Determining Mechanisms of Action and Optimum Use of Polyphenols As Food Preservatives
Lead Research Organisation:
University of East Anglia
Department Name: Graduate Office
Abstract
Preservatives are crucial ingredients in a wide range of foods which are present in order to control growth of pathogenic and spoilage organisms and maintain product quality and shelf life. A wide range of chemicals are currently used in this context including organic acids, salts and various nitrite compounds. Some of these additives, for example sodium and potassium nitrite, have potential health concerns1, 2 and there is great interest in industry in exploring alternatives3 which can retain antimicrobial activity but have a better safety profile. Polyphenols are a diverse group of natural products, extracted from plants which have multiple properties including antimicrobial activity4. Due to their perceived safety there has been great interest in using polyphenols as preservatives in various food stuffs5. Prosur is one commercially available polyphenol mixture currently being added to various foods or evaluated for incorporation in others. Despite the potential for polyphenols as antimicrobials there are major knowledge gaps regarding their mechanisms of action, spectrum of activity or potential bacterial resistance.
This project aims to identify active polyphenols from a well curated panel and determine their mechanisms of action and investigate how foodborne pathogens respond to polyphenol exposure. The ultimate goal is to inform development of improved polyphenol formulations to be used in food.
The project will incorporate analytical chemistry, microbiology and functional genomics to provide complementary approaches to these problems.
This project aims to identify active polyphenols from a well curated panel and determine their mechanisms of action and investigate how foodborne pathogens respond to polyphenol exposure. The ultimate goal is to inform development of improved polyphenol formulations to be used in food.
The project will incorporate analytical chemistry, microbiology and functional genomics to provide complementary approaches to these problems.
People |
ORCID iD |
| Mark Webber (Primary Supervisor) | |
| Ryan Sweet (Student) |
Publications
Sweet R
(2022)
Activity of antibacterial phytochemicals and their potential use as natural food preservatives.
in Critical reviews in food science and nutrition
| Description | Phenotypical Screening Findings- Via the use of rapid Optical Density Assays, Growth Curves and Membrane Permeability Assays numerous polyphenolic compounds were identified as possessing bioactive properties. Notable compounds which directly inhibited the growth of foodborne pathogens (Salmonella enterica, Staphylococcus aureus, Pseudomonas aeruginosa and Listeria monocytogenes) were Naringenin (with especially potent inhibitive effects against the Gram-positive micro-organism Staphylococcus aureus) and Thymol which significantly reduced the growth of all four tested pathogens. In terms of permeabilizing the bacterial cellular membrane, an indicator/mechanism for the compounds' capacities to synergize with presently used antibiotics and biocides, Caffeic Acid showed particular promise. Resistant Mutant Generation Frequency Rate Findings- To provide an indication of the potential longevity of the polyphenol compounds should they be utilized as antibiotic/food preservative substitutes, as well as to inform future genetic work being undertaken, the mutation frequencies of the tested pathogens was investigated using selective agar plating techniques. Across the four micro-organisms detailed above, an average mutation frequency comparable to that for presently used antibiotics was observed; although after further validation of the mutant strains it was discovered that the candidate strains presented tolerant, rather than resistant, phenotypes. While this in itself may prove promising for the real-world application of polyphenolic compounds, the tolerant mutants generated through this process are also being used further within this project. As of writing, further work is now being undertaken to investigate the primary mechanism of inhibitive action for Thymol, and the genetic adaptations the tested pathogens may induce in response to the presence of this monoterpenoid phenol. |
| Exploitation Route | These outcomes may provide others with a framework and comparison for their own research and data, while additionally establishing a platform for future scientific investigations. |
| Sectors | Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | From this project two papers have been synthesized and are awaiting final edits and submission for publication. The two papers are as follows- 1. Literature review, collating research produced on antimicrobial polyphenols from the last decade, with a particular focus on the advantages, disadvantages and application of these compounds within the food industry (and as a minor consideration, the clinic). 2. Experimental paper collating the first half of this project; the phenotypical screening (OD(600nm) assays, growth curves, membrane permeability assays) and finishing with the potential for thymol, in particular, to induce the generation of resistant/tolerant mutants of common foodborne pathogens at a rate similar to that for currently used antibiotic classes. |
| First Year Of Impact | 2021 |
| Sector | Other |
| Impact Types | Societal,Economic |
| Title | Listeria monocytogenes Membrane Permeability Assay SOP |
| Description | By taking advantage of the fluorescent properties of certain compounds (e.g.; Resazurin once internalised and metabolised by micro-organisms or Ethidium Bromide as it intercalates nucleic aids, usually restricted to the internal compartments of cells), one can quantify the capacity of tested compounds (Polyphenols, novel antimicrobials etc.) to permeabilise the outer-membranes/cell walls of, in this case, bacterial cells. While not a wholly novel method in and of itself, this method has been tweaked for the optimum use in Listeria monocytogenes, a psychotropic pathogen of some concern. Briefly; a 96-well plate is set up with the tested compound(s) and appropriate Negative and Positive Controls (For Positive Controls a known Efflux Pump Inhibitor which naturally enables the intracellular accumulation of various compounds is effective). The fluorescent indicator molecule (Resazurin or Ethidium Bromide; the latter in the case of Listeria) is added to all wells and finally a quantified bacterial suspension in Phosphate Buffered Saline (PBS) is added to each sample well (Excluding Blank wells used to normalise the fluorescence readings of the compounds/cells. These Blanks are instead inoculated with sterile PBS). The 96-well plate is then sealed with a transparent membrane and placed into a plate reader with the appropriate fluorescence filters and detectors. After an overnight run, measuring fluorescence and OD(600nm), the data is trimmed to an appropriate timeframe and can thus be utilised to quantify the effect of the tested compound(s) on the bacterial membrane/cell wall integrity of Listeria monocytogenes. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2020 |
