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
计量
- 文章访问数: 14
- HTML全文浏览量: 46
- PDF下载量: 0
- 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

HTML全文

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 4 牛皮胶原蛋白的紫外吸收光谱图

Figure 4. UV absorption spectrum of cowhide collagen

下载: 全尺寸图片幻灯片

图 5 EEC和UAEEC红外图谱

Figure 5. Infrared spectra of EEC and UAEEC

下载: 全尺寸图片幻灯片

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)

下载: 全尺寸图片幻灯片

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×)

下载: 全尺寸图片幻灯片

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

下载: 导出CSV

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

下载: 导出CSV

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。

下载: 导出CSV

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

下载: 导出CSV

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）。

下载: 导出CSV

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

下载: 导出CSV

参考文献(39)

[1]	李国英. 胶原的类型及其结构特征[J]. 中国皮革,2002(17):20−21. [LI G Y. Types of collagen and its structural characteristics[J]. China Leather,2002(17):20−21.
[2]	DAN W, CHEN Y, DAN N, et al. Multi-level collagen aggregates and their applications in biomedical applications[J]. International Journal of Polymer Analysis and Characterization,2019,24(8):1−17.
[3]	JAFARI H, LISTA A, SIEKAPEN M, et al. Fish collagen: Extraction, characterization, and applications for biomaterials engineering[J]. Polymers,2020,12(10):2230. doi: 10.3390/polym12102230
[4]	陈诗妍. 胶原蛋白在食品中的应用分析[J]. 现代食品,2019(18):27−28, 31. [CHEN S Y. Analysis of the application of collagen in food[J]. Modern Food,2019(18):27−28, 31.
[5]	曹兵海, 张越杰, 李俊雅, 等. 2021年肉牛牦牛产业技术发展报告[J]. 中国畜牧杂志,2022,58(3):245−250. [CAO B H, ZHANG Y J, LI J Y, et al. Technology development report of beef cattle and yak industry in 2021[J]. China Animal Husbandry Journal,2022,58(3):245−250.
[6]	余群力, 冯玉萍. 家畜副产物综合利用[M]. 北京: 中国轻工业出版社, 2014. YU Q L, FENG Y P. Comprehensive utilization of livestock by-products[M]. Beijing: China Light Industry Press, 2014.
[7]	金晓丹, 田永强, 吴昊, 等. 制革行业土壤铬污染特征及其影响因素[J]. 环境工程,2021,39(12):206−211, 219. [JIN X D, TIAN Y Q, WU H, et al. Characteristics and influencing factors of soil chromium pollution in the leather industry[J]. Environmental Engineering,2021,39(12):206−211, 219.
[8]	但卫华, 曾睿. 生物质与生物质工程[J]. 中国皮革,2002,31(11):5. [DAN W H, ZENG R. Biomass and biomass engineering[J]. China Leather,2002,31(11):5.
[9]	GEOW C H, TAN M C, YEAP S P, et al. A review on extraction techniques and its future applications in industry[J]. European Journal of Lipid Science and Technology,2021,123(4):2000302.
[10]	PATIST A, BATES D. Ultrasonic innovations in the food industry: From the laboratory to commercial production[J]. Innovative Food Science & Emerging Technologies,2008,9(2):147−154.
[11]	ZA A, SK B, ET B. Effects of sonication on the extraction of pepsin-soluble collagens from lamb feet and product characterization[J]. LWT,2022,159:113253. doi: 10.1016/j.lwt.2022.113253
[12]	廖伟, 夏光华, 李川, 等. 尖吻鲈鱼鳞和鱼皮胶原蛋白的提取及其理化特性分析[J]. 食品科学,2018,39(1):36−41. [LIAO W, XIA G H, LI C, et al. Extraction of barramundi fish scales and skin collagen and analysis of its physical and chemical properties[J]. Food Science,2018,39(1):36−41.
