三相萃取法纯化枸杞多糖
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摘要
利用三相萃取法可避开多糖的醇沉过程,达到短时间内分离多糖的同时,也可一定程度的分离子水提液中的蛋白质和黄酮的原理,本文采用此法分离枸杞多糖,考察了叔丁醇-(NH_4)_2SO_4-水提液体系的三相萃取性质,研究了提取液中(NH_4)_2SO_4的添加量、叔丁醇添加量、萃取液pH、温度对枸杞提取液多糖、蛋白质、黄酮的萃取率的影响,并通过正交试验优化了多糖的萃取条件,研究发现:多糖最佳萃取条件是:10 mL提取液中(NH_4)_2SO_4添加量为30%(w/v)、叔丁醇添加量为10 mL、温度为35℃、pH为6,此时多糖的萃取率可达95.31%,蛋白质萃取率可达83.45%,黄酮萃取率可达50.66%。通过多糖的红外光谱、抗氧化活性实验可发现三相萃取法所得多糖与乙醇沉淀法所得多糖的化学结构,抗氧化活性均无太大差异。因此,该体系是一种可行的高效分离纯化枸杞多糖的绿色方法。
The three phase extraction method can avoid the alcohol precipitation process of polysaccharides to separate polysaccharides in a short time. At the same time,the protein and flavonoid in the aqueous extract of ionic water can also be segregated in a certain extent. In this paper,Lycium barbarum polysaccharides were separated by this method,and the three phase extraction properties of tertiary butanol-(NH_4)_2SO_4-aqueous extract system were investigated. The effects of four factors,including the amount of(NH_4)_2SO_4 in the extract,the amount of tertiary butanol,pH and temperature of the extract,on the extraction rate of polysaccharides,protein and flavonoids from Lycium barbarum extract were studied,and the extraction conditions of polysaccharides were optimized by orthogonal experiment. The optimum extraction conditions were as follows:10 mL extract(NH_4)_2SO_4 was added to 30%(w/v),tertiary butanol was added to 10 mL,the temperature was 35 ℃ and pH was 6,the extraction rate of polysaccharides reached 95.31%,the protein extraction rate reached 83.45%,and the extraction rate of flavonoids reached 50.66%. The infrared spectra and antioxidant activities of polysaccharides showed that there was no significant difference in the chemical structure and antioxidant activity of polysaccharides obtained by three phase extraction and ethanol precipitation. Therefore,the system would be a feasible and efficient method for separating and purifying Lycium barbarum polysaccharides.
The three phase extraction method can avoid the alcohol precipitation process of polysaccharides to separate polysaccharides in a short time. At the same time,the protein and flavonoid in the aqueous extract of ionic water can also be segregated in a certain extent. In this paper,Lycium barbarum polysaccharides were separated by this method,and the three phase extraction properties of tertiary butanol-(NH_4)_2SO_4-aqueous extract system were investigated. The effects of four factors,including the amount of(NH_4)_2SO_4 in the extract,the amount of tertiary butanol,pH and temperature of the extract,on the extraction rate of polysaccharides,protein and flavonoids from Lycium barbarum extract were studied,and the extraction conditions of polysaccharides were optimized by orthogonal experiment. The optimum extraction conditions were as follows:10 mL extract(NH_4)_2SO_4 was added to 30%(w/v),tertiary butanol was added to 10 mL,the temperature was 35 ℃ and pH was 6,the extraction rate of polysaccharides reached 95.31%,the protein extraction rate reached 83.45%,and the extraction rate of flavonoids reached 50.66%. The infrared spectra and antioxidant activities of polysaccharides showed that there was no significant difference in the chemical structure and antioxidant activity of polysaccharides obtained by three phase extraction and ethanol precipitation. Therefore,the system would be a feasible and efficient method for separating and purifying Lycium barbarum polysaccharides.
引文
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[17]Jing-Kun Yan,Yao-Yao Wang,Wen-Yi Qiu,et al. Three-phase partitioning for efficient extraction and separation of polysaccharides from Corbicula fluminea[J].Carbohydrate Polymers,2017,163:10-19.
[18]韩苗苗,姚娟,易阳,等.龙眼果肉干燥过程中多糖的理化特征与活性变化规律[J].食品科学,2017,38(21):67-73.
[19]苏平,孙昕,宋思圆,等. 不同提取方法对黄秋葵花多糖的结构组成及抗氧化活性的影响[J].食品科学,2018,39(15):103-110.
