Study on the Influence of Carrageenan on the Fluid Stability and Emulsifying Ability of Rabbit Myofibril Protein Near the Isoelectric Point

SU Chang; LAN Zifan; HE Zhifei; LI Hongjun

doi:10.13718/j.cnki.xdzk.2024.07.002

2024 Volume 46 Issue 7

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SU Chang, LAN Zifan, HE Zhifei, et al. Study on the Influence of Carrageenan on the Fluid Stability and Emulsifying Ability of Rabbit Myofibril Protein Near the Isoelectric Point[J]. Journal of Southwest University Natural Science Edition, 2024, 46(7): 11-19. doi: 10.13718/j.cnki.xdzk.2024.07.002

Citation:

SU Chang, LAN Zifan, HE Zhifei, et al. Study on the Influence of Carrageenan on the Fluid Stability and Emulsifying Ability of Rabbit Myofibril Protein Near the Isoelectric Point[J]. Journal of Southwest University Natural Science Edition, 2024, 46(7): 11-19. doi: 10.13718/j.cnki.xdzk.2024.07.002

Study on the Influence of Carrageenan on the Fluid Stability and Emulsifying Ability of Rabbit Myofibril Protein Near the Isoelectric Point

1.
College of Food Science, Southwest University, Chongqing 400715, China
2.
Chongqing Key Laboratory of Special Food Co-built by Sichuan and Chongqing, Chongqing 400715, China

More Information

Corresponding author: LI Hongjun
Received Date: 25/06/2023
Available Online: 20/07/2024
MSC: S965

Abstract

Meat protein is a kind of protein with high nutritional value. However, its poor dispersibility under low ionic strength medium and slightly acidic (pH 4.0~7.0) conditions hinders its further development and utilization. In this study, high intensity ultrasound (25 kHz, 600 W for 15 min) was firstly used to improve the dispersion ability of myofibrillar protein, the main protein of meat protein, under low ionic strength and neutral conditions (pH 7.0). By complexing with Carrageenan, a kind of hydrophilic polysaccharide, the dispersibility and emulsifying ability of the protein were further improved under slightly acidic conditions. The results showed that ultrasound treatment could significantly improve the solubility of the protein (from 23.51% to 77.32%, p < 0.05). Under slightly acidic conditions (pH 5.0~6.0), because it is near the isoelectric point of the protein, the charge and solubility of the protein decreased significantly. The addition of Carrageenan complexed with protein can significantly improve the functional properties of protein under slightly acidic conditions. With the increase of Carrageenan concentration, the protein dispersion and emulsifying ability increased significantly. Low concentration of Carrageenan (the ratio of protein to Carrageenan is 8∶1) will form a complex with protein with larger molecular weight, resulting in a decline in emulsifying capacity. The addition of Carrageenan can significantly increase the surface charge of the protein near the isoelectric point, destroy the internal hydrogen bond of the molecule, promote the unwinding effect of the protein molecule, thus exposing the internal hydrophobic groups, improving the amphiphilic properties of the protein, so improving the functional properties of the protein near the isoelectric point.
- myofibril protein,
- Carrageenan,
- isoelectric point,
- dispersibility,
- emulsifying capacity

References

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肉类蛋白是一种高营养价值蛋白，与植物蛋白相比，肉类蛋白具有丰富的必需氨基酸和更高的消化率^[1]，然而这种高价值蛋白并没有像植物蛋白、牛奶蛋白那样得到充分的利用和开发，部分原因是肉类蛋白中的主要蛋白——肌原纤维蛋白在低离子介质或等电点附近具有较差的功能特性(分散、乳化、凝胶)^[2-3]. 在低离子介质中肌原纤维蛋白中的最主要蛋白——肌球蛋白容易通过尾部静电吸引缠绕自组装形成螺旋结构，导致蛋白聚集、沉降，致使蛋白功能特性下降^[4]. 在等电点附近，蛋白分子间由于电荷屏蔽效应也会引起肌球蛋白自组装及聚集^[5]，因此，调节分子间的电荷作用能一定程度改善肌原纤维蛋白在低离子介质或等电点附近的功能特性.

