超声辅助酶改性小球藻蛋白工艺优化及其乳化特性的分析

杨清馨; 刘少伟; 韩莉君

doi:10.13386/j.issn1002-0306.2022080075

超声辅助酶改性小球藻蛋白工艺优化及其乳化特性的分析

doi: 10.13386/j.issn1002-0306.2022080075

华东理工大学食品科学与工程系生物反应器工程国家重点实验室，上海 200237

详细信息

作者简介:
杨清馨（1998−），女，硕士研究生，研究方向：食品蛋白加工、食品生物技术等，E-mail：dorayqx@163.com

通讯作者:
刘少伟（1972−），男，博士，教授，研究方向：食品加工、谷物、乳制品食品开发等，E-mail：swliu@ecust.edu

中图分类号: TS201.2
计量
- 文章访问数: 22
- HTML全文浏览量: 32
- PDF下载量: 0
- 被引次数: 0
出版历程
- 收稿日期: 2022-08-09
- 刊出日期: 2023-05-15

Optimization of Ultrasound-Assisted Enzyme Modification Process of Chlorella Protein and Analysis of Its Emulsification Properties

The State Key Laboratory of Bioreactor Engineering, Department of Food Science and Engineering, East China University of Science and Technology, Shanghai 200237, China

摘要

摘要: 本文以小球藻蛋白为研究对象，探究了超声辅助酶改性条件优化及改性后乳化特性的变化。选择超声时间、超声功率、酶添加量、酶反应时间为考察因素，使用层次分析法对乳化活性、乳化稳定性和乳析指数分别赋予权重，并以综合加权得分为响应值，采用四因素三水平的响应面法探究超声辅助酶改性小球藻蛋白乳化特性的最佳改性条件。结果表明，最佳改性工艺条件为：超声时间31 min、超声功率209 W、酶添加量3.1%、酶反应时间3 h。在此优化条件下，乳化性能的最佳评分可以达到98.99，与预测值接近。同时，改性小球藻蛋白的溶解性和起泡性与原蛋白相比分别提高了68.30%和49.44%，但持水性和持油性的改善效果并不明显。综上，该研究为小球藻蛋白在食品乳化体系中的应用提供了一定的理论基础。
- 小球藻蛋白 /
- 乳化特性 /
- 超声改性 /
- 酶改性 /
- 响应面 /
- 层次分析法
Abstract: The optimized conditions of ultrasound-assisted enzyme modification and the effects of modification on the emulsification properties of Chlorella protein were investigated in this paper. The ultrasonic time, ultrasonic power, enzyme addition and enzyme reaction time were selected as the investigating factors. The analytic hierarchy process (AHP) was used to weight the emulsification activity, emulsification stability and creaming index, and the comprehensive weighted score was used as the response value. A four-factor, three-level response surface method was used to investigate the optimal modification conditions for the emulsification properties of ultrasound-assisted enzyme-modified Chlorella protein. The results showed that the optimal modification conditions were as follows: Ultrasonic time 31 min, ultrasonic power 209 W, enzyme addition 3.1% and enzyme reaction time 3 h. Under the optimized conditions, the best score of emulsification performance was 98.99, which was close to the predicted value. Compared with the original protein, the solubility and foaming ability of modified Chlorella protein increased by 68.30% and 49.44%, respectively. However, the modification of water-holding and oil-holding properties showed no significant effect. In summary, this study could provide certain theoretical basis for the application of Chlorella protein in food emulsification systems.
- Chlorella protein /
- emulsifying characteristics /
- ultrasonic modification /
- enzymatic modification /
- response surface methodology /
- analytical hierarchy process

HTML全文

图 1 超声时间对小球藻蛋白乳化性能及其综合得分的影响

Figure 1. Effects of ultrasonic time on the emulsifying property and comprehensive score of Chlorella protein

下载: 全尺寸图片幻灯片

图 2 超声功率对小球藻蛋白乳化性能及其综合得分的影响

Figure 2. Effects of ultrasonic power on the emulsifying property and comprehensive score of Chlorella protein

下载: 全尺寸图片幻灯片

图 3 酶添加量对小球藻蛋白乳化性能及其综合得分的影响

Figure 3. Effects of enzyme addition on the emulsifying property and comprehensive score of Chlorella protein

下载: 全尺寸图片幻灯片

图 4 酶反应时间对小球藻蛋白乳化性能及其综合得分的影响

Figure 4. Effects of enzyme reaction time on the emulsifying property and comprehensive score of Chlorella protein

