Elsevier

Food Chemistry

Volume 168, 1 February 2015, Pages 115-123
Food Chemistry

Targeted separation of antibacterial peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis

https://doi.org/10.1016/j.foodchem.2014.07.027Get rights and content

Highlights

Abstract

Anchovy (Engraulis japonicus) cooking wastewater (ACWW) is a by-product resulted from the production of boiled–dried anchovies in the seafood processing industry. In this study, the protein hydrolysate of ACWW (ACWWPH) was found to have antimicrobial activity after enzymatic hydrolysis with Protamex. For the targeted screening of antibacterial peptides, liposomes constructed from Staphylococcus aureus membrane lipids were used in an equilibrium dialysis system. The hydrolysate was further purified by liposome equilibrium dialysis combined with high performance liquid chromatography. The purified antimicrobial peptide (ACWWP1) was determined to be GLSRLFTALK, with a molecular weight of 1104.6622 Da. The peptide exhibited no haemolytic activity up to a concentration of 512 μg/ml. It displayed a dose-dependent bactericidal effect in reconstituted milk. The change in cell surface hydrophobicity and membrane-permeable action of the purified ACWWP1 may have contributed to the antibacterial effect. This study suggests that liposome equilibrium dialysis can be used for the targeted screening of antimicrobial peptides.

Introduction

The increasing problem of multiple drug resistance in bacteria and the potential health risks of chemical food preservatives have lead to a demand for new antimicrobial strategies. With action modes markedly different from that of traditional antibiotics, it has been proposed that antimicrobial peptides (AMPs) might form the foundation for a new class of antimicrobials that are effective against antibiotic-resistant bacteria (Brogden, 2005, Parisien et al., 2008, Reddy et al., 2004). An increasing number of AMPs have been isolated from various different species (Zasloff, 2002). These have helped in the discovery of new antibacterial drugs and in elucidating the exact action mechanism of AMPs. However, the conventional procedure for screening AMPs is an activity-guided chromatographic protocol, in which the multiple-step extraction and isolation of peptides from complex sources are followed by the analysis of the purified individual constituents (Pichu et al., 2009, Salampessy et al., 2010, Tan et al., 2013). These methods are arduous and time-consuming. Thus, an effective method for targeted-separation of AMPs is needed.

Equilibrium dialysis, a technique used to characterise the interaction properties of drugs with biological systems (Gunn et al., 2012, Hou et al., 2013, Varghese et al., 2011), has been proposed for the screening (Qi et al., 2006) and analysis of the multiple bioactive compounds found in traditional Chinese medicines (Deng et al., 2011). In equilibrium dialysis, each component that is below a certain molecular weight threshold can pass freely through a dialysis membrane. The size of the liposomes used in equilibrium dialysis is larger than the pore size of the dialysis membrane. Components that combine with the liposomes can thus not diffuse out (Ottiger & Wunderli-Allenspach, 1997). As a result, equilibrium dialysis can be used for targeted separation of the active components.

Liposomes have previously been used as model membranes to study the interactions between AMPs and the bacterial cell membrane (Bonev et al., 2000, Epand et al., 2003, Yu et al., 2009). Traditional liposomes were prepared by one or several specific polar lipids at a certain ration. This cannot mimic the highly regulated phospholipid composition and content in real bacterial cellular membranes. Our group constructed liposomes from bacterial membranes which were used in immobilized bacterial membrane liposome chromatography for the separation of AMPs (Tang, Zhang, Wang, & Qian, 2014). This kind of liposome was much finer than conventional liposomes and more accurately reflected the interaction between AMPs and the bacterial cell membrane. Liposomes constructed from bacterial membrane lipids have not previously been used for equilibrium dialysis. In addition, equilibrium dialysis has not yet been used for the screening of potential AMPs.

