小球藻胞内多糖提取纯化及其抗氧化活性

李思雨; 刘红全; 孙寒; 徐琰杰; 黄磊恒; 龙寒; 凌宏林; 成江弈; 杨堃峰

doi:10.13386/j.issn1002-0306.2021110051

小球藻胞内多糖提取纯化及其抗氧化活性

doi: 10.13386/j.issn1002-0306.2021110051

1.
广西民族大学海洋与生物技术学院，广西多糖材料与改性重点实验室，广西民族大学海洋生物资源保护与利用重点实验室，广西南宁 530007
2.
中国海洋大学海洋生命学院，山东青岛 266003

基金项目: 广西自然科学基金项目（2018GXNSFAA294032）。

详细信息

作者简介:
李思雨（1994−），女，硕士研究生，研究方向：植物分子生物学，E-mail：lisiyu7788@163.com

通讯作者:
刘红全（1975−），男，博士，教授，研究方向：植物分子生物学，E-mail：lhongquan@163.com

杨堃峰（1978−），男，博士，实验师，研究方向：细胞生物学，E-mail：yhykf@ouc.edu.cn

中图分类号: TS201.2
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- 文章访问数: 21
- HTML全文浏览量: 24
- PDF下载量: 4
- 被引次数: 0
出版历程
- 收稿日期: 2021-11-08
- 网络出版日期: 2022-06-19
- 刊出日期: 2022-08-03

Extraction and Purification of Intracellular Polysaccharide from Chlorella vulgaris and Its Antioxidant Activity

1.
Guangxi Key Laboratory of Polysaccharide Materials and Their Modification, Key Laboratory of Protection and Utilization of Marine Resources, College of Marine and Biotechnology, Guangxi University for Nationalities, Nanning 530007, China
2.
Ocean University of China College of Marine Life Sciences, Qingdao 266003, China

摘要

摘要: 为提高小球藻胞内粗多糖得率，并探究小球藻胞内多糖纯化组分的抗氧化作用。本研究采取超声波破碎和热水浸提相结合的方法提取小球藻胞内粗多糖，利用响应面法进行提取条件优化，在此基础上，采用阴离子交换柱和葡聚糖凝胶柱层析对提取得到的粗多糖进行分离纯化，并进行结构表征和抗氧化试验。响应面结果显示，小球藻胞内粗多糖最优提取条件为：NaOH质量分数为2.0%，料液比为1:25（g/mL），超声功率为200 W，超声时间为20 min，提取温度为80 ℃，提取时间为1.5 h，在此条件下，小球藻胞内粗多糖的得率为18.086%±0.143%。结构表征和体外抗氧化结果显示，纯化多糖（Purification of intracellular polysaccharide，SCIP），主要由葡萄糖、鼠李糖及半乳糖成分组成，是一种含有糖醛酸的吡喃糖。其在20 mg/mL时，对1,1-二苯基-2-苦基肼（1,1-Diphenyl-2-picrylhydrazyl，DPPH）自由基的清除率最大，为75.64%±1.56%，在25 mg/mL时，对羟基自由基清除率最大，为71.08%±0.58%，IC₅₀分别6.42 mg/mL和8.59 mg/mL。研究结果为深入了解小球藻胞内多糖理化性质及小球藻多糖的开发利用提供基础。
- 小球藻 /
- 胞内多糖 /
- 提取优化 /
- 分离纯化 /
- 抗氧化
Abstract: In order to improve the yield of crude polysaccharide from Chlorella vulgaris and explore the antioxidant effect of purified intracellular polysaccharide from Chlorella vulgaris, this study adopted the method of ultrasonic crushing and hot water extraction to extract intracellular crude polysaccharides from Chlorella vulgaris. Response surface method was used to optimize the extraction conditions. On this basis, anion exchange column and dextran gel column chromatography were used to separate and purify the extracted crude polysaccharides, and the characterization and antioxidant test were carried out. Response surface results showed that the optimal extraction conditions of intracellular crude polysaccharides from Chlorella vulgaris were as follows: The mass fraction of NaOH 2.0%, the material-liquid ratio 1:25 (g/mL), the ultrasonic power 200 W, the ultrasonic time 20 min, the extraction temperature 80 ℃, the extraction time 1.5 h. Under these conditions, the yield of intracellular crude polysaccharide was 18.086%±0.143%. The results of characterization and antioxidant activity in vitro showed that SCIP was a pyranose sugar containing uronic acid and was composed of glucose, rhamnose and galactose. At 20 mg/mL, the DPPH radical scavenging rate was the largest of 75.64%±1.56%, and at 25 mg/mL, the hydroxyl radical scavenging rate was the largest of 71.08%±0.58%. IC₅₀ were 6.42 mg/mL and 8.59 mg/mL, respectively. The results provided a basis for further understanding of the physicochemical properties and biological activities of polysaccharides from Chlorella sp.
- Chlorella vulgaris /
- intracellular polysaccharide /
- extraction optimization /
- separation and purification /
- antioxidant