下载: 全尺寸图片幻灯片

图 5 各因素交互作用对小球藻蛋白乳化特性影响的响应面图

Figure 5. Response surface diagram of the interaction of various factors on the emulsifying characteristics of Chlorella protein

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 for response surface test

水平	因素
水平	A超声时间（min）	B超声功率（W）	C酶浓度（%）	D酶反应时间（h）
−1	20	150	2	2.5
0	30	200	3	3.0
1	40	250	4	3.5

下载: 导出CSV

表 2 目标树层次评分标准^[23]

Table 2. Evaluation standard of target tree at different levels^[23]

相对重要性	定义
1	同等重要
3	稍微重要
5	明显重要
7	强烈重要
9	绝对重要
2,4,6,8	以上相邻指标的中间值
倒数	若i与j的重要性之比为A_ij，则j与i的重要性之比为1/A_ij

下载: 导出CSV

表 3 3种指标的比较矩阵表

Table 3. Decision matrix of paried comparison on three indexes

指标	乳化活性	乳化稳定性	乳析指数	特征向量	权重值（%）	最大特征值	CI值	CR值
乳化活性	1	1	5/3	1.154	38.462	3.001	0.0005	0.0009
乳化稳定性	1	1	5/3	1.154	38.462
乳析指数	3/5	3/5	1	0.692	23.077
注：CI=（最大特征根−n）/（n−1）；CR=CI/RI。

下载: 导出CSV

表 4 响应面设计方案与结果

Table 4. The design and results of response surface

实验编号	A	B	C	D	EAI（m²/g）	ESI（min）	CI（%）	综合得分
1	−1	−1	0	0	25.77	109.22	25.55	75.13
2	1	−1	0	0	28.21	115.23	23.17	82.42
3	−1	1	0	0	29.54	111.74	22.73	83.29
4	1	1	0	0	30.13	115.49	20.97	87.20
5	0	0	−1	−1	24.88	112.78	24.72	76.30
6	0	0	1	−1	28.83	114.92	22.64	83.63
7	0	0	−1	1	26.06	112.69	22.42	80.32
8	0	0	1	1	28.86	114.17	20.28	85.42
9	−1	0	0	−1	26.33	116.78	22.49	81.76
10	1	0	0	−1	27.91	114.96	22.08	83.36
11	−1	0	0	1	28.64	111.49	22.02	83.14
12	1	0	0	1	30.19	113.96	19.81	88.22
13	0	−1	−1	0	24.72	110.34	25.52	74.72
14	0	1	−1	0	25.44	112.24	23.13	78.67
15	0	−1	1	0	27.90	113.02	23.98	80.44
16	0	1	1	0	30.69	116.74	20.13	89.19
17	−1	0	−1	0	25.55	112.98	25.76	75.79
18	1	0	−1	0	28.43	114.77	23.77	81.78
19	−1	0	1	0	29.16	115.34	22.15	84.70
20	1	0	1	0	27.02	117.01	20.13	85.44
21	0	−1	0	−1	26.83	109.77	25.65	76.28
22	0	1	0	−1	29.73	111.84	22.43	83.88
23	0	−1	0	1	24.12	110.77	19.97	80.70
24	0	1	0	1	29.07	112.67	19.85	86.60
25	0	0	0	0	36.79	121.82	19.18	98.28
26	0	0	0	0	35.62	126.22	19.56	97.93
27	0	0	0	0	36.15	125.71	19.03	98.97
28	0	0	0	0	35.08	125.13	18.87	97.88
29	0	0	0	0	35.27	124.98	19.14	97.70

下载: 导出CSV

表 5 响应面方差分析

Table 5. Variance analysis of response surface

方差来源	平方和	自由度	均方	F	P	显著性
模型	1472.82	14	105.13	158.33	<0.0001	**
A-超声时间	50.47	1	50.47	76.01	<0.0001	**
B-超声功率	127.66	1	127.66	192.26	<0.0001	**
C-酶添加量	141.73	1	141.73	213.44	<0.0001	**
D-酶反应时间	30.69	1	30.69	46.22	<0.0001	**
AB	2.86	1	2.86	4.30	0.0570
AC	6.89	1	6.89	10.38	0.0062	*
AD	3.03	1	3.03	4.56	0.0509
BC	5.76	1	5.76	8.67	0.0106	*
BD	0.72	1	0.72	1.09	0.3146
CD	1.24	1	1.24	1.87	0.1928
A²	323.59	1	323.59	487.33	<0.0001	**
B²	499.67	1	499.67	752.51	<0.0001	**
C²	530.30	1	530.30	798.64	<0.0001	**
D²	354.30	1	354.30	533.58	<0.0001	**
残差	9.30	14	0.67
失拟项	8.28	10	0.83	3.27	0.1356
纯误差	1.01	4	0.25
总误差	1481.12	28
R²=0.9937 R²_Adj=0.9874 CV（%）=0.9609
Adequate Precision：39.7870
注：*为P<0.0001，差异极显著；为P<0.05，差异显著。