卡拉胶是一种硫酸化阴离子多糖，这种亲水胶体分子侧链带有大量负电荷，当它与蛋白分子形成络合物后，多糖提供的电荷能改善蛋白的功能特性^[6-7]. 此外，多糖分子自身的空间位阻和黏性也能在一定程度上阻碍蛋白的聚集，改善蛋白的功能，例如蛋白的溶解能力^[8]等. 卡拉胶也常被作为一种稳定剂用来控制乳品流体的质地特性^[9]，广泛用于控制乳清蛋白^[10]、牛奶蛋白^[11]、大豆分离蛋白^[12]乳液流体品质，然而卡拉胶如何影响肉类蛋白的乳化能力还鲜有报道. 此外，流体食品的开发、设计过程中往往要考虑消费者的适口性，而微酸的蛋白饮料(pH值为4.0~6.0)具有较好的口感和风味^[13]. 对于肌原纤维蛋白来说，其等电点在pH值为5.5附近，此时蛋白因电荷的减少而导致功能特性受到负面影响，因此，本研究以促进肌原纤维蛋白在低离子介质和等电点附近分散能力为目的，通过超声改性以及多糖混合，进一步提高蛋白在微酸条件下的分散和乳化能力，为肉类蛋白的进一步开发流态食品提供理论依据.

1. 材料与方法

1.1. 材料和试剂

实验所用兔肉样本来自重庆阿兴记兔场，均为雄性伊拉兔(2.3~2.5 kg，75日龄). 按照商业屠杀标准，去皮和内脏后将胴体放入6 ℃冰箱，1 h内运往实验室，并贮藏于-20 ℃冰箱中冷冻保存. 卡拉胶(纯度99%)，法国Louis Francois公司；磷酸氢二钠、磷酸二氢钠、乙二胺四酸钠、酒石酸钾钠、五水硫酸铜、氢氧化钠、盐酸、氯化钠、氯化镁、十二烷基硫酸钠均为分析纯，重庆跃翔科技有限公司；大豆油，益海嘉里金龙鱼粮油食品股份有限公司.

1.2. 仪器和设备

绞肉机(MC-30X1-101A1)，美的集团；匀浆均质机、冷冻干燥机，宁波新芝生物科技股份有限公司；pH计，上海仪电科学仪器股份有限公司；台式高速离心机(5810型)，德国Eppendorf公司；冷冻离心机，美国贝克曼库尔特公司；光学显微镜，日本奥林巴斯株式会社；紫外-可见分光光度计，日本岛津仪器有限公司；Zeta电位仪，英国马尔文公司；磁力搅拌器，德国艾卡；超声波细胞破碎仪，南京舜玛仪器设备有限公司.

1.3. 实验方法

1.3.1. 兔肉肌原纤维蛋白的提取

将兔后腿肉在4 ℃下解冻12 h，并将解冻的兔肉用绞肉机搅成肉泥状. 将25 g肉泥与100 mL的磷酸盐(PBS)缓冲液混合(20 mmol/L中含100 mmol/L NaCl，2 mmol/L MgCl₂，1 mmol/L EDTA-2Na)；然后在8 000 r/min的条件下均质1 min，间歇1 min，再均质1 min；匀浆后，将肉糜在5 000 r/min，4 ℃条件下离心15 min，并移除上清液；随后将100 mL PBS缓冲液再次加入肉糜沉淀重复均质、离心操作2次；去除上清液后，100 mL PBS缓冲液(20 mmol/L，pH值为6.25，100 mmol/L NaCl)加入并按照相同的参数均质、离心，最后去除上清液，向沉淀中加入100 mL超纯水(Milli-Q). 混合物按上述条件进行2次均质、离心、去除上清液操作，再用4层纱布过滤. 将样品溶解于超纯水中，并调节其浓度为10 mg/mL.

