响应面法优化日本蛇菰多糖脱色、脱蛋白工艺

秦宇; 华宗; 张敏; 姜薇薇

doi:10.13386/j.issn1002-0306.2022070206

响应面法优化日本蛇菰多糖脱色、脱蛋白工艺

doi: 10.13386/j.issn1002-0306.2022070206

秦宇^1,,
华宗¹,
张敏¹,
姜薇薇^{1, 2, 3, 4,*, ,}

1.
云南农业大学理学院，云南昆明 650201
2.
云南省药用植物生物学重点实验室，云南昆明 650201
3.
西南中药材种质创新与利用国家地方联合工程研究中心，云南昆明 650201
4.
云南特色植物提取实验室，云南昆明 650201

基金项目: 云南省重大科技专项（202002AA1000051-2）；云南特色植物筛选与研发服务CXO平台建设（2022YKZY001）。

详细信息

作者简介:
秦宇（2001−），女，本科，研究方向：多糖提取分离纯化鉴定，E-mail：2753076190@qq.com

通讯作者:
姜薇薇（1982−），女，博士，讲师，研究方向：天然产物开发与研究，E-mail：17366529@163.com

中图分类号: O629.12
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- 文章访问数: 32
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出版历程
- 收稿日期: 2022-07-19
- 刊出日期: 2023-05-15

Optimization of Decolorization and Deproteinization of Balanophora japonica Makino Polysaccharide by Response Surface Methodology

QIN Yu^1
,,
HUA Zong¹,
ZHANG Min¹,
JIANG Weiwei^{1, 2, 3, 4,*
, ,}

1.
College of Science, Yunnan Agricultural University, Kunming 650201, China
2.
Key Laboratory of Medicinal Plant Biology, Yunnan Agricultural University, Kunming 650201, China
3.
National & Local Joint Engineering Research Center on Germplasm Innovation and Utilization of Chinese Medicinal Materials in Southwestern China, Kunming 650201, China
4.
Yunnan Characteristic Plant Extraction Laboratory, Kunming 650201, China

摘要

摘要: 研究日本蛇菰多糖双氧水脱色、Sevag法脱蛋白的条件并对其进行工艺优化。在单因素的基础上，以多糖脱色率为指标，采用响应面法设计三因素三水平实验对蛇菰多糖的脱色条件进行优化；以蛋白质脱除率、多糖保留率为指标，选取Sevag试剂比例（氯仿:正丁醇）、样液:Sevag试剂、振荡时间为因素水平设计响应面试验，得到最佳工艺条件。结果表明，在pH为8的条件下双氧水脱色最佳工艺为：双氧水用量为20%、脱色时间为46 min、脱色温度为60 ℃，在此条件下蛇菰多糖的脱色率为84.21%。脱蛋白最佳工艺条件为：氯仿:正丁醇5:1、样液:Sevag试剂4:1、振荡时间15 min，在此条件下，多糖保留率为81.03%，蛋白质脱除率为40.44%。双氧水和Sevag法脱色脱蛋白工艺稳定，方法可行操作简单，适用于蛇菰多糖的脱色、脱蛋白。
- 日本蛇菰多糖 /
- 脱色 /
- 脱蛋白 /
- 双氧水 /
- Sevag法 /
- 响应面
Abstract: To study the decolorization and deproteinization conditions of Balanophora japonica polysaccharide by hydrogen peroxide and Sevag method, and to optimize the process. Based on single factor experiments, the response surface methodology was used to design a three factor and three level experiments to optimize the decolorization conditions of Balanophora japonica polysaccharides using the decolorization rate of polysaccharides as an index. Using protein removal rate and polysaccharide retention rate as indicators, a response surface experiment was designed at the factor level of Sevag reagent ratio (chloroform:n-butanol), sample solution: Sevag reagent and oscillation time to obtain the optimal process conditions. Results showed that, under the condition of pH8, the optimal decolorization process was as follows: The dosage of hydrogen peroxide was 20%, the decolorization time was 46 min, and the decolorization temperature was 60 ℃. Under this condition, the decolorization rate of polysaccharides was 84.21%. The optimal deproteinization conditions were chloroform:n-butanol 5:1, sample solution:Sevag reagent 4:1, and shock time 15 min, under this condition, the polysaccharide retention rate was 81.03%, and the protein removal rate was 40.44%. The decolorization and deproteinization of Balanophora japonica polysaccharide by hydrogen peroxide and Sevag method was stable, feasible and simple, and would be suitable for decolorization and deproteinization of Balanophora japonica polysaccharide.
- Balanophora japonica Makino polysaccharide /
- decolorization /
- take off the protein /
- hydrogen peroxide /
- Sevag method /
- response surface