下载: 导出CSV

表 6 超声辅助酶改性对小球藻蛋白功能特性的影响

Table 6. Effect of ultrasound assisted enzymatic modification on the functional properties of Chlorella protein

样品	溶解性（%）	持水性（g·g⁻¹）	持油性（g·g⁻¹）	起泡性（%）	泡沫稳定性（%）
小球藻蛋白	32.75±1.83	1.51±0.03	3.11±0.03	45.83±1.45	133.33±3.98
改性小球藻蛋白	55.12±2.47	1.74±0.01	3.42±0.04	68.49±3.01	120.97±2.66
注：以上经t检验均在P<0.05水平上差异显著。

下载: 导出CSV

参考文献(40)

[1]	岳海艳, 刘小杰, 孙敏, 等. 蛋白核小球藻的培养及应用研究[J]. 粮油食品科技,2022,30(4):178−184. [YUE H Y, LIU X J, SUN M, et al. Cultivation and application of Chlorella pyrenoidosa[J]. Science and Technology of Cereals, Oils and Foods,2022,30(4):178−184.
[2]	ADMASSU H, GASMALLA M A A, YANG R, et al. Bioactive peptides derived from seaweed protein and their health benefits: Antihypertensive, antioxidant, and antidiabetic properties[J]. Journal of Food Science,2018,83(1):6−16. doi: 10.1111/1750-3841.14011
[3]	THYS R, DIPRAT A B, RODRIGUES E, et al. Chlorella sorokiniana: A new alternative source of carotenoids and proteins for gluten-free bread[J]. LWT-Food Science and Technology,2020,134:109974. doi: 10.1016/j.lwt.2020.109974
[4]	ZHANG R L, CHEN J, ZHANG X W. Extraction of intracellular protein from Chlorella pyrenoidosa using a combination of ethanol soaking, enzyme digest, ultrasonication and homogenization techniques[J]. Bioresource Technology Biomass Bioenergy Biowastes Conversion Technologies Biotransformations Production Technologies,2018,247:267−272.
[5]	LIAN H J, WHEN C T, ZHANG J X, et al. Effects of simultaneous dual-frequency divergent ultrasound-assisted extraction on the structure, thermal and antioxidant properties of protein from Chlorella pyrenoidosa[J]. Algal Research,2021,56:102294. doi: 10.1016/j.algal.2021.102294
[6]	马石霞, 马咸莹, 丁功涛, 等. 小球藻叶绿素脱除工艺优化及蛋白提取[J]. 西北民族大学学报(自然科学版),2020,41(4):74−79. [MA S X, MA X Y, DING G T, et al. Optimization of chlorophyll removal process and protein extraction from chlorella[J]. Journal of Northwest Minzu University (Natural Science Edition),2020,41(4):74−79. doi: 10.3969/j.issn.1009-2102.2020.04.015
[7]	DAI L X, HINRICHS J, WEISS J. Emulsifying properties of acid-hydrolyzed insoluble protein fraction from Chlorella protothecoides: Formation and storage stability of emulsions[J]. Food Hydrocolloids,2020,108:105954. doi: 10.1016/j.foodhyd.2020.105954
[8]	MNN A, ASD A, RM B. Modification approaches of plant-based proteins to improve their techno-functionality and use in food products[J]. Food Hydrocolloids,2021,118:106789. doi: 10.1016/j.foodhyd.2021.106789
[9]	RESENDIZ-VAZQUEZ J A, ULLOA J A, URíAS-SILVAS J, et al. Effect of high-intensity ultrasound on the technofunctional properties and structure of jackfruit (Artocarpus heterophyllus) seed protein isolate[J]. Ultrasonics Sonochemistry,2017,37:436−444. doi: 10.1016/j.ultsonch.2017.01.042
[10]	常慧敏. 超声和木瓜蛋白酶改善米糠蛋白乳化性的研究[D]. 郑州: 河南工业大学, 2019. CHANG H M. Improving emulsibility properties of rice bran protein with ultrasonic and papain hydrolysis methods[D]. Zhengzhou: Henan University of Technology, 2019.
[11]	ABISMAL B, CANSELIER J P, WILHELM A M, et al. Emulsification by ultrasound: Drop size distribution and stability[J]. Ultrasonics Sonochemistry, 1999, 6(1–2): 75-83.
[12]	李杨, 陈凡凡, 王中江, 等. 超声预处理对大豆蛋白聚集体结构和乳化特性的影响[J]. 农业机械学报,2020,51(6):366−374. [LI Y, CHEN F F, WANG Z J, et al. Effect of ultrasonic pretreatment on structure and functional properties of soy protein aggregates[J]. Transactions of the Chinese Society for Agricultural Machinery,2020,51(6):366−374.