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图 1 不同提取因素对多糖得率的影响

Figure 1. Effects of different extraction factors on the yield of polysaccharides

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图 2 NaOH质量分数、料液比、超声功率、提取时间交互作用对多糖得率影响的响应面图

Figure 2. Response surface map of the effects of NaOH concentration, solid-liquid ratio, ultrasonic power and extraction time on the yield of polysaccharides

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图 3 DEAE-52纤维素柱洗脱图

Figure 3. Eluent diagram of DEAE-52 cellulose column

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图 4 CIP-3葡聚糖凝胶柱洗脱图

Figure 4. Elution diagram of CIP-3 by Sephadex G-100

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图 5 SCIP紫外光谱图

Figure 5. UV spectrum of SCIP

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图 6 SCIP红外光谱图

Figure 6. IR spectrum of SCIP

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图 7 SCIP分子量分布图

Figure 7. Molecular weight distribution of SCIP

注：46.1 min处为流动相的峰。

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图 8 SCIP单糖组成分析图

Figure 8. Monosaccharide composition analysis of SCIP

注：溶剂峰：2.0 min为氢氧化钠的峰，40 min乙酸钠的峰。

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图 9 SCIP体外抗氧化能力

Figure 9. Antioxidant activity of SCIP in vitro

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表 1 单因素设计因素及水平

Table 1. Factors and levels of univariate design

因素	参数设置
A NaOH质量分数（%）	1.0	1.5	2.0	2.5	3.0	3.5
B 料液比（g/mL）	1:15	1:20	1:25	1:30	1:35	1:40
C 超声功率（W）	0	100	200	300	400	500
D 超声时间（min）	10	15	20	25	30	35
E 提取温度（℃）	50	60	70	80	90	100
F 提取时间（h）	1	2	3	4	5	6

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表 2 Plackett-Burman试验因素及水平

Table 2. Factors and levels of PB design experiment

水平	因素
水平	A（%）	B（g/mL）	C（W）	D（min）	E（℃）	F（h）
−1	2.5	1:30	300	20	80	3
1	3.5	1:40	400	25	100	4

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表 3 响应面试验因素及水平

Table 3. Factors and level of response surface experiment

水平	因素
水平	NaOH质量分数X₁（%）	料液比X₂（g/mL）	超声功率X₃（W）	提取时间X₄（h）
−1	1.5	1:20	150	1.5
0	2.0	1:25	200	2.0
1	2.5	1:30	250	2.5

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表 4 Plackett-Burman试验设计及结果

Table 4. Design and results of PB experiment

试验号	A	B	C	D	E	F	粗多糖得率（%）
1	−1	−1	−1	1	−1	1	16.094
2	−1	1	−1	1	1	−1	14.567
3	−1	1	1	−1	1	1	7.900
4	−1	−1	−1	−1	−1	−1	17.422
5	1	−1	1	1	−1	1	8.100
6	−1	−1	1	−1	1	1	12.190
7	1	1	−1	1	1	1	8.777
8	1	1	1	−1	−1	−1	9.460
9	−1	1	1	1	−1	−1	9.056
10	1	−1	1	1	1	−1	9.016
11	1	1	−1	−1	−1	1	9.215
12	1	−1	−1	−1	1	−1	15.058