[13]	杨恒. 超声辅助对鸡肺胶原蛋白提取工艺、理化性质及其酶解产物抗氧化活性的影响[D]. 南京: 南京财经大学, 2020. YANG H. The effect of ultrasound-assisted on the extraction process, physical and chemical properties of chicken lung collagen and the antioxidant activity of its enzymatic hydrolysis products[D]. Nanjing: Nanjing University of Finance and Economics, 2020.
[14]	齐越. 超声波辅助法酶解鹿皮胶原蛋白及其产品研发[D]. 长春: 吉林大学, 2014. QI Y. Ultrasonic-assisted enzymatic hydrolysis of deerskin collagen and its product development[D]. Changchun: Jilin University, 2014.
[15]	孟月志, 翁佩芳. 胃蛋白酶提取牛皮胶原蛋白的工艺研究[J]. 精细化工,2010,27(12):1187−1190, 1200. [MENG Y Z, WENG P F. Study on the extraction process of bovine bovine collagen by pepsin[J]. Fine Chemicals,2010,27(12):1187−1190, 1200.
[16]	沈菊泉, 汤俊, 沈亚领, 等. 酸法制备牛皮胶原蛋白及其结构性质研究[J]. 食品工业,2009,30(4):43−46. [SHEN J Q, TANG J, SHEN Y L, et al. Preparation of bovine bovine collagen by acid method and its structural properties[J]. Food Industry,2009,30(4):43−46.
[17]	徐毓谦, 郭美, 刘贵珊, 等. 牛皮胶原蛋白在牛肉饼中的抗冻特性评价[J]. 食品与发酵工业,2022,48(2):182−188. [XU Y Q, GUO M, LIU G S, et al. Evaluation of antifreeze properties of cowhide collagen in beef patties[J]. Food and Fermentation Industry,2022,48(2):182−188.
[18]	KHALAJI S, EBRAHIMI N G, HOSSEINKHANI H. Enhancement of biocompatibility of PVA/HTCC blend polymer with collagen for skin care application[J]. International Journal of Polymeric Materials,2020:1−10.
[19]	瞿怡, 苏寒雨, 刘文涛, 等. 超声波辅助法提取牛皮胶原蛋白[J]. 皮革科学与工程,2018,28(2):5−9. [QU Y, SU H Y, LIU W T, et al. Ultrasound-assisted extraction of bovine collagen[J]. Leather Science and Engineering,2018,28(2):5−9.
[20]	ETHERINGTON D J, SIMS T J. Detection and estimation of collagen[J]. Journal of the Science of Food and Agriculture,2010,32(6):539−546.
[21]	SAHNI P, SHARMA S, SURASANI V. Influence of processing and pH on amino acid profile, morphology, electrophoretic pattern, bioactive potential and functional characteristics of alfalfa protein isolates[J]. Food Chemistry,2020,333:127503. doi: 10.1016/j.foodchem.2020.127503
[22]	ZHU S, YUAN Q, YANG M, et al. A quantitative comparable study on multi-hierarchy conformation of acid and pepsin-solubilized collagens from the skin of grass carp (Ctenopharyngodon idella)[J]. Materials Science and Engineering C,2018,96(MAR.):446−457.
[23]	国家食品药品监督管理总局. 食品安全国家标准食品中氨基酸的测定: GB 5009.124-2016[S]. 北京: 中国标准出版, 2016. State Food and Drug Administration. National food safety standard determination of amino acids in food: GB 5009.124-2016[S]. Beijing: China Standard Publishing, 2016.
[24]	LIU J, LUO D, LI X, et al. Effects of inulin on the structure and emulsifying properties of protein components in dough[J]. Food Chemistry,2016,210(Nov.1):235−241.
[25]	UMEGO E, HE R, REN W, et al. Ultrasonic-assisted enzymolysis: Principle and applications[J]. Process Biochemistry,2020,100:59−68.
[26]	LIANG X, FENG S, AHMED S, et al. Effect of potassium sorbate and ultrasonic treatment on the properties of fish scale collagen/polyvinyl alcohol composite film[J]. Molecules,2019,24(13):2363. doi: 10.3390/molecules24132363