1.3.2. 超声处理中性pH条件下肌原纤维蛋白分散液

将超声探头浸入蛋白样品溶液距底部1.5 cm处，使用外加冷却装置使分散液温度保持在(4±0.5) ℃. 超声波细胞破碎仪(SM-1000A)采用间歇工作状态(2 s打开，3 s关闭)，超声频率为25 kHz，功率为600 W处理15 min，处理结束后蛋白样品作为对照. 将对照蛋白溶液分别用0.6 mol/L盐酸调节pH值至6.5，6.0，5.5，5.0，4.0，在测定蛋白溶解度之前将样品放置4 ℃冰箱保存. 独立处理3批样品.

1.3.3. 蛋白溶解度测定

将蛋白分散液浓度稀释至4 mg/mL，然后在10 000 r/min的条件下离心10 min，取蛋白上清液并用双缩脲试剂测定其浓度. 蛋白溶解度(S)按公式计算：

式中：P_上清为上清液蛋白浓度；P_总为总蛋白浓度.

1.3.4. 卡拉胶-肌原纤维蛋白络合物构建

卡拉胶-肌原纤维蛋白络合物构建参照Chen等^[3]的研究稍作修改，蛋白浓度为10 mg/mL，卡拉胶与蛋白按照3∶7的体积比混合，使得最终混合体系中蛋白与卡拉胶的质量比分别为8∶1，4∶1，2∶1. 用磁力搅拌器将蛋白与卡拉胶混合均匀，同时用0.6 mol/L盐酸溶液调节混合液pH至蛋白等电点附近(5.0，5.5，6.0)，该过程独立处理3批样品.

1.3.5. 等电点附近蛋白Zeta电位分析

将蛋白分散液分别用对应pH值的盐酸-氢氧化钠溶液稀释至0.5 mg/mL，将稀释液进行Zeta电位分析. 测试过程采用SOD模式，平衡时间为120 s，测试温度为25 ℃.

1.3.6. 等电点附近蛋白乳化能力测试

蛋白乳化能力和稳定性的测定与计算参照Agyare等^[14]的研究，将20 mL蛋白溶液和5 mL大豆油加入50 mL塑料离心管中，并在10 000 r/min的条件下均质1 min，然后取10 mL溶液加入平底顶空瓶中并开始计时，在0 min和10 min时从瓶底(约0.5 cm处)取出50 μL的样品分散于5 mL 0.1% SDS溶液中.

1.3.7. 乳液形态观察

利用光学显微镜观测蛋白乳液的形态，分别取不同处理组50 μL乳液样品转移到载玻片上，然后用盖玻片覆盖，在室温下用400倍放大镜进行观测.

1.3.8. 红外测定

将不同处理组的蛋白冷冻干燥，并研磨成细粉末用于红外光谱分析，光谱范围为400~4 000 cm^-1，使用Origin 8.1软件确定区间峰的位置.

1.3.9. 数据处理

实验独立处理3批样品，每次重复测量3次. 数据采用SPSS 22软件进行处理，采用方差分析(ANOVA) 对结果的差异显著性进行评价，p＜0.05为差异有统计学意义，图形用Origin 8.1进行绘制.

3. 结论

超声处理能够显著改善兔肉肌原纤维蛋白在中性pH、低离子介质中的分散能力，在等电点附近由于表面电荷减少，分散能力显著下降. 卡拉胶与蛋白络合后会提高蛋白的表面电荷，破坏分子氢键，促进蛋白分子发生解螺旋效应，从而暴露内部的疏水基团，提高蛋白的两亲性，因此显著改善了蛋白在等电点附近的分散及乳化能力. 这种变化受到卡拉胶添加量的影响，在等电点附近(pH值为5.5)，较低的卡拉胶浓度(蛋白与卡拉胶比例为8∶1)会与蛋白形成大分子量的络合物，降低了蛋白的乳化能力；随着卡拉胶浓度的升高，蛋白在等电点附近的乳化能力显著改善. 该研究为兔肉肌原纤维蛋白流态食品的设计和开发提供了一定的理论基础.

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Study on the Influence of Carrageenan on the Fluid Stability and Emulsifying Ability of Rabbit Myofibril Protein Near the Isoelectric Point