[13]	LUISA A, GASPAR C, DE GOES-FAVONI S P. Action of microbial transglutaminase (MTGase) in the modification of food proteins: A review[J]. Food Chemistry,2015,171(15):315−322.
[14]	FOEGEDING E A, DAVIS J P. Food protein functionality: A comprehensive approach[J]. Food Hydrocolloids,2011,25(8):1853−1864. doi: 10.1016/j.foodhyd.2011.05.008
[15]	YONG Y H, YAMAGUCHI S, MATSUMURA Y. Effects of enzymatic deamidation by protein-glutaminase on structure and functional properties of r-zein[J]. Food Chemistry,2004,52(23):7094−7100. doi: 10.1021/jf040133u
[16]	薛雨菲, 张玥, 程怡媚, 等. 酶法改性对核桃谷蛋白微观结构及功能特性的影响[J]. 食品与机械,2019,35(10):18−23. [XUE Y F, ZHANG Y, CHENG Y M, et al. Effect of enzymatic modification on microstructure and functional properties of walnut glutenin[J]. Food & Machinery,2019,35(10):18−23.
[17]	杨倩, 蔡茜茜, 郭淋凯, 等. 响应面优化提取小球藻蛋白质及其性质研究[J]. 中国食品学报,2018,18(6):183−190. [YANG Q, CAI Q Q, GUO L K, et al. Optimization of protein extraction from chlorella vulgaris using response surface methodology and study on its property[J]. Journal of Chinese Institute of Food Science and Technology,2018,18(6):183−190.
[18]	吴振起, 高畅, 杨璐, 等. 基于层次分析法结合Box-Behnken设计-响应面法优选养阴清肺汤加味提取工艺[J]. 中草药,2019,50(12):2862−2867. [WU Z Q, GAO C, YANG L, et al. Optimization of extraction process of modified Yangyin Qingfei Decoction by Box-Behnken response surface methodology based on analytic hierarchy process[J]. Chinese Traditional and Herbal Drugs,2019,50(12):2862−2867. doi: 10.7501/j.issn.0253-2670.2019.12.018
[19]	胡守珍. 异养小球藻粗蛋白质提取方法研究[D]. 南阳: 南阳师范学院, 2019. HU S Z. Study on extraction method of crude protein from heterotrophic chlorella[D]. Nanyang: Nanyang Normal University, 2019.
[20]	丁欣悦. 超声辅助转谷氨酰胺酶及美拉德反应对大豆分离蛋白结构和功能性质的影响[D]. 南昌: 南昌大学, 2018. DING X Y. Effects of ultrasound-assisted transglutaminase crosslinking and Maillard reaction on structure and functional properties of soy protein isolate[D]. Nanchang: Nanchang University, 2018.
[21]	HUI J, ZHENG X, YANG W, et al. Preparation of allicin-whey protein isolate conjugates: Allicin extraction by water, conjugates' ultrasound-assisted binding and its stability, solubility and emulsibility analysis[J]. Ultrasonics Sonochemistry,2020,63:104981. doi: 10.1016/j.ultsonch.2020.104981
[22]	SHAO Y, TANG C H. Characteristics and oxidative stability of soy protein-stabilized oil-in-water emulsions: Influence of ionic strength and heat pretreatment[J]. Food Hydrocolloids,2014,37:149−158. doi: 10.1016/j.foodhyd.2013.10.030
[23]	余天华, 张杰, 许婷婷, 等. 层次分析及响应面法优选猕猴桃枝条中纤维素提取工艺[J]. 贵州师范大学学报(自然科学版),2021,39(5):7−14, 28. [YU T H, ZHANG J, XU T T, et al. Optimization of extraction process of cellulose from kiwifruit branches with analytic hierarchy and response surface methodology[J]. Journal of Guizhou Normal University (Natural Sciences),2021,39(5):7−14, 28.
[24]	孙乾, 张爱琴, 薛雨菲, 等. 化学改性对核桃谷蛋白结构表征及功能特性的影响[J]. 食品科学,2019,40(20):87−93. [SUN Q, ZHANG A Q, XUE Y F, et al. Effect of chemical modification on structural characteristics and functional properties of walnut glutenin[J]. Food Science,2019,40(20):87−93.
[25]	MN A, NV A, NM A, et al. Pulsed ultrasound assisted extraction of protein from defatted Bitter melon seeds (Momardica charantia L. ) meal: Kinetics and quality measurements[J]. LWT,2022,155:112997. doi: 10.1016/j.lwt.2021.112997
