超声波辅助酶法提取牛皮胶原蛋白及其结构表征

赵改名; 王壮壮; 祝超智; 余小领; 张秋会; 祁兴山

doi:10.13386/j.issn1002-0306.2022070219

超声波辅助酶法提取牛皮胶原蛋白及其结构表征

doi: 10.13386/j.issn1002-0306.2022070219

1.
河南农业大学食品科学技术学院，河南郑州 450002
2.
恒都综合试验站，河南驻马店 463000

基金项目: 财政部和农业农村部：国家现代农业（肉牛牦牛）产业技术体系建设专项（CARS-37）

详细信息

作者简介:
赵改名（1965−），男，博士，教授，研究方向：肉类加工与产品质量安全控制，E-mail：gmzhao@126.com

通讯作者:
祝超智（1985−），女，博士，副教授，研究方向：肉品加工与质量控制，E-mail：zhuchaozhi66@163.com

中图分类号: TS251.92
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- 文章访问数: 12
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- 被引次数: 0
出版历程
- 收稿日期: 2022-07-19
- 刊出日期: 2023-05-01

Ultrasound-Assisted Enzymatic Extraction and Structural Characterization of Cowhide Collagen

1.
College of Food Science and Technology, Henan Agricultural University, Zhengzhou 450002, China
2.
Hengdu Comprehensive Experimental Station, Zhumadian 463000, China

摘要

摘要: 以牛皮为原料，优化超声波辅助酶提取牛皮中胶原蛋白的工艺。在单因素实验的基础上设计响应面试验，以牛皮胶原蛋白提取率为响应值，优化得到胶原蛋白的最佳提取工艺，并对其进行结构表征。结果表明：牛皮中胶原蛋白的最佳提取工艺条件为超声波功率161 W、超声波处理时间64 min、胃蛋白酶添加量109 U/g、料液比1:16 g/mL，在此条件下胶原蛋白的提取率为63.77%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、紫外光谱（UV）和傅立叶红外光谱（FTIR）分析表明，超声波辅助酶提取的胶原蛋白符合Ⅰ型胶原蛋白的特征，保持了其完整的三螺旋结构，氨基酸组成和扫描电镜（SEM）分析得到超声波辅助酶提取的胶原蛋白三螺旋稳定程度略微下降。本研究为牛皮中胶原蛋白的提取优化了一种有效的工艺，且很大程度上缩短了胶原蛋白的提取时间，并且具有较高的应用价值。
- 牛皮胶原蛋白 /
- 超声波 /
- 提取率 /
- 工艺优化 /
- 结构表征
Abstract: Using cowhide as raw material, the process of ultrasonic-assisted enzyme extraction of collagen from cowhide was optimized. Based on the single factor experiment, a response surface test was designed, and the extraction rate of cowhide collagen was used as the response value to optimize the optimal extraction process of collagen, and its structure was characterized. The results showed that the optimal extraction conditions of collagen from cowhide were ultrasonic power 161 W, ultrasonic treatment time 64 min, pepsin addition 109 U/g, and solid-liquid ratio 1:16 g/mL. Under these conditions, the extraction rate of collagen was 63.77%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) analysis showed that the collagen extracted by ultrasound-assisted enzyme was in line with the characteristics of type I collagen, maintaining its complete triple helix structure, amino acid composition and scanning electron microscope (SEM) analysis showed that the collagen triple helix extracted by ultrasonic assisted enzyme was relatively stable. In this study, an effective process is optimized for the extraction of collagen from cowhide, besides, the extraction time of collagen is greatly shortened and it has high utilization value.
- cowhide collagen /
- ultrasound /
- extraction rate /
- process optimization /
- structure characterization

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图 1 不同单因素对牛皮胶原蛋白提取率的影响

注：不同小写字母表示差异显著（P<0.05）。

Figure 1. Effects of different single factors on the extraction rate of cowhide collagen

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图 2 各因素交互作用对牛皮胶原蛋白提取率的影响

Figure 2. Effects of interaction of various factors on the extraction rate of cowhide collagen

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图 3 牛皮胶原蛋白的SDS-PAGE凝胶电泳图

注：M为Marker；a为单一酶法提取；b为超声波辅助酶法提取。

Figure 3. SDS-PAGE gel electrophoresis image of cowhide collagen

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图 4 牛皮胶原蛋白的紫外吸收光谱图

Figure 4. UV absorption spectrum of cowhide collagen

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图 5 EEC和UAEEC红外图谱

Figure 5. Infrared spectra of EEC and UAEEC

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图 6 EEC（a）和UAEEC（b）酰胺I去卷积图谱

Figure 6. Amide I deconvolution map of EEC (a) and UAEEC (b)

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图 7 牛皮胶原蛋白的扫描电镜图（100×）

注：a：单一酶法提取；b：超声波辅助酶法提取。

Figure 7. SEM of cowhide collagen (100×)

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表 1 响应面试验因素水平表

Table 1. Factors and levels table of response surface experiment

水平	因素
水平	A超声功率（W）	B超声时间（min）	C酶添加量（U/g）	D料液比（g/mL）
−1	120	50	87	1:10
0	150	60	105	1:15
1	180	70	123	1:20

