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Work Packages

Coordination and Project management

Validation of the developed packaging films on real-food applications

Regulatory evaluation for the elements of A-BLOCK

Establishing and monitoring of A-BLOCK dissemination, communication and exploitation plan
Production of diatiom frustule, microcapsules and microemulsions embedded packaging films

Characterization of the antimicrobial, barrier, migration, optical, and mechanical properties of developed packaging films

Regulations on the recycling of developed packaging films
Isolation, production, formulation, characterization and bioactivity evaluation of natural agents

Upscaling of the production for natural agents
Design, characterization and production of biomimetic antimicrobial packaging films

Design, characterization and production of nanopatterned films combined with frustules, micro-capsules, and micro-emulsions

Exploitation Management

Data Management
Design, formulation and characterization of microemulsions

Validation of the antimicrobial performance of developed packaging films

Life Cycle Assessment of developed packaging materials
Validation of the antimicrobial performance of developed packaging films

WP 1: Coordination and Project management, WP Lead: IBUN

WP2: Isolation and formulation of natural antimicrobial/antioxidant (AM/AO) agents, WP Lead: NKUA

WP3: Biomimetic Bactericidal Food Package, WP Lead: LIST

WP4: Identification of individual and synergistic combinations for the novel packaging materials, WP Lead: UCBL

WP5: Validation in-situ, WP Lead: UCBL

WP6: Regulatory and environmental considerations, WP Lead: ÜB

WP2: Isolation and formulation of natural antimicrobial/antioxidant (AM/AO) agents, WP Lead: NKUA

NKUA
  1. Selected agri-waste shown to be rich sources of AM/AO agents will be provided by relevant industries located in the partner countries.
  2. The biomass will be extracted with green solvents (eg. water or ethanol that will be recycled, deep eutectic solvents) using different techniques (maceration, microwave and ultrasonic assisted extraction).
  3. The resulting extracts will be fractionated using standard chromatographic techniques with solvents acceptable in the food packaging industry to obtain fractions enriched in bioactive compounds, displaying more pronounced activity.
  4. The extracts will be evaluated for their bioactivity. The antimicrobial activity will be investigated in vitro using methods, such as the disk/well diffusion, agar dilution or broth dilution against several foodborne-related microbial strains.
  5. Potential synergistic antimicrobial effects of selected combinations against specific target microorganisms may be evaluated by determination of fractional inhibitory concentrations (FICs).
  6. The antioxidant activity will be assessed using methods, such as DPPH, FRAP or TEAC methods. The antimicrobial and antioxidant activity will be compared to that of synthetic agents currently in use for food products.
  1. Micro/nanoemulsions, from the lipophilic and hydrophilic fractions of the AM/AO agents, will be designed by using different techniques.
  2. Coarse emulsions will be formed by using high-pressure homogenization. Then, micro/nanoemulsions will be obtained by microfluidizing the coarse emulsions. The number of homogenization and microfluidizing cycles will be optimized as well as the pressure of each.
  3. The suitable emulsifier types will be selected to offer better stability of the micro/nanoemulsions, in environmental stresses such as pH, ionic strength, heating, cooling, or long-term storage. Other techniques will be investigated, including membrane emulsification, which offers low energy consumption, tuneable droplet size, monomodal distribution and high encapsulation efficiency without shear or thermal degradation.
  4. Microstructure, size distribution and stability of the micro/nanoemulsions, will be analysed via SEM, dynamic light scattering (DLS) and zeta potential.
  5. Creaming stability will be investigated to provide indirect information about the extent of droplet flocculation and aggregation in the micro/nanoemulsions systems.
  6. The micro/nanoemulsions stability to environmental stress such heating stability, cooling stability and salt stability will be investigated. Chemical analyses will be conducted using HPLC to determine the chemical compositions before and after emulsification.
  7. The microcapsules will be produced from the micro/nanoemulsions by mixing them with a suitable wall material solution having the same pH and then drying them in a laboratory and pilot scale spray-dryer or freeze-dryer. For the spray dryer, the inlet and outlet temperatures and air pressure will be optimized. For the freeze dryer, the vacuum pressure and condensing temperature will be optimized.
  8. The effect of emulsifier type, pH, and drying method on the particle size of dried microcapsules will be evaluated. Encapsulation efficiency is the main parameter to evaluate the best combination of the three factors to encapsulate the micro/nanoemulsions, namely acidity, emulsifier type, and drying method. Encapsulation efficiency of the microcapsules will be measure
  9. Surface morphology by SEM, water content, thermogravimetric analysis (TGA) and structural characterization using Fourier Transform Infrared Spectroscopy (FTIR) will be carried out for the microcapsules.
  10. The antimicrobial activity of micro/nanoemulsions and microcapsules will be investigated by the agar well diffusion method on a Gram-positive bacteria and a Gram-negative bacteria as well as selected spoilage bacteria based on the specific food applications.
  11. The antioxidant activity of micro/nanoemulsions and microcapsules will be measured by DDPH or ABTS methods and by conducting absorbance measurements using a spectrophotometer. The objective of this work package is to reach at least 90% of encapsulation efficiency for both micro/nanoemulsions and microcapsules.
  1. To produce AM/AO agents-loaded diatom frustules, live diatom samples will be obtained from the Mediterranean coast of Türkiye by taking samples of subsurface water. Larger species, such as Cyclotella sp. and Thalassiosira sp. that has a cylindrical capsule structure will be targeted for cultivation upscaling.
  2. Diatom specie with highest production yield will be selected based on specific growth rate, doubling time and biomass productivity.
  3. Diatom specie will be characterized genetically from the strain’s 18S rRNA gene sequence with using D512F/D978R primers. Results of the DNA sequencing will be prepared in SnapGeneViewer programme and will be analysed by BLAST (Basic Local Alignment Search Tool) using NCBI (National Center for Biotechnology) nucleotide data bank.
  4. If the growth speed and production yield of selected diatom specie will not be enough to collect necessary amounts of frustules to be used in the project, live diatom specie Thalassiosira sp., which is a member of Mediterranean phytoplankton community, will be purchased (Carolina Biological Supply).
  5. After cultivation, diatoms will be aggregated, organic cell structures will be removed by washing and oven heating. Then, diatom frustules will be obtained as a solid powder to be used as capsules for AM/AO agents loading.
  6. Morphology and size distribution of frustules will be analysed via SEM and chemical structure of diatoms will be studied by XRD. Chemical analysis will show if there is any contamination and further washing steps can be applied to purify the sample.
  7. NKUA will provide AM/AO agents and the solvents that are able to dissolve each agent will be used for encapsulation by absorption or vacuum techniques. The most efficient technique will be selected for loading.
  8. The loading will be analysed by TGA and EDX analysis and for the release study, antimicrobial agents will be detected with appropriate techniques (UV-VIS spectroscopy, HPLC).
  9. The antimicrobial efficacy of samples (antimicrobial emulsions, antimicrobial loaded diatom frustules) throughout their development and optimization phase will be evaluated against a collection of target microorganisms in-vitro with high-throughput minimum inhibitory concentration (MIC) assays using the microdilution method or headspace release methods in microtiter plates.
  10. In case of water-insoluble components, solubilization in appropriate concentrations of solvents like dimethyl sulfoxide (DMSO) or different alcohols will be evaluated in advance in order to select those which do not affect the growth of the microorganisms of interest.
  11. In order to evaluate the effect of pH on the activity of the above-mentioned formulations, the MIC determination will be done in at least 2 different pH levels relevant to the target microorganisms and the food application.
  12. Comparatively, in cases where needed, the antimicrobial efficacy of the antimicrobial blended biomimetic packaging films through release of the antimicrobial in the headspace will be done in-vitro by microbial target growth kinetic studies in wells of microtiter plates filled with nutrient medium by attachment of the film samples on the lid of the plates and by monitoring the effect on the cell counts of the target at regular time intervals as described earlier.