[20]Christine M,Oliver,Ashwini,et al. Use of FTIR and mass spectrometry for characterization of glycated caseins[J].The Journal of Dairy Research,2009,76(1):105-110.
[21]江艳,武培怡.大豆蛋白的中红外和近红外光谱研究[J].化学进展,2009,21(4):705-714.
[22]Ling-Ling Fu,Hai-Xia Chen,Peng Dong,et al. Effects of ultrasonic treatment on the physicochemical properties and DPPH radical scavenging activity of polysaccharides from mushroom Inonotus obliquus[J].Journal of Food Science,2010,75(4):C322-C327.
[2]董汝晶.多糖提取方法的研究进展[J].农产品加工(学刊),2014(8):46-48,51.
[3]Dennison C,Lovrien R. Three phase partitioning:Concentration and purification of proteins[J]. Protein Expression and Purification,1997,11:149-161.
[4]胡静波,张玉军,谷利军.三相分离技术提取大豆油[J].河南工业大学学报:自然科学版,2009,30(4):32-35.
[5]魏胜华,周清华,钱伟,等.三相法制备木瓜蛋白酶的过程研究[J].现代化工,2017,37(10):114-117.
[6]Roy I,Gupta M N. Three-phase partitioning of proteins[J]. Analytical Chemistry,2002,300:11-14.
[7]Tan Z J,Wang C Y,Yi Y J,et al. Three phase partitionning for simultaneous purification of aloe polysaccharide and protein using a single-step extraction[J]. Process Biochemistry,2015,50:482-486.
[8]王文平,郭祀远,李琳,等.考马斯亮蓝法测定野木瓜多糖中蛋白质的含量[J].食品研究与开发,2008(1):115-117.
[9]白生文,汤超,田京,等.沙棘果渣总黄酮提取工艺及抗氧化活性分析[J].食品科学,2015,36(10):59-64.
[10]Yang C X,Gou Y Q,Chen J Y,et al. Structural characterization and antitumor activity of a pectic polysaccharide from Codonopsis pilosula[J]. Carbohyd Polym,2013,98(15):886-895.
[11]Krisna C,Duong L Y,Gabelli S B. Chapter seven-salting out of proteinsusing ammonium sulfate precipitation[J]. Methods in Enzymology,2014,541:85-94.
[12]Pakhale S V,Bhagwat S S. Purification of serration peptidase from Serratia marcescens NRRL B23112 using ultrasound assisted three phase partitioning[J]. Ultrasonics Sonochemistry,2016,31:532-538.
[13]Tan Z,Yang Z,Yi Y,et al. Extraction of oil from flax seed(Linum usitatissimum L.)using enzyme-assisted three-phase partitioning[J]. Applied Biochemistry and Biotechnology,2016,179:1325-1335.
[14]Sharma A,Gupta M N. Three phase partitioning of carbohydrate polymers:Separation and purification of alginates[J].Carbohydrate Polymers,2002,48:391-395.
[15]Sharma A,Gupta M N. Three phase partitioning as a large-scale separation method for purification of a wheat germ bifunctional protease/amylase inhibitor[J]. Process Biochemistry,2001,37:193-196.
[16]Coimbra C G O,Lopes C E,Calazans G M T. Three-phase partitioning of hydrolyzed levan[J]. Bioresource Technology,2010,101:4725-4728.
[17]Jing-Kun Yan,Yao-Yao Wang,Wen-Yi Qiu,et al. Three-phase partitioning for efficient extraction and separation of polysaccharides from Corbicula fluminea[J].Carbohydrate Polymers,2017,163:10-19.
[18]韩苗苗,姚娟,易阳,等.龙眼果肉干燥过程中多糖的理化特征与活性变化规律[J].食品科学,2017,38(21):67-73.
[19]苏平,孙昕,宋思圆,等. 不同提取方法对黄秋葵花多糖的结构组成及抗氧化活性的影响[J].食品科学,2018,39(15):103-110.
[20]Christine M,Oliver,Ashwini,et al. Use of FTIR and mass spectrometry for characterization of glycated caseins[J].The Journal of Dairy Research,2009,76(1):105-110.
[21]江艳,武培怡.大豆蛋白的中红外和近红外光谱研究[J].化学进展,2009,21(4):705-714.
[22]Ling-Ling Fu,Hai-Xia Chen,Peng Dong,et al. Effects of ultrasonic treatment on the physicochemical properties and DPPH radical scavenging activity of polysaccharides from mushroom Inonotus obliquus[J].Journal of Food Science,2010,75(4):C322-C327.