Anchovy cooking wastewater (ACWW) is a by-product resulted from the production of boiled–dried anchovies, a traditional Chinese seafood known as “haiyan”. The cooking operation gives rise to approximately 1.5 tons of liquid waste for each ton of canned sardine. Generally, the wastewater generated by fish processing contains valuable amount of protein, peptides and amino acids and lipids (Amado et al., 2013, Chowdhury et al., 2010, Ferraro et al., 2013). Therefore, the use of this waste for the extraction of valuable compounds has great potentials for the medical, pharmaceutical and food industries. However, ACWW is generally discarded as waste into the sea. The high nutrient content of ACWW means that inappropriate disposal of ACWW could not only waste resources, but also cause eutrophication in coastal waters and environmental pollution. Hydrolysis of fish cooking water has been used to convert waste into value-added forms (Choi et al., 2012, H-Kittikun et al., 2012, Hsu et al., 2009). To our present knowledge, isolation and characterisation of AMPs from the protein hydrolysate of ACWW (ACWWPH) have been seldom reported.

In this study, a new antibacterial peptide was purified from the protein hydrolysate of ACWW by equilibrium dialysis combined with HPLC. In addition, the antimicrobial spectrum, haemolytic effect, and mode of action of the peptide were studied.

Section snippets

Materials

Frozen anchovies (Engraulis japonicus) were purchased from local aquatic wholesale market (Qingdao, China), and stored at −20 °C until use. Protamex (food-grade) was a Bacillus protease complex from Novozymes (Bagsvaerd, Denmark). Full-fat powdered milk was from Inner Mongolia Yili Industrial Group Co., Ltd. (Huhhot, China). Propidium iodide (PI), acetonitrile (ACN), formic acid and trifluoroacetic acid (TFA) for HPLC were purchased from Sigma (St. Louis, MO, USA). All other chemicals and

Preparation of ACWWPH

The knowledge of the composition is important for the utilisation of ACWW. The waste water contained 5.07 ± 0.08 g/l crude protein, 12.91 ± 0.10 g/l ash, and 0.12 ± 0.02 g/l total lipids. The amino acid analysis result of ACWW (Supporting information, Table S1) showed that the total content of essential amino acids was 56.7% of the total amino acids. The chemical score based on the reference protein of hen egg amino acids indicated that the protein in ACWW was of high nutritive value. These together

Conclusion

An antimicrobial peptide termed ACWWP1 was successfully targeted isolated from anchovy cooking wastewater hydrolysate by equilibrium dialysis in which the liposomes were constructed from S. aureus membrane lipids. The molecular weight of ACWWP1 was determined to be 1104.6622 Da and the amino acid sequence was GLSRLFTALK. The peptide possessed antibacterial activity against the tested pathogenic or food-spoilage-related bacteria in bacterial growth medium. It also exhibited a dose-dependent

Acknowledgments

This study was supported by the National Nature Science Foundation of China (NSFC, No. 31271934), the National High-tech Research and Development Program of China (863 Program, Nos. 2013AA102207 and 2013AA102203-07), the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Project of China National Key Technology Research and Development Program for the 12th Five-year Plan (No. 2012BAD37B08-3).

References (58)

  • R.E. Hancock et al.

    Role of membranes in the activities of antimicrobial cationic peptides

    FEMS Microbiology Letters

    (2002)
  • A. H-Kittikun et al.

    Hydrolysis of surimi wastewater for production of transglutaminase by Enterobacter sp. C2361 and Providencia sp. C1112

    Food Chemistry

    (2012)
  • G. Hou et al.

    Studies on the interactions between ginsenosides and liposome by equilibrium dialysis combined with ultrahigh performance liquid chromatography–tandem mass spectrometry

    Journal of Chromatography B

    (2013)
  • L. Hou et al.

    Inhibition of foodborne pathogens by Hf-1, a novel antibacterial peptide from the larvae of the housefly (Musca domestica) in medium and orange juice

    Food Control

    (2007)
  • K.-C. Hsu et al.

    Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase (Bacillus subtilis)

    Food Research International

    (2009)
  • A.S. Ladokhin et al.

    Bilayer interactions of indolicidin, a small antimicrobial peptide rich in tryptophan, proline, and basic amino acids

    Biophysical Journal

    (1997)
  • L. Li et al.

    Selectivity for and destruction of Salmonella typhimurium via a membrane damage mechanism of a cell-penetrating peptide ppTG20 analogue

    International Journal of Antimicrobial Agents

    (2012)
  • H. Liao et al.

    Analysis of Escherichia coli cell damage induced by HPCD using microscopies and fluorescent staining

    International Journal of Food Microbiology

    (2010)
  • B. Liaset et al.

    Chemical composition and theoretical nutritional evaluation of the produced fractions from enzymic hydrolysis of salmon frames with Protamex™

    Process Biochemistry

    (2003)
  • Y. Liu et al.

    Characterization of structural and functional properties of fish protein hydrolysates from surimi processing by-products

    Food Chemistry

    (2014)
  • C. Ottiger et al.

    Partition behaviour of acids and bases in a phosphatidylcholine liposome–buffer equilibrium dialysis system

    European Journal of Pharmaceutical Sciences

    (1997)
  • N. Papo et al.

    Can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes?

    Peptides

    (2003)
  • S. Pichu et al.

    Purification and characterization of a novel salivary antimicrobial peptide from the tick, Ixodes scapularis

    Biochemical and Biophysical Research Communications

    (2009)
  • K. Reddy et al.

    Antimicrobial peptides: Premises and promises

    International Journal of Antimicrobial Agents

    (2004)
  • J. Salampessy et al.

    Release of antimicrobial peptides through bromelain hydrolysis of leatherjacket (Meuschenia sp.) insoluble proteins

    Food Chemistry

    (2010)
  • N. Song et al.

    Transglutaminase cross-linking effect on sensory characteristics and antioxidant activities of Maillard reaction products from soybean protein hydrolysates

    Food Chemistry

    (2013)
  • Y. Su

    Isolation and identification of pelteobagrin, a novel antimicrobial peptide from the skin mucus of yellow catfish (Pelteobagrus fulvidraco)

    Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology

    (2011)
  • Y.N. Tan et al.

    Purification and characterisation of antibacterial peptide-containing compound derived from palm kernel cake

    Food Chemistry

    (2013)
  • S.J. Varghese et al.

    In vitro interaction study of retinoic acid isomers with telmisartan and amlodipine by equilibrium dialysis method using UV spectroscopy

    Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

    (2011)
  • Cited by (65)

    • Exploration of bioactive peptides from various origin as promising nutraceutical treasures: In vitro, in silico and in vivo studies

      2022, Food Chemistry
      Citation Excerpt :

      In literature, protein hydrolysates of anchovy cooking wastewater produced using protamex were found to have strong antibacterial activity against S. aureus, B. subtilis, S. pneumoniae, E. coli, S. dysenteriae, P. aeruginosa, and S. typhimurium. The hydrolysates were effective in changing cell surface hydrophobicity and permeability of cell membrane (Tang et al., 2015). Ma, Guo, Fu, and Jin (2020) identified a novel antimicrobial peptide OVTp12 from protein hydrolysates of egg white ovotransferrin.

    • NMR spectroscopy of wastewater: A review, case study, and future potential

      2021, Progress in Nuclear Magnetic Resonance Spectroscopy
      Citation Excerpt :

      As an example, the analysis of macromolecules may employ dialysis to remove salts and other low molecular weight components [77,114]. After such pre-treatment, liquid–liquid extractions using an organic solvent [114–116] may be employed to concentrate the specific analyte or analytes and reduce undesired compounds. That said, it is important to realize that extraction (and its efficiency) creates a number of variables.

    View all citing articles on Scopus
    View full text