Elsevier

Food Hydrocolloids

Volume 101, April 2020, 105547
Food Hydrocolloids

Effect of pulsed electric field on assembly structure of α-amylase and pectin electrostatic complexes

https://doi.org/10.1016/j.foodhyd.2019.105547Get rights and content

Highlights

  • Electrostatic binding of α-amylase to pectin increased the enzyme sensitivity to PEF.

  • Zeta potential of α-amylase/pectin complexes did not change significantly after PEF treatment.

  • Particle size of α-amylase/pectin complexes rose gradually after PEF treatment.

  • Final structure of complexes after PEF treatment showed the branched, ring, or circles-like shape.

Abstract

Pulsed electric field (PEF) could change the charge distribution of proteins and polysaccharides and affect their interactions and complexes aggregation, but those influences are not enough evaluated. Here, the effects of PEF on the complexes of α-amylase and pectin driven by electrostatic binding were studied. Changes in molecular conformation of α-amylase and assembly structure of α-amylase/pectin complexes were orderly assessed by fluorescence, FTIR, DSC, enzyme activities, particle size, ζ-potential, CLSM, and SEM. After PEF treatment (E~20 kV/cm, texp~1 ms, and 5 cycles), the intrinsic fluorescence of α-amylase was quenched, the content of β-sheet increased, enzyme activities lose almost 80%, and the denatured temperature increased. Ζeta-potential of α-amylase/pectin complexes did not change significantly, but the particle size rose gradually. The particle revolution of α-amylase/pectin complexes was recorded by Turbiscan, and the size growth model fit the Allometric function well. Finally, the complexes of α-amylase and pectin after PEF treatment tended to the branched, ring, or circles-like shape.

Graphical abstract

Assembling behavior of alpha-amylase and pectin complexes after pulsed electric field treatment.

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Introduction

Electrostatic interaction between proteins and polysaccharides commonly results in the formation of complexes in food. Formation of complexes influences the stability, texture, and functional properties of proteins or polysaccharides (Comert & Dubin, 2016), including the encapsulation of bioactive compounds (Yan, Qiu, Wang, & Wu, 2017), forming novelty gels (Le, Rioux, & Turgeon, 2017), etc. Many factors could affect this electrostatic binding, such as pH, ionic strength, mixing ratio, charge density, and charge distribution et al. (Souza & Garcia-Rojas, 2016). However, the influence of pulsed electric field, which is widely applied in food processing, on the electrostatic interaction remains unclear. Electric field is able to change the charge distribution of biomacromolecules (Giteru, Oey, & Ali, 2018). Especially for proteins, they possess the polyampholyte character. If they are applied in the external electric field, the proton migrates from the NH3+ to the COO groups, and results in an additional ion-dipole interaction (Mattison, Dubin, & Brittain, 1998). Usually, when the pH of protein is close to the isoelectric point (pI), the charge-induced attraction is stronger than the polarization-induced attraction (Silva, Da Mikael, Bo, & Torbjörn, 2006). However, polysaccharides belong to polyelectrolytes, which are capable to adjust their charge distribution when they moved to an oppositely charged micelle (Cooper et al., 2006).

Pulsed electric field (PEF), one of the non-thermal processing techniques, applies a high voltage and short time to the material between two electrodes. It has received an increasing attention to keep fresh and to minimize the destruction of food quality. Compared with the traditional thermal processing techniques, PEF has advantages in the lower processing temperature, short processing (or treatment) time (μs-ms), less energy consumption, and no pollution (Yang, Kang, Lyu, & Wang, 2016). So, PEF has been widely applied in food industry for pasteurization (Martín, Qin, Chang, Barbosa-Cánovas, & Swanson, 2010; Pina-Pérez, Rivas, Martínez, & Rodrigo, 2018), inactivation enzymes (Ruijin, Si, & Howard, 2004; Wei, Ruijin, Rongrong, Yali, & Wenbin, 2007), assisting extraction for target compounds (Barbosa-Pereira, Guglielmetti, & Zeppa, 2018; Turk, Baron, & Vorobiev, 2010), and modification of biomacromolecules (Johnson et al., 2010; Qian et al., 2010).