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表 2 Box-Behnken 设计方案及结果

Table 2. Design scheme and results of Box-Behnken

试验号	A 超声功率	B 超声时间	C 酶添加量	D 料液比	Y 胶原蛋白提取率（%）
1	−1	1	0	0	52.00
2	0	−1	−1	0	48.35
3	0	−1	1	0	54.40
4	−1	0	−1	0	50.03
5	0	0	0	0	63.24
6	0	0	−1	1	56.99
7	1	−1	0	0	55.43
8	0	0	1	−1	57.32
9	0	0	1	1	59.33
10	1	0	1	0	59.09
11	0	1	−1	0	55.32
12	0	−1	0	1	51.24
13	0	0	0	0	63.62
14	0	0	−1	−1	47.24
15	0	−1	0	−1	55.25
16	1	0	0	1	58.96
17	−1	−1	0	0	53.50
18	−1	0	1	0	54.60
19	0	0	0	0	62.83
20	0	1	1	0	58.85
21	1	0	0	−1	53.24
22	0	0	0	0	61.81
23	−1	0	0	1	55.14
24	0	0	0	0	61.77
25	−1	0	0	−1	51.96
26	1	0	−1	0	53.46
27	0	1	0	−1	54.21
28	0	1	0	1	59.59
29	1	1	0	0	60.87

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表 3 回归模型方差分析

Table 3. Variance analysis of regression model

方差来源	平方和	自由度	均方	F值	P值	显著性
模型	520.11	11	47.28	22.42	<0.0001	**
A-超声功率	47.28	1	47.28	22.42	0.0005	**
B-超声时间	42.86	1	42.86	20.32	0.0007	**
C-酶添加量	86.45	1	86.45	40.99	<0.0001	**
D-料液比	40.45	1	40.45	19.18	0.0009	**
AB	12.06	1	12.06	5.72	0.0385	*
AC	0.28	1	0.28	0.12	0.7343
AD	1.62	1	1.62	0.70	0.4170
BC	1.59	1	1.59	0.69	0.4204
BD	22.02	1	22.02	10.44	0.0081	**
CD	15	1	15	7.11	0.0232	*
A²	98.07	1	98.07	46.5	<0.0001	**
B²	93.59	1	93.59	44.37	<0.0001	**
C²	120.08	1	120.08	56.93	<0.0001	**
D²	84.89	1	84.89	40.25	<0.0001	**
残差	35.86	17	2.11
失拟项	33.04	13	2.54	3.61	0.0898	不显著
纯误差	2.82	4	0.7
总误差	555.97	28
R²=0.93 R²_Adj=0.89 变异系数=1.45
注：*表示影响极显著，P<0.01；表示影响显著，P<0.05。

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表 4 FTIR红外光谱谱峰位置、峰值波数及说明

Table 4. FTIR infrared spectrum peak position, peak wave number and description

谱峰位置	峰值波数（cm⁻¹）		说明
谱峰位置	EEC	UAEEC	说明
酰胺A	3308.22	3305.34	N—H伸缩振动
酰胺B	3079.28	3076.40	CH₂基团的反对称收缩振动
酰胺Ⅰ	1638.37	1634.07	C=O伸缩振动
酰胺Ⅱ	1548.23	1545.36	N—H弯曲振动和C—N伸缩振动
酰胺Ⅲ	1240.58	1234.42	N—H 形变振动

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表 5 EEC和UAEEC二级结构含量

Table 5. Secondary structure content of EEC and UAEEC

处理方式	α-螺旋（%）	β-折叠（%）	β-转角（%）	无规则卷曲（%）
EEC	33.70±1.44^a	37.11±2.29^b	8.11±0.17^a	18.73±0.67^b
UAEEC	20.93±0.40^b	44.23±1.21^a	7.23±0.11^b	28.37±0.68^a
注：同列不同字母表示具有显著性差异（P<0.05）。

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表 6 牛皮胶原蛋白的氨基酸组成分析

Table 6. Analysis of the amino acid composition of cowhide collagen

序号	氨基酸种类	氨基酸含量（%）
序号	氨基酸种类	EEC	UAEEC
1	天门冬氨酸	0.02±0.00	0.04±0.00
2	谷氨酸	1.36±0.06	2.43±0.04
3	丝氨酸	2.95±0.20	3.61±0.06
4	甘氨酸	29.96±0.03	27.14±0.46
5	组氨酸	1.10±0.01	0.93±0.01
6	精氨酸	7.49±0.13	6.97±0.08
7	苏氨酸	1.92±0.14	1.09±0.05
8	丙氨酸	10.16±0.27	8.22±0.04
9	脯氨酸	29.39±0.41	35.96±0.22
10	酪氨酸	2.82±0.00	2.14±0.02
11	缬氨酸	0.28±0.00	0.24±0.00
12	蛋氨酸	2.96±0.06	2.51±0.05
13	胱氨酸	0.13±0.01	0.46±0.02
14	异亮氨酸	1.54±0.01	1.44±0.01
15	亮氨酸	3.01±0.01	2.64±0.11
16	苯丙氨酸	1.93±0.05	2.00±0.21
17	赖氨酸	2.98±0.02	2.16±0.08

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