[26]	AGYARE K K, ADDO K, XIONG Y L. Emulsifying and foaming properties of transglutaminase-treated wheat gluten hydrolysate as influenced by pH, temperature and salt[J]. Food Hydrocolloids,2009,23(1):72−81. doi: 10.1016/j.foodhyd.2007.11.012
[27]	YU C, LI S, SUN S, et al. Modification of emulsifying properties of mussel myofibrillar proteins by high-intensity ultrasonication treatment and the stability of O/W emulsion[J]. Colloids and Surfaces, A. Physicochemical and Engineering Aspects, 2022, 641: 128511.
[28]	SORIA A C, VILLAMIEL M. Effect of ultrasound on the technological properties and bioactivity of food: A review[J]. Trends in Food Science & Technology,2010,21(7):323−331.
[29]	LI Y, WANG D, ZHANG S, et al. Stability andin vitro simulated release characteristics of ultrasonically modified soybean lipophilic protein emulsion[J]. Food & Function,2020,11:3800−3810.
[30]	SHA L, KOOSIS A O, WANG Q, et al. Interfacial dilatational and emulsifying properties of ultrasound-treated pea protein[J]. Food Chemistry,2021,350(2):129271.
[31]	DABBOUR M I, XIANG J, MINTAH B, et al. Localized enzymolysis and sonochemically modified sunflower protein: Physical, functional and structure attributes[J]. Ultrasonics Sonochemistry,2020,63:104957. doi: 10.1016/j.ultsonch.2019.104957
[32]	ZHANG L, XIAO Q, WANG Y, et al. Effects of sequential enzymatic hydrolysis and transglutaminase crosslinking on functional, rheological, and structural properties of whey protein isolate[J]. LWT-Food Science and Technology,2021,1:112415.
[33]	HRYNETS Y, NDAGIJIMANA M, BETTI M. Transglutaminase-catalyzed glycosylation of natural actomyosin (NAM) using glucosamine as amine donor: Functionality and gel microstructure[J]. Food Hydrocolloids,2014,36(may):26−36.
[34]	ALAM P, SIDDIQUI N A, REHMAN M T, et al. Box-behnken design (BBD)-based optimization of microwave-assisted extraction of parthenolide from the stems of Tarconanthus camphoratus and cytotoxic analysis[J]. Molecules,2021,26(7):1876. doi: 10.3390/molecules26071876
[35]	ZHU Z, ZHU W, YI J, et al. Effects of sonication on the physicochemical and functional properties of walnut protein isolate[J]. Food Research International,2018,106(Apr.):853−861.
[36]	JIANG L, WANG J, LI Y, et al. Effects of ultrasound on the structure and physical properties of black bean protein isolates[J]. Food Research International,2014,62:595−601. doi: 10.1016/j.foodres.2014.04.022
[37]	LI Y, CHENG Y, ZHANG Z, et al. Modification of rapeseed protein by ultrasound-assisted pH shift treatment: Ultrasonic mode and frequency screening, changes in protein solubility and structural characteristics[J]. Ultrasonics Sonochemistry,2020,69:105240. doi: 10.1016/j.ultsonch.2020.105240
[38]	LIU Y X, ZHANG Y F, GUO Z H, et al. Enhancing the functional characteristics of soy protein isolate via cross-linking catalyzed byBacillus subtilis transglutaminase[J]. Journal of the Science of Food and Agriculture,2020,101(10):4154−4160.
[39]	GLUSAC J, ISASCHAR-OVDAT S, FISHMAN A. Transglutaminase modifies the physical stability and digestibility of chickpea protein-stabilized oil-in-water emulsions[J]. Food Chemistry,2020,315:126301. doi: 10.1016/j.foodchem.2020.126301
[40]	GAO K, ZHA F C, YANG Z Y, et al. Structure characteristics and functionality of water-soluble fraction from high-intensity ultrasound treated pea protein isolate[J]. Food Hydrocolloids,2021,125:107409.