Elsevier

Process Biochemistry

Volume 62, November 2017, Pages 223-230
Process Biochemistry

Characterization and antibacterial activity of the nanocomposite of half-fin anchovy (Setipinna taty) hydrolysates/zinc oxide nanoparticles

https://doi.org/10.1016/j.procbio.2017.07.002Get rights and content

Highlights

  • An antibacterial nanocomposite was prepared using a hydrothermal method.

  • The ZnO NPs were functionalized with half-fin anchovy hydrolysates (HAHp).

  • The nanocomposite of HAHp(3.0)/ZnO NPs caused disorganization in the cytomembrane.

  • HAHp(3.0)/ZnO NPs induced the formation of ROS in intracellular cells.

Abstract

Half-fin anchovy (Setipinna taty) hydrolysates (HAHp) was conjugated with zinc oxide nanoparticles (ZnO NPs) using a hydrothermal method to develop a novel antibacterial nanocomposite. The generated supernatants of the conjugate, designated as HAHp(3.0)/ZnO NPs, were characterized by transmission electron microscopy, high resolution transmission electron microscopy, and inductively coupled plasma-optical emission spectrometer. Results showed that HAHp(3.0) was absorbed on the surface of the ZnO NPs. The total content of zinc element was 9127.4 mg/kg in HAHp(3.0)/ZnO NPs. The increased antibacterial effects were observed for the HAHp(3.0)/ZnO NPs with the minimal inhibitory concentration of 3.5 μgprotein/mL against Escherichia coli (E. coli), Pseudomonas fluorescens, Salmonella and Staphylococcus aureus, compared to the bare HAHp(3.0). The antibacterial activity of HAHp(3.0)/ZnO NPs was further evaluated using E. coli as the model strain. The incubation of HAHp(3.0)/ZnO NPs increased the outer and inner membrane permeability in E. coli cells, and the leakages of potassium ions and the cytoplasmic β-galactosidase were detected during the process. Furthermore, porous structures were observed on the membrane of E. coli cells by scanning electron microscopy. In addition, the formation of intracellular reactive oxygen species was detected using fluorescence microscopy. The results suggested that the HAHp(3.0)/ZnO NPs could be a promising antibacterial nanocomposite.

Introduction

Zinc oxide (ZnO) is a kind of wide band gap (3.37 eV) semiconductor with large exciton binding energy (60 meV) at room temperature [1]. ZnO is generally regarded as a safe (GRAS) material by the United States Food and Drug Administration (USFDA, 21CFR182.8991) [2]. The worldwide production of ZnO is estimated at 0.1–1.2 million tons per year [3], [4]. Due to physical and chemical stabilities, high biocompatibility, lower cost, and nontoxicity, ZnO and their nanoparticles (ZnO NPs) have unique advantages over other nano metal oxides, and thereby are extensively used in daily life products such as cosmetics, medical devices, electronic sensor, lubricants, etc. [5].

Nano structured materials have been used in food industry to inhibit the growth of bacteria present on the surface of food products [6]. The most commonly used polymers are polysaccharides and proteins in biocomposite [7]. The properties of polymer, such as thermal, mechanical, and gas barrier, can be enhanced by addition of nanoparticles [8], [9], [10], [11]. The antimicrobial property of ZnO NPs would make them a viable approach to prevent infectious diseases [12]. The large specific surface area and high surface energy of ZnO NPs will contribute to their excellent interfacial interactions on polymer branches [13]. To date, ZnO NPs have been explored as functional nanofiller incorporated into packaging and food contact materials to inhibit the target bacteria [14], [15], [16]. For example, the polymer incorporation of ZnO NPs as active packaging has been reported to extend the shelf life or to enhance quality for fresh orange juice [17], fresh-cut ‘Fuji’ apple [18], convenient vegetable dishes [19], postharvest strawberry [20], soft white cheese [21], [22], and ready-to-eat poultry meat [23].

Recently, researchers have been focusing on the conjugates of nanoparticles with antimicrobial peptides (AMPs) as a promising approach to achieve superior antimicrobial activity. Mei et al. found that the silver nanoparticles (AgNPs) functionalized with both bacitracin A and polymyxin E (AgNPs-BA&PE) easily attached and penetrated into the bacterial cell membrane, resulting in up to 10-fold increase in the antibacterial activity without the emergence of bacterial resistance [24]. In a latest study by Pal et al., who confirmed the activity and stability of cysteine containing AMPs were enhanced after conjugation with AgNPs [25]. The conjugate of gold nanoparticles (AuNPs) and peptide PEP (H-Cys-Ala-Cys-Trp-Gln-Val-Ser-Arg-Arg-Arg-Arg-Gly-OH) integrated the advantages of cationic AuNPs and AMPs as unique non-viral vectors for gene delivery to mesenchymal stem cells [26]. The functionalized ZnO NPs with glucose oxidase for specific biosensors [27], and with β-galactosidase or α-amylase for enhanced heat-resistance and stability [28], [29] have been reported compared to the corresponding free enzymes. However, to the best of our knowledge, there are few studies about antibacterial protein hydrolysates or peptides incorporated with ZnO NPs.

