体外模拟消化过程中大豆分离蛋白拉曼光谱和荧光光谱分析
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摘要
采用胃蛋白酶在37℃和pH 1.2条件下对大豆分离蛋白(SPI)进行体外模拟消化,对体外模拟消化过程中SPI进行拉曼光谱和荧光光谱分析。随着消化时间的延长,蛋白质的消化程度逐渐增大,当消化反应超过2 h后,蛋白质的消化速率减慢。消化反应使SPI的α-螺旋含量升高,β-折叠含量降低,β-转角含量先增加后降低,而无规卷曲含量变化不规律。经长时间的消化反应使无规卷曲含量有所增加。在整个消化过程中,酪氨酸和色氨酸残基趋向于"暴露式",这与消化过程中蛋白质变性以及分子结构展开有关。体外模拟消化反应使SPI的荧光强度降低,最大吸收波长(λmax)增加,即λmax发生红移,这表明色氨酸残基的暴露程度增大,同时使SPI三级结构变的松散。此外,随着消化时间的延长,SPI消化产物λmax的红移程度增加。
Raman spectroscopy and fluorescence spectroscopy of SPI during vitro simulated digestion process were analyzed by using pepsin in vitro digestion of soybean protein isolate(SPI) at 37 ℃ and pH of 1.2. The digestion degree of protein increased gradually with the extension of digestion time. However, the digestion rate of protein slowed down when digestion reaction time was more than 2 h. The digestion reaction could increase α-helix content and decrease β-sheet content, while β-turn content increased at first and then decreased. The changes of random coil content were irregular, however, random coil content increased slightly after digestion reaction for long time. The tyrosine and tryptophan residues tended to be ‘exposed' in the entire process of digestion, which was related to protein denaturation and unfolding of molecular structure. The digestion reaction was able to decrease fluorescence intensity of SPI and increase maximum absorption wavelength(λmax). The bathochromic-shift of λmaxindicated that the degree of exposure for tryptophan residues increased and the tertiary structure of SPI became looser. In addition, the degree of bathochromic-shift of λmax for SPI digestion products increased with the extension of digestion time.
Raman spectroscopy and fluorescence spectroscopy of SPI during vitro simulated digestion process were analyzed by using pepsin in vitro digestion of soybean protein isolate(SPI) at 37 ℃ and pH of 1.2. The digestion degree of protein increased gradually with the extension of digestion time. However, the digestion rate of protein slowed down when digestion reaction time was more than 2 h. The digestion reaction could increase α-helix content and decrease β-sheet content, while β-turn content increased at first and then decreased. The changes of random coil content were irregular, however, random coil content increased slightly after digestion reaction for long time. The tyrosine and tryptophan residues tended to be ‘exposed' in the entire process of digestion, which was related to protein denaturation and unfolding of molecular structure. The digestion reaction was able to decrease fluorescence intensity of SPI and increase maximum absorption wavelength(λmax). The bathochromic-shift of λmaxindicated that the degree of exposure for tryptophan residues increased and the tertiary structure of SPI became looser. In addition, the degree of bathochromic-shift of λmax for SPI digestion products increased with the extension of digestion time.
引文
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[2] LIU C, WANG X S, MA H, et al. Functional properties of protein isolates from soybeans stored under various conditions[J]. Food Chemistry, 2008,111(1):29-37.
[3] MOURE A, SINEIRO J, DOMINGUES H, et al.Functionality of oilseed protein products:a review[J].Food Research International, 2006, 39(9):945-963.
[4]张艳芳,董平,梁兴国.食物成分消化模型的研究进展[J].食品工业科技, 2013, 34(18):356-361.
[5] RAGAB D D M, BABIKER E E, ELTINAY A H,et al. Fractionation, solubility and functional properties of cowpea(Vigna unguiculata)proteins as affected by pH and/or salt concent ration[J]. Food Chemistry, 2004, 84(2):207-212.
