• EI
    • CSA
    • CABI
    • 卓越期刊
    • CA
    • Scopus
    • CSCD
    • 核心期刊

水热预处理提高花生分离蛋白酶解效率及其机理分析

陈林, 陈建设, 于泓鹏, 吴克刚

陈林, 陈建设, 于泓鹏, 吴克刚. 水热预处理提高花生分离蛋白酶解效率及其机理分析[J]. 农业工程学报, 2017, 33(1): 278-284. DOI: 10.11975/j.issn.1002-6819.2017.01.038
引用本文: 陈林, 陈建设, 于泓鹏, 吴克刚. 水热预处理提高花生分离蛋白酶解效率及其机理分析[J]. 农业工程学报, 2017, 33(1): 278-284. DOI: 10.11975/j.issn.1002-6819.2017.01.038
Chen Lin, Chen Jianshe, Yu Hongpeng, Wu Kegang. Hydrothermal pretreatment improving proteolysis efficiency of peanut protein isolates and its mechanism analysis[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2017, 33(1): 278-284. DOI: 10.11975/j.issn.1002-6819.2017.01.038
Citation: Chen Lin, Chen Jianshe, Yu Hongpeng, Wu Kegang. Hydrothermal pretreatment improving proteolysis efficiency of peanut protein isolates and its mechanism analysis[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2017, 33(1): 278-284. DOI: 10.11975/j.issn.1002-6819.2017.01.038

水热预处理提高花生分离蛋白酶解效率及其机理分析

基金项目: 广东省科技计划项目(2016A020210113);高等学校博士学科点专项科研基金项目(20134420120008)。

Hydrothermal pretreatment improving proteolysis efficiency of peanut protein isolates and its mechanism analysis

