25	9.40	0.0132
乙醇质量分数 Concentration of ethanol	8.12	1	8.12	17.96	0.0082
硫酸铵质量分数 Concentration of ammonium sulphate	7.32	1	7.32	16.18	0.0101
液固比 Solvent-to-material ratio	0.10	1	0.10	0.23	0.6538
提取温度 Temperature	0.58	1	0.58	1.28	0.3101
功率 Ultrasonic power	0.84	1	0.84	1.85	0.2316
超声时间 Ultrasonic time	8.55	1	8.55	18.92	0.0074
残差 Residual	2.26	5	0.45
总差 Total	27.76	11
R²	0.9186	R²_Adj	0.8209	R²_Pred	0.5310

参数 Parameter	平方和 Quadratic sum	自由度 DOF	均方 Mean square	F值 F value	P值 P value	显著性 Significance
模型Model	44.46	9	4.94	61.17	<0.0001	**
x₁	1.71	1	1.71	21.19	0.0025	**
x₂	1.14	1	1.14	14.12	0.0071	**
x₃	0.52	1	0.52	6.44	0.0388	*
x₁x₂	7.65	1	7.65	94.68	<0.0001	**
x₁x₃	1.05	1	1.05	13.01	0.0087	**
x₂x₃	0.055	1	0.055	0.68	0.4355
x₁²	11.07	1	11.07	137.05	<0.0001	**
x₂²	15.70	1	15.70	194.48	<0.0001	**
x₃²	2.60	1	2.60	32.23	0.0008	**
残差 Residual	0.57	7	0.081
失拟项 Lack of fit	0.14	3	0.045	0.42	0.7464
纯误差 Pure error	0.43	4	0.11
总和 Total	45.02	16

方法 Method	溶剂 Solvent	提取条件 Extraction condition			得率 Yield (%)	纯度 Purity (%)
方法 Method	溶剂 Solvent	时间 Time (min)	温度 Temperature (℃)	液固比 Solvent-to-material	得率 Yield (%)	纯度 Purity (%)
乙醇提取法 Ethanol extraction	80%乙醇 80% ethanol	60	80	10:1	27.02±0.37a	62.01±0.03b
水提法 Water extraction	H₂O	60	80	10:1	22.26±0.99b	47.13±0.06c
超声波辅助双水相提取法 UAATPE	27.50%乙醇/19.60%硫酸铵 27.50% ethanol/19.60% ammonium sulphate	32	60	50:1	26.21±0.54a	66.09±0.04a

Optimization and Mechanism of Ultrasonic-Assisted Two-Phase Extraction of Tea Saponin

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流动相 Mobile phase	时间 Time (min)
流动相 Mobile phase	0	25	35	40	45
A：甲醇 A: Methanol	10	60	62	70	72
B：0.4%乙酸溶液 B: 0.4% acetic acid solution	90	40	38	30	28

因素 Factor	水平 Level
因素 Factor	-1	0	+1
乙醇质量分数 Concentration of ethanol (x₁, %)	26	28	30
硫酸铵质量分数 Concentration of ammonium sulphate (x₂, %)	18	20	22
超声时间 Ultrasonic time (x₃, min)	20	30	40

标准顺序 Standard	试验顺序 Test no.	乙醇质量分数 Concentration of ethanol x₁	盐质量分数 Concentration of ammonium sulphate x₂	超声时间 Ultrasonic time x₃	茶皂素得率 Tea saponin yield (%)
11	1	30	20	20	24.04
15	2	28	22	20	23.20
13	3	28	20	30	26.85
14	4	28	20	30	26.35
5	5	28	20	30	26.75
6	6	28	20	30	26.12
10	7	28	20	30	26.83
3	8	28	22	40	24.01
9	9	26	18	30	25.41
2	10	30	18	30	21.64
12	11	28	18	20	23.95
7	12	26	20	20	23.86
8	13	26	20	40	25.33
17	14	30	22	30	23.41
4	15	26	22	30	21.65
16	16	30	20	40	23.46
1	17	28	18	40	24.29

[1]	LIU ZhenRong,ZHAO WuQi,HU XinZhong,HE LiuCheng,CHEN YueYuan. Optimization of Drying Process in Oat Noodle Production [J]. Scientia Agricultura Sinica, 2022, 55(24): 4927-4942.
[2]	ZHANG QingAn,XU BoWen,CHEN BoYu,ZHANG BaoShan,CHENG Shuang. Changes of Wine Flavor Properties from the Decreased Higher Alcohols Induced by Ultrasound Irradiation [J]. Scientia Agricultura Sinica, 2021, 54(8): 1772-1786.
[3]	GU MingHui,YANG ZeSha,MA Ping,GE XinYu,LIU YongFeng. Application of Ultrasound-Assisted Thawing in the Role of Maintaining Physicochemical Properties and Reducing Protein Loss in Mutton During Multiple Freeze-Thaw Cycles [J]. Scientia Agricultura Sinica, 2021, 54(18): 3970-3983.
[4]	ZHAO Ying-Ying, ZOU Yu-Feng, WANG Peng, CHEN Lin, LI Ke, XU Xing-Lian. Effect of Pre-Emulsified Emulsion Treated with Ultrasound on Qualities of Low-Fat Frankfurter-Style Sausages [J]. Scientia Agricultura Sinica, 2014, 47(13): 2634-2642.
[5]	LI Zhen, HA Yi-Ming, LI An, LI Wei-Ming, WANG Feng, LI Qing-Peng. Optimization of Ultrasound-Assisted Extraction Technology of Polyphenols from Apple Pomace by Response Surface Methodology [J]. Scientia Agricultura Sinica, 2013, 46(21): 4569-4577.
[6]	WANG Hong-rong,CHEN Xu-wei,WANG Meng-zhi. Effect of Yucca schidigera Saponin and Tea Saponin Mixture on the Rumen Fermentation and Its Fibrolytic Bacterial Activity in the Rusitec Substrates with Different Concentrate to Forage Ratio [J]. Scientia Agricultura Sinica, 2011, 44(8): 1710-1719 .
[7]	WU Xiao-xia,LI Jian-ke,ZHANG Yan-yu . Ultrasound-Assisted Extraction and Antioxidation Stability of Silkworm Pupa Oil#br# [J]. Scientia Agricultura Sinica, 2010, 43(8): 1677-1687 .
[8]	,,. Optimization of Cultivation Conditions for Exopolysaccharide and Mycelial Biomass by Clitocybe sp. Using Box-Behnken Design [J]. Scientia Agricultura Sinica, 2005, 38(03): 634-638 .
[9]	,. Effect of Ultrasonic Treatment on the Extracting Activity of Kid Rennet [J]. Scientia Agricultura Sinica, 2004, 37(10): 1555-1559 .