Half-fin anchovy (Setipinna taty) hydrolysates (HAHp) derived from the digestion of half-fin anchovy are composed of peptides and amino acids [30]. HAHp demonstrated antibacterial, antioxidant, and antiproliferative activities [31], [32]. In this study, HAHp were conjugated with ZnO NPs via a simple and green hydrothermal method to fabricate a novel nanocomposite. The property of the nanocomposite was evaluated. Furthermore, the antibacterial effects of the nanocomposite were determined using Escherichia coli as the indicative strain. All the results will provide references for the adsorption of peptide and/or amino acids onto ZnO NPs, and further deplore a novel antibacterial agent derived from HAHp used in foods.

Section snippets

Materials

Half-fin anchovy (Setipinna taty), were obtained from Fengmao aquatic market in Zhoushan City, China. The bacteria strains of Escherichia coli (E. coli) (CGMCC 1.1100), Pseudomonas fluorescens (P. fluorescens) (CICC 20225), Staphylococcus aureus (S. aureus) (CMCC 26003), Salmonella (CICC 10982), and Bacillus cereus (B. cereus) (CICC 10809) were storage strains in College of Food and Pharmacy, Zhejiang Ocean University. Erythromycin was purchased from Aladdin in China. O-Nitro-phenyl-β-d

Micromorphology and total Zinc content of HAHp(3.0)/ZnO NPs

The ZnO crystals used in this study were synthesized by a low temperature wet chemical method. The TEM image of Fig. 1A revealed that spherical and rhomboid shapes were observed in ZnO crystals. The particle sizes were 10–40 nm. The pattern of these crystals displayed well-defined diffraction peaks at 2θ of 31.83°, 34.49°, 36.32°, 47.59°, 56.52°, 62.87°, 66.39°, 68.00°, 69.10°, 72.56°, and 76.95°, as observed in Fig. 1B, which are indicated as (100), (002), (101), (102), (110), (103), (200),

Conclusions

The nanocomposite of HAHp(3.0)/ZnO NPs was obtained by a simple, low cost and green method. Results showed that the HAHp(3.0)/ZnO NPS demonstrated stronger inhibition effects on E. coli, P. fluorescens, Salmonella and S. aureus compared with the bare HAHp(3.0), according to their MIC values. Mechanistic investigation for the antibacterial effect revealed that the HAHp(3.0)/ZnO NPs caused disorganization in the bacterial cytomembrane and led to leakage of cytoplasmic contents, hence exhibited

Acknowledgments

This work was supported by grants from the Zhejiang Natural Science Project (LY15C200018), the Public Welfare Project of Zhejiang Province (2016C32066), the Zhoushan Science and Technology Bureau Project (2014C41005), and the National Natural Science Foundation of China (31301568). We also thank Prof. Zuisu Yang for assisting the operation of fluorescence microscopy.

References (54)

  • I. Pal et al.

    Enhanced stability and activity of an antimicrobial peptide in conjugation with silver nanoparticle

    J. Colloid Interface Sci.

    (2016)
  • L.H. Peng et al.

    Integration of antimicrobial peptides with gold nanoparticles as unique non-viral vectors for gene delivery to mesenchymal stem cells with antibacterial activity

    Biomaterials

    (2016)
  • T. Kong et al.

    An amperometric glucose biosensor based on the immobilization of glucose oxidase on the ZnO nanotubes

    Sens. Actuators B

    (2009)
  • N. Antony et al.

    Immobilization of diastase α-amylase on nano zinc oxide

    Food Chem.

    (2016)
  • R. Song et al.

    Isolation and identification of antioxidative peptides from peptic hydrolysates of half-fin anchovy (Setipinna taty)

    LWT Food Sci. Technol.

    (2015)
  • H. Lee et al.

    Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism

    BBA Biomembranes

    (2015)
  • J. Miao et al.

    Inhibitory effects of a novel antimicrobial peptide from kefir against Escherichia coli

    Food Control

    (2016)
  • S.C. Sharma

    ZnO nano-flowers from Carica papaya milk: degradation of Alizarin Red-S dye and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus

    Optik

    (2016)
  • A.K. Bhunia et al.

    Synthesis, characterization of ZnO nanorods and its interaction with albumin protein

    Mater. Today Proc.

    (2016)
  • T. Togashi et al.

    Material-binding peptide application-ZnO crystal structure control by means of a ZnO-binding peptide

    J. Biosci. Bioeng.

    (2011)
  • T.P. Dasari et al.

    Determination of the mechanism of photo induced toxicity of selected metal oxide nanoparticles (ZnO, CuO, Co3O4 and TiO2) to E. coli bacteria

    J. Environ. Sci.

    (2013)
  • V. Svetlichnyi et al.

    ZnO nanoparticles obtained by pulsed laser ablation and their composite with cotton fabric: preparation and study of antibacterial activity

    Appl. Surf. Sci.

    (2016)
  • T. Jin et al.

    Antimicrobial efficacy of zinc oxide quantum dots against Listeria monocytogenes, Salmonella, Enteritidis, and Escherichia coli O157:H7

    J. Food Sci.

    (2009)
  • A. Das et al.

    Preparation of zinc oxide free, transparent rubber nanocomposites using a layered double hydroxide filler

    J. Mater. Chem.

    (2011)
  • A. Kumar et al.

    Genotoxic and carcinogenic potential of engineered nanoparticles: an update

    Arch. Toxicol.

    (2013)
  • S.M. Alfadul et al.

    Use of nanotechnology in food processing, packaging and safety review

    Afr. J. Food Agric. Nutr. Dev.

    (2010)
  • S.S. Ugur et al.

    Modifying of cotton fabric with nano-ZnO multilayer films by layer-by-layer deposition method

    Nanoscale Res. Lett.

    (2010)
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