  • 摘要: 为了提高花生分离蛋白的酶解效率,该文采用水热法对花生分离蛋白进行预处理,利用响应面试验设计优化预处理工艺,并研究比较了预处理前后花生分离蛋白酶解敏感性和空间构象的变化。结果表明:优化后的最佳预处理条件为水热压力90 MPa、水热温度85℃、水热时间20 min,此条件下酶解产物水解度达到16.3%,相对未经预处理的酶解产物提高了8.1个百分点。水热预处理提高了花生分离蛋白的主要组分花生球蛋白和伴球蛋白的酶解敏感性,使酶解产物蛋白质回收率提高了31.9个百分点。进行荧光光谱和红外光谱分析发现水热预处理使花生分离蛋白三级结构展开、二级结构紧密程度下降,可能是其酶解敏感性提高的主要原因。因此水热预处理是一种辅助提高花生分离蛋白酶解效率行之有效的方法。
    Abstract: Abstract: The use of peanut protein has attained an increasing attention, primarily attributed to its high nutrition value, steady supply, and low cost compared with other proteins with different sources. Peanut protein isolate (PPI) is the most refined peanut protein product, containing 90% protein on a moisture-free basis, and is used as a kind of important protein material in food industry. However, the poor protein solubility and functional properties of commercial PPI limit its application in food and non-food products. Because of small side reactions and safeness, proteolysis has been widely used to improve protein functionalities and to prepare bioactive peptides. However, peanut proteins are resistant to proteolysis due to their compact structures that protect many of the hydrolysis sites. Recently, several studies have reported that hydrothermal treatment could not only alter the spatial conformations of globular proteins, but also break up protein aggregates into smaller pieces, which may cause the exposure of previously buried hydrolysis sites. However, little work has been done so far to investigate this possibility. Therefore, this work aimed to investigate the influences of hydrothermal pretreatment on the proteolysis pattern and the structure of PPI. Hydrothermally cooked PPI (HPPI) was prepared using a CJ-200 autoclave, and protease Protamex was used for the preparation of PPI hydrolysates (PPIH) and HPPI hydrolysates (HPPIH). Response surface methodology (RSM) was used to optimize the processing conditions of hydrothermally cooking, and the optimal conditions were as follows: the pressure was 90 MPa, the temperature was 85 ℃, and the time was 20 min. The actual degree of hydrolysis (DH) of HPPIH obtained under this pretreatment condition was 16.3%±0.2%, which was not significantly different (P>0.05) from the predicted value (DH=16.2%). And the analysis of variance of the regression equation for the response surface quadratic model showed that the sequence of the importance for influential factors was pressure > temperature > time. The analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of PPI, HPPI, PPIH and HPPIH showed that hydrothermal pretreatment substantially improved the enzymatic accessibility of the major subunits of conarachin and arachin in PPI, which were initially resistant to Protamex hydrolysis. As a result, more peanut proteins in HPPI could be readily hydrolyzed and become soluble, causing a strong increase in protein recovery (PR) for HPPIH. Under the 90 MPa pressure, HPPIH showed a much higher PR of 73.4% than that of PPIH (PR=41.5%). In addition, it was somewhat surprising that the observations of SDS-PAGE profiles and DH measurement both showed that hydrothermal pretreatment enhanced the spontaneous hydrolysis of PPI. Analysis of the intrinsic emission fluorescence spectra of PPI and HPPI demonstrated that compared with the control PPI, HPPI showed a decrease in the intensity of the fluorescence peak at 323 nm, and a red-shift for the peak at 332 nm, which suggested that hydrothermal treatment caused the unfolding of tertiary structure for PPI. Analysis of Fourier transform infrared spectra (FTIR) of PPI and HPPI demonstrated that compared with the control PPI, HPPI showed a significant decrease in α-helix and β-turn, and an increase in β-sheet and random coil, which suggested that hydrothermal treatment could loosen the secondary structure of peanut protein. So it is inferred that the unfolded tertiary structure and the loosened secondary structure for HPPI after hydrothermal pretreatment may be the main causes for its improved enzymatic accessibility. In conclusion, this study shows that hydrothermal pretreatment is a highly effective technique to accelerate and enhance the proteolysis of PPI.
  • [1] Phillips G O, Williams P A. Handbook of Food Proteins[M]. Cambridge: Woodhead Publishers, 2011, 242-246.
    [2] 赵晓燕,孙秀平,陈峰亮,等. 花生蛋白的研究进展及开发利用现状[J]. 中国粮油学报,2011,26(12):118-122.Zhao Xiaoyan, Sun Xiuping, Chen Fengliang, et al. Research progress and application of peanut protein[J]. Journal of the Chinese Cereals and Oils Association, 2011, 26(12): 118-122. (in Chinese with English abstract)
    [3] Zhao X Y, Chen J, Du F L. Potential use of peanut by-products in food processing: a review[J]. International Journal of Food Science and Technology, 2012, 49: 521-529.
    [4] 魏红艳,卞科. 花生蛋白的研究与开发[J]. 农产品加工(创新版),2010,(5):46-51.Wei Hongyan, Bian Ke. Research and development of peanut protein[J]. Innovational Edition of Farm Products Processing, 2010, (5): 46-51. (in Chinese with English abstract)
    [5] Tavano O L. Protein hydrolysis using proteases: An important tool for food biotechnology[J]. Journal of Molecular Catalysis B: Enzymatic, 2013, 90: 1-11.
    [6] 陈林,吴克刚,柴向华,等. 物理预处理改善食品蛋白酶解特性的研究进展[J]. 食品与发酵工业,2013,39(10):33-38.Chen Lin, Wu Kegang, Chai Xianghua, et al. Progress on improving the enzymatic hydrolysis of food protein using physical pre-treatment[J]. Food and Fermentation Industries, 2013, 39(10): 33-38. (in Chinese with English abstract)
    [7] Chen L, Chen J S, Yu L, et al. Improved emulsifying capabilities of hydrolysates of soy protein isolate pretreated with high pressure microfluidization[J]. LWT-Food Scienceand Technology, 2016, 69: 1-8.