[27]	宋正规, 朱丽娜, 张洪超, 等. 胃蛋白酶提取马面鱼皮胶原蛋白及结构分析[J]. 食品科学,2018,39(20):260−267. [SONG Z G, ZHU L N, ZHANG H C, et al. Extraction of collagen from horse noodle fish skin by pepsin and structural analysis[J]. Food Science,2018,39(20):260−267. doi: 10.7506/spkx1002-6630-201820038
[28]	PRASANTH K S, STONE A K, GUO Q, et al. Effect of alkaline de-esterified pectin on the complex coacervation with pea protein isolate under different mixing conditions[J]. Food Chemistry,2019,284:227−235. doi: 10.1016/j.foodchem.2019.01.122
[29]	CAO S, WANG Y, XING L, et al. Structure and physical properties of gelatin from bovine bone collagen influenced by acid pretreatment and pepsin[J]. Food and Bioproducts Processing,2020:121.
[30]	BLANCO M, VÁZQUEZ J A, PÉREZ-MARTÍN R I, et al. Collagen extraction optimization from the skin of the small-spotted catshark (S. canicula) by response surface methodology[J]. Marine Drugs,2019,17(1):40. doi: 10.3390/md17010040
[31]	史睿, 何静, 吉日木图. 超声波辅助提取骆驼皮胶原蛋白的工艺及结构表征[J]. 中国食品学报,2022,22(2):213−223. [SHI R, HE J, JIRI M T. Ultrasonic-assisted extraction of collagen from camel skin and its structural characterization[J]. Chinese Journal of Foodstuffs,2022,22(2):213−223.
[32]	张坤, 邹烨, 王道营, 等. 高强度超声处理对鹅胸肉肌动球蛋白特性的影响[J]. 食品科学,2018,39(21):59−65. [ZHANG K, ZOU Y, WANG D Y, et al. Effects of high-intensity ultrasound treatment on the properties of actomyosin in goose breast meat[J]. Food Science,2018,39(21):59−65.
[33]	李八方, 郭鸣, 侯虎, 等. 胡子鲶鱼皮酸溶性胶原蛋白的理化性质研究[J]. 现代食品科技,2013,29(11):2580−2585. [LI B F, GUO M, HOU H, et al. Study on the physical and chemical properties of acid-soluble collagen from clarias catfish skin[J]. Modern Food Science and Technology,2013,29(11):2580−2585.
[34]	NALINANON S, BENJAKUL S, KISHIMURA H, et al. Type I collagen from the skin of ornate threadfin bream (Nemipterus hexodon): Characteristics and effect of pepsin hydrolysis[J]. Food Chemistry,2010,125(2):500−507.
[35]	CHEUNG H S, WANG F L, ONDETTI M A, et al. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence[J]. Journal of Biological Chemistry,1980,255(2):401−407. doi: 10.1016/S0021-9258(19)86187-2
[36]	MDA B, EAAA C, BM A, et al. Proteolysis kinetics and structural characterization of ultrasonic pretreated sunflower protein[J]. Process Biochemistry,2020,94:198−206. doi: 10.1016/j.procbio.2020.04.018
[37]	向安妮, 许爽, 鞠红梅, 等. 遏蓝菜硒蛋白的纯化、结构表征及抗氧化活性研究[J]. 西北农业学报,2022,31(3):299−309. [XIANG A N, XU S, JU H M, et al. Purification, structural characterization and antioxidant activity of selenoproteins from Rhizoma chinensis[J]. Northwest Agricultural Journal,2022,31(3):299−309. doi: 10.7606/j.issn.1004-1389.2022.03.005
[38]	杨恒, 陈银基, 邹烨, 等. 两种方法提取的鸡肺胶原蛋白性质及保湿性研究[J]. 食品工业科技,2020,41(17):1−8. [YANG H, CHEN Y J, ZOU Y, et al. Study on the properties and moisturizing properties of chicken lung collagen extracted by two methods[J]. Food Industry Science and Technology,2020,41(17):1−8.
[39]	SUN X, WANG J, WANG Y, et al. Collagen-based porous scaffolds containing PLGA microspheres for controlled kartogenin release in cartilage tissue engineering[J]. Artificial Cells Nanomedicine & Biotechnology,2017:1.