Electrochemical reactions and polarization of structural moieties are two primary mechanisms of PEF working on the enzyme inactivation in food matrix (Giteru et al., 2018). First, external electric field changes the surface charged patch (ionized COO or NH3+) of the enzymes, resulting in structure collapsing of the internal peptide chain (Zhao & Yang, 2012). Second, PEF induces the partial unfolding of enzymes and enhances the inter-/intramolecular noncovalent linkages as hydrogen bond, hydrophobic, and electrostatic interaction (Wei et al., 2007). Lots of factors would affect the efficacy of PEF on enzyme inactivation, such as electric field intensity (Espachs-Barroso, Loey, Hendrickx, & Martín-Belloso, 2006), processing time (Min & Zhang, 2010), pulse frequency, enzyme concentration (Zhao et al., 2012), source of enzyme (Giteru, et al., 2017), etc. Besides, co-existed substances, especially because food system has a complexity in the chemical composition, also has an important influence on the enzyme activity and aggregation behavior under PEF treatment. Ho et al. studied that complexes of protease with casein protected the protease from being inactivated by PEF (Ho, Mittal, & Cross, 1997). Interaction between lysozyme and ovalbumin inhibited the aggregation with an electric field intensity of 25 kV/cm for 800 μs (Wu, Zhao, Yang, & Yan, 2015). A similar phenomenon occurred in the mixture of ovalbumin and bovine serum albumin, resulting from the enhancement of protein-protein interaction and an increase of S–H groups. Thus, the presence of other components in matrix could regulate the aggregation behavior and activity of proteins or enzymes (Zhao et al., 2012).

Effects of PEF on single protein or polysaccharide solution has been investigated widely, but the studies of PEF on the protein/polysaccharide complexes are insufficient (Giteru et al., 2018). Gao et al. used capillary electrophoresis to study the effect of external electric field on the electrostatic interaction between proteins and polyelectrolytes (Gao, Dubin, & Muhoberac, 1997, 1998). Proteins and polysaccharides complexes formed by electrostatic interaction that is a short-range force (Jones & Mcclements, 2011), which might be affected by high voltage of electric field. Alpha-amylase (E.C. 3.2.1.1) is commonly used for hydrolysis of internal α-1, 4-glycosidic bonds in starch. Non-starch polysaccharides in food matrix could interact with α-amylase and change its enzyme activity. Here, the effect of PEF on the structure revolution of α-amylase/pectin electrostatic complexes was studied. Changes in the molecular conformation were detected by fluorescence quenching, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and enzyme activity. Aggregation behaviors after PEF treatment were investigated by measuring the particle size, ζ-potential, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). Results might provide the parameters for PEF inactivation of enzyme when it is utilized in the complicated food matrix.

Section snippets

Chemicals

Alpha-amylase was fermented by Bacilus licheniformis with the initial enzyme activity of ~105 U/mL. Before usage, α-amylase was dialyzed with Milli-Q water until the chloride ions did not detect by silver nitrate. Pectin, molecular weight is 4.63 × 105 g mol−1 and the degree of esterification is around 51.7%, was provided by Aladdin company (Shanghai, China). It was purified by the Amberlite IR-120 resin according to the former study (Renard, Thibault, Voragen, Broek, & Pilnik, 1993). Other

Optimized PEF parameters

First, to study the effect of electric field intensity on α-amylase, the pulse frequency, flow rate, and exposure time were set as 1 kHz, 50 mL/min, and 1.02 ms, respectively. Intrinsic fluorescence spectroscopy is based on monitoring changes of tryptophan, tyrosine, or phenylalanine for studying the structure, dynamics, and interactions of proteins in solution. Eleven residues might be involved in the structure and function of α-amylase, including 56 tyrosine and 263 tryptophan residues (M.

Conclusion

In the current work, the influence of pulsed electric field (PEF) on the structure and assemblies of α-amylase/pectin electrostatic complexes was investigated. Optimized parameters of PEF treatment were listed as followings: electric field intensity of 20 kV/cm, texp of 1.02 ms, and processing cycles of 5. It was found that PEF could inactive enzyme activity by changing its structure and assembly behaviors, which was attributed to induce the aggregation (an increase of size) and change the

Author Contribution

Weiping Jin: Conceptualization, Methodology, Writing original draft, Funding acquisition

Zhifeng Wang: Data curation, Formal analysis

Dengfeng Peng: Software, Writing review & editing

Wangyang Shen: Resources

Zhenzhou Zhu: Methodology

Shuiyuan Cheng: Project administration

Bin Li: Supervision

Qingrong Huang: Visualization, Writing review & editing.

Declaration of competing interest

● The work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part.

● We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work.

● No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. All the authors listed have approved the manuscript that is

Acknowledgment

This work financially supported by the National Natural Science Foundation of China (Grant No. 31801587) and the Natural Science Foundation of Hubei Province (Grant No. 2018CFB277).

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