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