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Improving the performance of bacteriocins as natural food additives through the use of nanotechnology

Principal Supervisor: Dr. Francisco Fernandez-Trillo, School of Chemistry

Co-supervisor: Dr Zhenyu Zhang, School of Chemical Engineering; Dr Kostas Gkatzionis, School of Chemical Engineering

PhD project title: Improving the performance of bacteriocins as natural food additives through the use of nanotechnology.

University of Registration: University of Birmingham

Project outline:

One of the major challenges to modern food industry is to meet the demand by consumers that products should look appealing for prolonged period of time. Also, there is an increasing preference by consumers for minimally processed products that do not contain “chemical” preservatives. Bacteriocins, a family of antimicrobial peptides of microbial origin, have been postulated as an attractive natural alternative for the preservation of food. Nisin, a polycyclic bacteriocin produced by the bacterium Lactococcus lactis, has been approved as a preservative for food since the 50s and this peptide is currently used as an additive for dairy products.

Nisin has a broad spectrum of activity and is stable against heat and shows no adverse effects when formulated in foods. However, proteolytic enzymes in the stomach readily degrade most bacteriocins. Additionally, when used in food packaging, bacteriocins’ antimicrobial activity is compromised by the slow diffusion of the peptides within the packaging polymeric matrix.

It is therefore highly desirable to develop novel formulations based on the above-mentioned natural food preservatives, to improve their stability and increase their spectrum of application, as additives in both food and packaging.

Aim: In this project we will demonstrate the use of polyelectrolyte complexes as carriers to develop novel formulations for nisin with potential applications in food processing. Thus, positively charged nisin will be conjugated with negatively charged polymers to form PolyIon Complex (PIC) particles where nisin will be shielded from the action of exogenous proteases. We will also explore the use of Layer-by-Layer (LbL) deposition to prepare polymeric films containing nisin. In both cases, negatively charged polymers will be selected from FDA approved additives. At later stages of the project, we will explore the use of enzyme responsive moieties to modulate the degradation of the polyelectrolyte complexes and optimise the delivery of nisin in the presence of the targeted pathogens.

Research Objectives:

O1. Synthesis of Nisin containing PIC particles: We will evaluate a library of FDA approved negatively charged polymeric additives (e.g. alginate, carrageenans) to identify the most appropriate candidates to complex positively charged nisin in solution and subsequently form well defined particles. The stability of these nisin-containing particles will be evaluated under relevant biological conditions, including a range of pHs (e.g. pH in cheese varies 4-7.5 depending on the type) and temperatures relevant to the food manufacturing (e.g. long term stability at 30-40 ºC during cheese manufacturing vs short exposure to 60-70 ºC during milk pasteurization).

O2. Synthesis of nisin containing LbL Films: Films will be prepared by depositing alternative layers of the polymeric additives and nisin onto glass slides. The number of layers and deposition time will be optimised to enhance surface coverage and film microstructure. Then, we will evaluate the ability of these films (with and without additives such as EDTA) to inhibit the growth of model pathogens: Staphylococcus aureus (gram-positive) and Salmonella enterica (gram-negative).

O3. Pathogen-responsive formulations: In the latter stages of the project we will investigate the synthesis of novel polyelectrolyte complexes that will only release the encapsulated nisin in the presence of the targeted pathogens. It is now well understood that most pathogens secrete exotoxins (e.g. proteases, lipases) to aid in colonisation and settlement. To demonstrate the feasibility of this approach, we will prepare a library of anionic peptides that can be selectively degraded by pathogenic proteases. We will then evaluate the ability of these peptides to form similar formulations to those prepared in O1 and O2. We anticipate that these pathogen responsive formulations will be inert when exposed to human proteases (e.g. liver protease) but release therapeutic concentrations of nisin when exposed to the targeted pathogen.

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

Due to the multidisciplinary approach, the PhD student will develop a wide range of experimental skills described in the following training objectives. These training objectives will be tailored to the student’s background, skills and interests:

  • Polymer Chemistry and Nanotechnology: The PhD Student will receive basic training in polymer chemistry, particle synthesis and nanotechnology. Training in standard analytical techniques (such as Nuclear Magnetic Resonance (NMR), Mass (MS), Infrared (IR), Ultra-Violet and Visible (UV-Vis), Fluorescence spectroscopy, High Performance Liquid Chromatography (HPLC), Gel Permeation Chromatography (GPC) and Dynamic Light Scattering (DLS) is expected.
  •  Surface Chemistry: The PhD student will receive training on the synthesis and characterisation of LbL films and their interphase with biological media. Training in surface analysis techniques such as scanning electron microscopy (SEM), atomic force microscopy (AFM), single molecule force spectroscopy, quartz crystal microbalance (QCM) and ellipsometry is expected.
  •  Microbiology and Molecular Biology Training: The PhD Student will receive training in standard techniques for microbial culture, identification and viability, including on class 2 pathogens.

 Contact: Dr. Francisco Fernandez-Trillo, School of Chemistry