| Provided To Others? | No |
| Impact | This Research Method will enable other researchers to quantify the in vitro effect of experimental compounds on the bacterial cell wall/membrane integrity of Listeria monocytogenes; this will be particularly useful in the fields of Antimicrobial Resistance Mechanisms (ARM) elucidation and the coupled sector of novel antimicrobial research. |
| Title | Polyphenol Initial Screening Methodology |
| Description | A simple, quick and efficient 96-well screening method for the assaying of Polyphenol bioactivity vs. a micro-organism of choice. This assay is flexible in that it can be modified to test for the inhibitory activity of isolated polyphenols, and for the potentiative activity of polyphenols in conjunction with a known, efflux-pump ejected antibiotic (Chloramphenicol in this case). Assay relies on OD(600nm) measurements at the end-point of an incubation period (usually O/N). |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | Assay was generated during the first year of my "new" project (i.e; after my transfer from the IFR's Peck Group to the QIB's Webber Group). Allows for a quick, efficient and middling-throughput initial screening of Polyphenols; used in this project to identifiy Polpyhenols with sufficient bioactvity to take through to the next stage of more detailed, quantitative screening. |
| Description | Norwich Science Fair, Staffing a QIB Stand on Biocides, Antimicrobials and Antimicrobial Resistance |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | Myself and the Webber Research Group manned a stand at the annual Norwich Science Fair at The Forum. Our stall was designed to educate the general public about biocides, antimicrobials and their overuse, antimicrobial resistance and for my particular part the potential of plant-based polyphenols as substitutes for the above. We engaged with well over 500+ members of the general public, many of which were young school children, to educate them about the appropriate use of biocides/antimicrobials and to hopefully inspire young children towards a career in scientific research. Many members of the public left feedback after attending our stall, all of which was positive and complimentary in nature. Children in particular seemed to enjoy our stall and its' accompanied activities. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Poster Presentation and Attendance: ASM and FEMS Joint World Microbe Forum, June 20th-24th 2021 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | The ASM and FEMS jointly-held World Microbe Forum, held online in 2021 due to the ongoing COVID-19 pandemic, was one of the world's largest gatherings of scientists from across the globe, again intended to bring together scientists and students together to discuss and present novel and current research within multiple disciplines across the spectrum of Microbiology. As one of the world's largest conferences, networking and opportunities for discussion were unparalleled. While attending this 5 day event, I engaged with relevant presentations, networked and discussed ideas with experts in the field and with current students. As of writing, I have also submitted a poster abstract to present the first large section of my work (phenotypical screenings and mutation frequency rate investigations as described in the recent Key Findings update of this award). |
| Year(s) Of Engagement Activity | 2021 |
| Description | Poster Presentation and Attendance: Microbiology Society's Annual Meeting, April 26th-30th 2021 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | The Microbiology Society's Annual Meeting, held online in 2021 due to the ongoing COVID-19 pandemic, was a conference intended to bring together scientists and students from around the UK, and further afield, to discuss and present novel and current research within various disciplines across the spectrum of Microbiology; of particular interest and relevance were those talks and presentations on Antimicrobial Resistance (AMR), Career Development and Microbial Biotechnology. While attending this 5 day event, I engaged with relevant presentations, networked and discussed ideas surrounding experimental designs, potentially untapped mines of novel compounds, etc., with experts in the field and with current students. As of writing, I have also submitted a poster abstract to present the first large section of my work (phenotypical screenings and mutation frequency rate investigations as described in the recent Key Findings update of this award). |
| Year(s) Of Engagement Activity | 2021 |
| Description | Poster Presentation: BSAC ARM Workshop, Birmingham Nov. 2019. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | The British Society of Antimicrobial Chemotherapy's Antimicrobial Resistance Mechanisms (BSAC ARM) Workshop, held in Birmingham in 2019, was a conference intended to bring together scientists and students from around the UK, and potentially further, to discuss and present novel and current research within the fields of antimicrobial resistant epidemiology, antimicrobial resistance mechanisms and the development of novel antimicrobial substitutes (Analogues of presently used antibiotics, substitute compounds such as polyphenols etc.). While attending this 1-2 day event, I networked and discussed ideas surrounding experimental design, potentially untapped mines of novel compounds, etc., with experts in the field and with current students. I also presented a poster detailing a section of my work studying the capacity for polyphenols (Secondary plant metabolites) to disrupt bacterial membranes and synergise with currently used antibiotics. This poster gained much attention and sparked a plethora of discussion. |
| Year(s) Of Engagement Activity | 2019 |