HTML全文

图 1 双氧水用量对脱色效果的影响

Figure 1. Effect of hydrogen peroxide dosage on decolorization effect

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图 2 脱色温度对脱色率的影响

Figure 2. Effect of decolorization temperature on decolorization rate

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图 3 脱色时间对脱色率的影响

Figure 3. Effect of decolorization time on decolorization rate

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图 5 Sevag试剂比例对多糖脱蛋白的影响

Figure 5. Influence of Sevag reagent ratio on polysaccharide deproteinization

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图 6 样液:Sevag试剂对多糖脱蛋白的影响

Figure 6. Effect of sample solution:Sevag reagent on polysaccharide deproteinization

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图 7 振荡时间对多糖脱蛋白的影响

Figure 7. Effect of shock time on polysaccharide deproteinization

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图 8 脱蛋白次数对多糖脱蛋白的影响

Figure 8. Influence of deproteinization times on polysaccharide deproteinization

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图 4 脱色时间与脱色温度对脱色率的交互影响

Figure 4. Interaction of decolorization time and decolorization temperature on decolorization rate

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图 9 Sevag试剂比例与样液:Sevag试剂比例对蛋白质脱除率的交互影响

Figure 9. Interaction of Sevag reagent ratio and sample solution:Sevag reagent on protein removal rate

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图 11 氯仿：正丁醇与振荡时间对蛋白质脱除率的交互影响

Figure 11. Interaction of Sevag reagent ratio and shock time on protein removal rate

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图 10 氯仿：正丁醇与振荡时间对蛋白质脱除率的交互影响

Figure 10. Interaction of Sevag reagent ratio and oscillation time on protein removal rate

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表 1 双氧水脱色工艺响应面试验因素和水平设计

Table 1. Response surface test factors and horizontal design of hydrogen peroxide decolorization process

水平	因素
水平	A双氧水用量（%）	B脱色时间（min）	C脱色温度（℃）
−1	10	40	40
0	15	60	50
1	20	80	60

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表 2 Sevag法脱蛋白工艺响应面试验因素与水平设计

Table 2. Response surface test factors and horizontal design of Sevag deproteinization process

水平	因素
水平	A 氯仿:正丁醇	B 样液:Sevag试剂	C振荡时间（℃）
−1	3:1	2:1	10
0	4:1	3:1	15
1	5:1	4:1	20

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表 3 双氧水响应面试验结果

Table 3. Experimental results of hydrogen peroxide response surface

实验号	A	B	C	Y：脱色率%
1	0	1	1	82.52
2	0	0	0	78.2
3	−1	−1	0	75.99
4	0	1	−1	77.74
5	−1	0	−1	72.59
6	0	0	0	79.02
7	0	0	0	79.12
8	1	0	−1	74.61
9	−1	0	1	80.22
10	1	−1	0	79.67
11	−1	1	0	78.66
12	0	1	1	81.14
13	0	0	0	79.67
14	1	0	1	81.42
15	0	−1	−1	71.02
16	1	1	0	81.23
17	0	0	0	79.58