    [8] 陈林,吴克刚,柴向华,等. 微射流均质预处理提高大豆分离蛋白酶解效率及酶解产物乳化性能[J]. 农业工程学报,2015,31(5):331-338.Chen Lin, Wu Kegang, Chai Xianghua, et al. Microfluidization pretreatment improving enzymatic hydrolysis of soy isolated protein and emulsifying properties of hydrolysates[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2015, 31(5): 331-338. (in Chinese with English abstract)
    [9] 张宇昊,王强. Alcalase 酶水解花生蛋白制备花生短肽的研究[J]. 农业工程学报,2007,23(4):258-263.Zhao Yuhao, Wang Qiang. Peanut protein hydrolyzing by Alcalase to prepare peanut oligopeptides[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2007, 23(4): 258-263. (in Chinese with English abstract)
    [10] Tsou, M J, Lin W T, Lu H C, et al. The effect of limited hydrolysis with Neutrase and ultrafiltration on the anti- adipogenic activity of soy protein[J]. Process Biochemistry, 2009, 45(2): 217-222.
    [11] Gall M L, Guguen J, Sve B, et al. Effects of grinding and thermal treatments on hydrolysis susceptibility of pea proteins (Pisum sativum L.)
    [J]. Journal of Agricultural and Food Chemistry, 2005, 53: 3057-3064.
    [12] Govindaraju H, Srinivas H. Studies on the effects of enzymatic hydrolysis on functional physico-chemical properties of arachin[J]. LWT-Food Science and Technology, 2006, 39(1): 54-56.
    [13] Zhao G L, Liu Y, Zhao M M, et al. Enzymatic hydrolysis and their effects on conformational and functional properties of peanut protein isolate[J]. Food Chemistry, 2011, 127(4): 1438-1443.
    [14] Stanojevic S P, Barac M B, Pesic M B, et al. Bioactive proteins and energy value of Okara as a byproduct in hydrothermal processing of soy milk[J]. Journal of Agricultural and Food Chemistry, 2013, 61(38): 9210-9219.
    [15] 叶荣飞,杨晓泉,郑田要,等. 热变性和热聚集对大豆分离蛋白溶解性的影响[J]. 食品科学,2008,29(7):106-108.Ye Rongfei, Yang Xiaoquan, Zheng Tianyao, et al. Effects of thermal denaturation and aggregation on solubility of soy protein isolates[J]. Food Science, 2008, 29(7): 106-108. (in Chinese with English abstract)
    [16] Tedford L A, Kelly S M, Price N C, et al. Combined effects of thermal and pressure processing on food protein structure[J]. Food and Bioproducts Processing, 1998, 76(2): 80-86.
    [17] Tedford L A, Kelly S M, Price N C, et al. Interactive effects of pressure, temperature and time on the molecular structure of beta-lactoglobulin[J]. Journal of Food Science, 1999, 64(3): 396-399.
    [18] Wang H, Wang T, Johnson L A. Mechanism for refunctionalizing heat-denatured soy protein by alkaline hydrothermal cooking[J]. Journal of the American Chemists' Society, 2006, 83(1): 39-45.
    [19] Aouzelleg A, Bull L A, Price N C, et al. Molecular studies of pressure/temperature-induced structural changes in bovine beta-lactoglobulin[J]. Journal of the Science of Food and Agriculture, 2004, 84(5): 398-404.
    [20] Adler-Nissen J. Enzymic Hydrolysis of Food Proteins[M]. London: Applied Science Publishers, 1986.
    [21] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227: 680-685.
    [22] 魏慧敏,张年辉,杜林方. 用蛋白内源荧光考察溶液pH对PSII两种外周蛋白构象的影响[J]. 四川大学学报:自然科学版,2004,41(5):1059-1063.Wei Huimin, Zhang Nianhui, Du Linfang. Effects of pH on conformations of the two extrinsic proteins in photosystem II examined by the influorescence[J]. Journal of Sichuan University: Natural Science Edition, 2004, 41(5): 1059- 1063. (in Chinese with English abstract)
    [23] Cruz-Angeles J, Martinez L M, Videa M. Application of ATR-FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state[J]. Biopolymers, 2015, 103(10): 574-484.
    [24] 卢雁,张玮玮,王公轲. FTIR用于变性蛋白质二级结构的研究进展[J]. 光谱学与光谱分析,2008,28(1):88-93.Lu Yan, Zhang Weiwei, Wang Gongke. Progress in study of secondary structure of denaturized protein by FTIR[J]. Spectroscopy and Spectral Analysis, 2008, 28(1): 88-93. (in Chinese with English abstract)
    [25] Yu J M, Ahmedna M, Goktepe I. Peanut protein concentrate: production and functional properties as affected by processing[J]. Food Chemistry, 2007, 103(1): 121-129.
    [26] 仇超颖,孙为正,崔春,等. 干燥方式对脱酰胺小麦面筋蛋白特性的影响[J]. 华南理工大学学报:自然科学版,2014,42(6):129-135.Qiu Chaoying, Sun Weizheng, Cui Chun, et al. Effects of drying methods on characteristics of deamidated wheat gluten[J]. Journal of South China University of Technology:Natural Science Edition, 2014, 42(6): 129-135. (in Chinese with English abstract)
    [27] Li X J, Liu T H, Song L J. Influence of high-molecular-weight glutenin subunit composition at Glu-A1 and Glu-D1 loci on secondary and micro structures of gluten in wheat (Triticum aestivum L.)
    [J]. Food Chemistry, 2016, 213: 728-734.
    [28] Zhang Q T, Tu Z C, Xiao H, et al. Influence of ultrasonic treatment on the structure and emulsifying properties of peanut protein isolate[J]. Food and Bioproducts Processing, 2014, 92: 30-37.
    [29] Lee S H, Lefèvre T, Subirade M, et al. Changes and roles of secondary structures of whey protein for the formation of protein membrane at soy oil/water interface under high- pressure homogenization[J]. Journal of Agricultural and Food Chemistry, 2007, 55(26), 10924-10931.
    [30] Chen L, Chen J S, Ren J Y, et al. Effects of ultrasound pretreatment on the enzymatic hydrolysis of soy protein isolates and on the emulsifying properties of hydrolysates[J]. Journal of Agricultural and Food Chemistry, 2011, 59(6): 2600-2609.
    [31] O'Loughlin I B, Murray B A, Kelly P M, et al. Enzymatic hydrolysis of heated aggregates of whey protein isolate[J]. Journal of Agricultural and Food Chemistry, 2012, 60(12): 4895-4904.
计量
  • 文章访问数:  2347
  • HTML全文浏览量:  0
  • PDF下载量:  1112
  • 被引次数: 0
出版历程
  • 收稿日期:  2016-07-08
  • 修回日期:  2016-10-29
  • 发布日期:  2016-12-31

目录

    /

    返回文章
    返回