[6] LO W M Y, LI-CHAN E C Y. Angiotensin I converting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein[J]. Journal of Agricultural and Food Chemistry, 2005, 53(9):3369-3376.
[7] ABDEL-AAL E S M. Effects of baking on protein digestibility of organic spelt products determined by two in vitro digestion methods[J]. LWT-Food Science and Technology, 2008, 41(7):1282-1288.
[8] WOOD B R, MCNAUGHTON D. Raman excitation wavelength investigation of single red blood cells in vivo[J]. Journal of Raman Spectroscopy, 2002, 33(7):517-523.
[9] PETRUCCELLI S, A譙魷N M. Relationship between the method of obtention and the structural and functional properties of soy proteins isolates. 1.Structural and hydration properties[J]. Journal of Agricultural and Food Chemistry, 1994, 42(10):2161-2169.
[10] ZHANG S, VARDHANABHUTI B. Effect of initial protein concentration and pH on in vitro gastric digestion of heated whey proteins[J]. Food Chemistry,2014, 145(145C):473-480.
[11] ADLER-NISSEN J. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid[J]. Journal of Agricultural and Food Chemistry, 1979, 27(6):1256-1262.
[12]张萍,郑大威,刘晶,等.基于表面增强拉曼光谱技术的豆芽6-BA残留快速检测方法[J].光谱学与光谱分析, 2012, 32(5):1266-1269.
[13]王中江,江连洲,魏冬旭,等. pH值对大豆分离蛋白构象及表面疏水性的影响[J].食品科学, 2012,33(11):47-51.
[14]江连洲,王瑞,李秋慧,等.体外模拟消化过程中大豆蛋白的亚基组成及分子质量分布[J].中国食品学报, 2015, 15(10):65-72.
[15] OVERMAN S A, THOMAS G J. Raman spectroscopy of the filamentous virus Ff(fd, fl, M13):structural interpretation for coat protein aromatics[J].Biochemistry, 1995, 34(16):5400-5451.
[16] HERRERO A. Raman spectroscopy for monitoring protein structure in muscle food systems[J]. Critical Reviews in Food Science and Nutrition, 2008, 48(6):512-523.
[17]王中江,江连洲.大豆分离蛋白在不同pH下的拉曼光谱分析[J].食品工业科技, 2012, 33(11):63-66.
[18] HERRERO A M, JIMENEZ-COLMENERO F,CARMONA P. Elucidation of structural changes in soy protein isolate upon heating by Raman spectroscopy[J]. International Journal of Food Science&Technology, 2009, 44(4):711-717.
[19] ZHAO J, XIONG Y L, MCNEAR D H. Changes in structural characteristics of antioxidative soy protein hydrolysates resulting from scavenging of hydroxyl radicals[J]. Journal of Food Science, 2013, 78(2):152-159.
[20] WONG H W, CHOI S M, PHILLIPS D L, et al.Raman spectroscopic study of deamidated food proteins[J]. Food Chemistry, 2009, 113(2):363-370.
[21] LI-CHAN E C Y. The applications of Raman spectroscopy in food science[J]. Trends in Food Science and Technology, 1996, 7(11):361-370.
[22] PALLAR魬S I, VENDRELL J, AVIL魬S F X, et al.Amyloid fibril formation by a partially structured intermediate state ofα-chymotrypsin[J]. Journal of Molecular Biology, 2004, 342(1):321-331.
[23] NATARAJAN S, XU C P, BAE H, et al. Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes[J]. Plant Physiology and Biochemistry, 2007, 45(6/7):436-444.
[24] VIVIAN J T, CALLIS P R. Mechanisms of tryptophan fluorescence shifts in proteins[J]. Biophysical Journal, 2001, 80(5):2093-2109.
[25]源博恩.亚基解离与重聚集对大豆蛋白结构和功能特性的影响[D].广州:华南理工大学, 2012.