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表 4 方差分析结果

Table 4. Analysis of variance results

方差来源	离差平方和	自由度	均方	F值	P值	显著性
模型	156.67	9	17.41	45.16	<0.0001	**
A双氧水用量	11.21	1	11.21	29.08	0.0010	**
B脱色时间	11.45	1	11.45	29.70	0.0010	**
C脱色温度	107.60	1	107.60	279.12	<0.0001	**
AB	0.31	1	0.31	0.80	0.4011
AC	0.17	1	0.17	0.44	0.5302
BC	16.40	1	16.40	42.55	0.0003	**
A²	1.33	1	1.33	3.46	0.1052
B²	0.46	1	0.46	1.21	0.3085
C²	7.62	1	7.62	19.77	0.0030	**
残差	2.70	7	0.39
失拟项	1.33	3	0.44	1.29	0.3917
纯误差	1.37	4	0.34
总离差	159.37	16
注：R²=0.9831；“”表示P<0.05显著；“*”表示P<0.01极显著。

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表 5 脱蛋白响应面剂验结果

Table 5. Results of deproteinization response surface experiment

实验号	A	B	C	Y1：多糖保留率（%）	Y2：蛋白质脱除率（%）
1	0	−1	0	55.35	27.82
2	0	−1	1	56.11	42.33
3	0	1	1	62.96	38.4
4	0	1	−1	55.86	33.26
5	1	0	1	60.43	36.89
6	0	0	0	73.11	33.07
7	0	0	0	74.38	32.92
8	0	0	0	78.18	32.86
9	−1	0	−1	49.01	26.61
10	1	1	0	84.53	39.92
11	0	0	0	70.57	33.47
12	1	−1	0	72.6	45.36
13	−1	0	1	59.16	42.64
14	0	0	0	70.32	33.61
15	1	0	−1	63.22	35.08
16	−1	1	0	66.01	44.75
17	−1	−1	0	59.41	39.61

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表 6 多糖保留率方差分析结果

Table 6. Analysis of variance of polysaccharide retention rate results

方差来源	离差平方和	自由度	均方	F值	P值	显著性
模型	1339.77	9	148.86	11.59	0.0020	**
A 氯仿:正丁醇	278.36	1	278.36	21.67	0.0023	**
B样液:Sevag试剂	83.79	1	83.79	6.52	0.0379	*
C振荡时间	28.96	1	28.96	2.25	0.1769
AB	7.10	1	7.10	0.55	0.4813
AC	41.86	1	41.86	3.26	0.1140
BC	10.05	1	10.05	0.78	0.4058
A²	5.52	1	5.52	0.43	0.5331
B²	9.85	1	9.85	0.77	0.4101
C²	850.48	1	850.48	66.22	<0.0001	**
残差	89.90	7	12.84
失拟项	48.55	3	16.18	1.57	0.3293
纯误差	41.35	4	10.34
总离差	1429.67	16
注：R²=0.9371；“”表示P<0.05显著；“*”表示P<0.01极显著。

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表 7 蛋白质脱除率方差分析表

Table 7. Analysis of variance of protein removal rate

方差来源	离差平方和	自由度	均方	F值	P值	显著性
模型	482.53	9	53.61	224.07	<0.0001	**
A 氯仿:正丁醇	1.66	1	1.66	6.92	0.0339	*
B样液:Sevag试剂	0.18	1	0.18	0.76	0.4108
C振荡时间	175.69	1	175.69	734.24	<0.0001	**
AB	27.98	1	27.98	116.95	<0.0001	**
AC	50.55	1	50.55	211.27	<0.0001	**
BC	21.95	1	21.95	91.73	<0.0001	**
A²	86.72	1	86.72	362.42	<0.0001	**
B²	92.45	1	92.45	386.36	<0.0001	**
C²	24.64	1	24.64	102.99	<0.0001	**
残差	1.67	7	0.24
失拟项	1.22	3	0.41	3.62	0.1230
纯误差	0.45	4	0.11
总离差	484.20	16
注：R²=0.9965；“”表示P<0.05显著；“*”表示P<0.01极显著。

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