Effect of enzymolysis alginate oligosaccharide on myofibrillar protein in simulated oral digestion
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摘要: 将鲢 (Hypophthalmichthys molitrix) 加工成肉糜制品能增加消费者的接受度从而提高其经济价值，酶解褐藻寡糖 (Enzymolysis alginate oligosaccharide, EAO) 能与鲢肌原纤维蛋白 (Myofibrillar protein, MP) 快速反应，提高食品的功能特性。食物经口腔加工后会发生明显的物理、化学变化。为了解肌原纤维蛋白在鲢消化过程中的结构变化，明确口腔消化对褐藻寡糖修饰后的肌原纤维蛋白的影响，从新鲜鲢中提取了肌原纤维蛋白，并向其中添加0.45 mg·mL−1的酶解褐藻寡糖获得寡糖-蛋白复合物 (EAO-MP)，考察了鲢肌原纤维蛋白 (M组) 与寡糖-蛋白复合物 (A组) 在模拟口腔消化中的傅里叶红外光谱、内源性荧光光谱、紫外吸收光谱、巯基含量、氢键含量、表面疏水性的变化差异。结果显示：经过模拟口腔消化后，M组无规卷曲结构、总巯基含量增加，表面疏水性显著降低 (P<0.05)，说明肌原纤维蛋白由于模拟口腔消化液的作用，二级、三级结构发生了改变；添加酶解褐藻寡糖后的A组无规卷曲结构下降了2.97%，A组在3个不同消化时间点 (0、5、15 s) α-螺旋结构相比M组分别增加了7.29%、2.73%、5.55%；氢键含量显著增加 (P<0.05)，说明肌原纤维蛋白与酶解褐藻寡糖通过氢键作用结合为共价聚合物；巯基含量显著降低，表面疏水性呈升高趋势，说明酶解褐藻寡糖的加入促进肌原纤维蛋白在模拟口腔消化液中展开蛋白结构。综上，酶解褐藻寡糖的添加能促使鲢肌原纤维蛋白分子结构在模拟口腔消化液中展开，推测这可能有利于蛋白后续的消化吸收。Abstract: Processing silver carp (Hypophthalmichthys molitrix) into surimi products can improve consumers' acceptance and its economic value. Enzymolysis alginate oligosaccharide (EAO) can quickly react with myofibrillar protein (MP) extracted from silver carp to improve functional characteristics. Food will undergo physical and chemical changes after oral processing. To better understand the effects of oral digestion on the EAO-modified MP, we extracted the MP from fresh silver carp which was added 0.45 mg·mL−1 EAO to create a complex (EAO-MP). Then we investigated the variations of Fourier infrared spectroscopy, endogenous fluorescence spectroscopy, UV absorption spectroscopy, contents of sulfhydryl and active sulfhydryl, surface hydrophobicity, and hydrogen bonds in the simulated oral digestion of silver carp MP (Group M) and EAO-MP (Group A). The results show that, after the simulated oral digestion, the random coil structure and total sulfhydryl content increased, while the surface hydrophobicity decreased significantly in Group M (P<0.05), which demonstrates that the secondary and tertiary structures of MP had changed due to the effect of simulated oral digestive fluid. Besides, the random coil in Group A decreased by 2.97% after adding EAO. At three different digestion times, the α-helix increased by 7.29%, 2.73% and 5.55%, respectively, compared with Group M. Additionally, the hydrogen bond content increased significantly in Group A (P<0.05), revealing that MP and EAO were bonded to a covalent polymer by the hydrogen bond force. The significant increase in surface hydrophobicity and decrease in sulfhydryl content show that the addition of EAO promotes the expansion of the protein structure of MP in simulated oral digestive fluid. In conclusion, the addition of EAO can promote the molecular structure of silver carp MP to expand in the simulated oral digestive fluid, which may be conducive to the subsequent digestion and absorption of protein.
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图 1 肌原纤维蛋白和寡糖-蛋白复合物口腔消化产物傅里叶红外光谱 (a) 和二级结构含量 (b)
注：下标1是α-螺旋显著性，下标2是β-折叠显著性，下标3是β-转角显著性，下标4是无规卷曲显著性。
Figure 1. Fourier infrared spectroscopy (a) and secondary structure content (b) of MP and EAO-MP oral digestive products
Note: Endnote subscript 1 is α-helix significance; endnote subscript 2 is β-helix significance; endnote subscript 3 is β-turn significance; endnote subscript 4 is random coil significance.
图 2 肌原纤维蛋白和寡糖-蛋白复合物口腔消化产物内源荧光光谱 (a) 和最大荧光强度 (b)
注：不同大写字母表示在同一模拟口腔消化时间中，不同消化产物之间存在显著性差异 (P<0.05)；不同小写字母表示在同组消化产物中，不同模拟口腔消化时间之间存在显著性差异 (P<0.05)；表2、图4—图6同此。
Figure 2. Endogenous fluorescence spectra (a) and maximum fluorescence intensity (b) of MP and EAO-MP oral digestive products
Note:Values with different capital letters indicate significant differences among different digestive products at the same simulated oral digestion time (P<0.05); values with different lowercase letters indicate significant differences in the same group of digestion products at different simulated oral digestion time (P<0.05). The same case in Table 2 and Fig. 4−Fig. 6.
图 3 肌原纤维蛋白和寡糖-蛋白复合物口腔消化产物紫外吸收光谱 (a) 和紫外二阶导数光谱 (b)
Figure 3. UV absorption spectra (a) and second order derivative spectra (b) of MP and EAO-MP oral digestive products
图 4 肌原纤维蛋白和寡糖-蛋白复合物口腔消化产物总巯基 (a) 和活性巯基质量摩尔浓度 (b)
Figure 4. Molality of total sulfhydryl (a) and active sulfhydryl (b) of MP and EAO-MP oral digestive products
图 5 肌原纤维蛋白和寡糖-蛋白复合物口腔消化产物氢键质量浓度
Figure 5. Hydrogen bond mass comcentration of MP and EAO-MP oral digestive products
图 6 肌原纤维蛋白和寡糖-蛋白复合物口腔消化产物蛋白表面疏水性
Figure 6. Surface hydrophobicity of MP and EAO-MP oral digestive products
表 1 口腔消化组别
Table 1. Oral digestion group
组别 Group 名称 Name 未消化鲢肌原纤维蛋白Undigested silver carp myofibrillar protein M0 鲢肌原纤维蛋白模拟口腔消化5 sSilver carp myofibrillar protein after simulated oral digestion for 5 s M5 鲢肌原纤维蛋白模拟口腔消化15 sSilver carp myofibrillar protein after simulated oral digestion for 15 s M15 未消化鲢肌原纤维蛋白-寡糖复合物Undigested silver carp myofibrillar protein-alginate oligosaccharide complex A0 鲢肌原纤维蛋白-寡糖复合物模拟口腔消化5 sSilver carp myofibrillar protein-alginate oligosaccharide complex after simulated oral digestion for 5 s A5 鲢肌原纤维蛋白-寡糖复合物模拟口腔消化15 sSilver carp myofibrillar protein-alginate oligosaccharide complex after simulated oral digestion for 15 s A15 
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表 2 紫外二阶导数生成 r值
Table 2. UV second order derivative generated r value
模拟口腔消化时间Simulated oral digestion time/s 鲢肌原纤维
蛋白组 (M组) MP group寡糖-蛋白
复合物组 (A组)EAO-MP group0 1.11±0.18Aa 1.62±0.88Aa 5 0.98±0.27Aa 1.12±0.16Aa 15 0.97±0.16Aa 1.55±0.31Aa
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