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表 5 Plackett-Burman试验方差分析

Table 5. Variance analysis of PB experiment

来源	平方和	自由度	均方	F值	P值	T值	显著性
模型	124.97	6	20.83	14.64	0.0049		**
A	25.82	1	25.82	18.15	0.0080	−1.47	**
B	29.78	1	29.78	20.94	0.0060	−1.58	**
C	53.81	1	53.81	37.83	0.0017	−2.12	**
D	2.75	1	2.65	1.86	0.2307	−0.47
E	0.28	1	0.28	0.20	0.6749	−0.15
F	12.62	1	12.62	8.87	0.0309	−1.03	*
残差	7.11	5	1.42
总和	132.08	11
注：表示对结果影响显著（P<0.05）；*表示对结果影响极显著（P<0.01）。

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表 6 最陡爬坡试验设计及结果

Table 6. Design and results of the steepest ascent experiment

试验号	NaOH质量分数X₁（%）	料液比X₂ （g/mL）	超声功率 X₃（W）	提取时间 X₄（h）	粗多糖得率（%）
1	0.5	1:10	50	0.5	12.860
2	1.0	1:15	100	1.0	14.879
3	1.5	1:20	150	1.5	16.964
4	2.0	1:25	200	2.0	17.893
5	2.5	1:30	250	2.5	16.107
6	3.0	1:35	300	3.0	14.288

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表 7 Box-Behnken试验设计与结果

Table 7. Design and results of Box-Behnken experiment

试验号	X₁	X₂	X₃	X₄	粗多糖得率（%）
1	0	0	0	0	18.073
2	−1	−1	0	0	14.195
3	0	0	−1	1	12.296
4	0	−1	0	−1	15.443
5	0	1	1	0	7.010
6	0	1	0	−1	14.939
7	0	0	1	1	11.897
8	0	0	0	0	17.781
9	−1	0	−1	0	11.539
10	1	0	0	−1	15.908
11	0	0	0	0	17.887
12	0	0	−1	−1	12.455
13	0	0	0	0	18.099
14	−1	0	0	−1	16.399
15	0	−1	0	1	15.204
16	0	1	0	1	14.646
17	−1	0	1	0	10.795
18	−1	1	0	0	13.411
19	0	0	0	0	17.223
20	1	0	0	1	15.696
21	0	0	1	−1	12.057
22	1	0	−1	0	11.287
23	0	−1	−1	0	9.786
24	1	−1	0	0	13.929
25	1	1	0	0	13.146
26	0	−1	1	0	7.727
27	1	0	1	0	10.530
28	0	1	−1	0	8.484
29	−1	0	0	1	16.147

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表 8 回归方程及方差分析

Table 8. Variance analysis of regression equation

方差来源	平方和	自由度	均方	F值	P值	显著性
模型	271.15	14	19.37	49.34	<0.0001	**
X₁	0.33	1	0.33	0.84	0.3742
X₂	1.80	1	1.80	4.59	0.0503
X₃	2.83	1	2.83	7.22	0.0177	*
X₄	0.14	1	0.14	0.37	0.5543
X₁X₂	0.000	1	0.000	0.000	1.0000
X₁X₃	4.409×10⁻⁵	1	4.409×10⁻⁵	1.123×10⁻⁴	0.9917
X₁X₄	3.968×10⁻⁴	1	3.968×10⁻⁴	1.011×10⁻³	0.9751
X₂X₃	0.085	1	0.085	0.22	0.6481
X₂X₄	7.055×10⁻⁴	1	7.055×10⁻⁴	1.797×10⁻³	0.9668
X₃X₄	0.000	1	0.000	0.000	1.0000
X₁²	9.95	1	9.95	25.36	0.0002	**
X₂²	63.19	1	63.19	161.00	<0.0001	**
X₃²	224.16	1	224.16	571.09	<0.0001	**
X₄²	3.880×10⁻³	1	3.880×10⁻³	9.885×10⁻³	0.9222
残差	5.50	14	0.39
失拟项	4.99	10	0.50	3.96	0.0984
误差	0.50	4	0.13
总和	276.64	28
注：“”表示对结果影响显著（P<0.05）；“*”表示对结果影响极显著（P<0.01）。

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表 9 SCIP单糖组成

Table 9. Monosaccharide composition of SCIP

名称	出峰时间（min）	摩尔比
GalN	8.684	0.051
Rha	9.225	0.190
Ara	9.792	0.062
GlcN	10.809	0.096
Gal	12.259	0.163
Glc	13.875	0.300
Xyl	16.184	0.041
GalA	43.759	0.049
GlcA	46.584	0.047

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