WP3: Biomimetic Bactericidal Food Package, WP Lead: LIST

  1. Self-assembly of diblock copolymers will be used to prepare well-defined nanoarray template. The characteristics of the template, mainly the feature size and pitch will be varied systematically by controlling the self-assembly (by solvent-quality) and/or self-organization (by evaporation control) processes.
  2. The templates will be transferred into the underlying substrate to produce nanopillar arrays by reactive ion etching. Nanopillar arrays would be produced on Si (for NIL stamps, or gradients), or QCM substrates (for nano-QCM investigations)
  3. Nanopillar arrays on packaging films will be produced using NIL in a 2-step process (a) producing Si nanopillar stamps, (b) which will be used to subsequently derive a conformal NIL stamp (high Tg or thermosetting polymers) with hole arrays (c) These conformal stamps will be used to produce nanopillar arrays on the chosen thermoplastic food packaging films by thermal/UV imprinting.
  4. Unidimensional surface gradients presenting multitude of nanopillar characteristics on a single surface will be developed for combinatorial screening of the nanostructure variables to identify nanopillar geometries that yield the most favorable anti-microbial properties.
  5. Design of experiments (DoE) approach will be adopted in two steps to optimize the bactericidal properties of the nanopillar structures. In the first step, geometry factors will be varied according a 2-level factorial design with center points in order to identify the ones significantly affecting the response. In the second step, Response Surface Methodology (RSM) will be used to optimize the activity by varying the significant geometry levels according to a Central Composite Design (CCD).
  6. The determination of the antimicrobial efficacy and mode of action of biomimetic packaging films will be done by use of methods specifically designed to test contact-killing, like the JIS Z 2801 or the Petrifilm® method. These methods will be used for initial screening and optimization of the killing efficiency of larger numbers of nanostructured film samples.
  7. The density and viability of the target microorganisms on a limited number of optimized samples of nanostructured packaging films may be evaluated with live-dead staining and fluorescent microscopy
  8. The identified nanopillar arrays will be reproduced on a QCM sensor to study anti-microbial performance in further detail, and to identify the conditions under which they could fail. Nano-QCM is an innovation from LIST that allows leveraging the label-free, highly sensitive and real-time detection capabilities of QCM sensor, to the context of measuring nanopillar-bacterial interactions.
  9. The nano-QCM results will be validated against the antibacterial tests performed by DIL.
  1. The nanostructured films prepared by LIST will be benchmarked against standard flexible packaging tests such as grammage, ink adhesion, coating adhesion, Oxygen Transmission Rate (OTR) and Water Vapor Transmission Rate (WVTR).
  2. Standard flexible film tests ASTM F1249, ASTM D3985, ASTM D1003, ASTI D2578, ASTM D1204, ASTM D882, ASTM D1894 and PPC internal methods will be made on 6” circular films (as output of T3.3).

WP4: Identification of individual and synergistic combinations for the novel packaging materials, WP Lead: UCBL

The production of the AM/AO agents (top 3 per food product category) that have the highest activity and the most pleasant organoleptic properties, while at the same time abiding by safety and regulatory requirements will be upscaled and adequate quantities will be provided for incorporation in the new packaging films orientated for use in raw cut meats, fresh cut vegetables and cakes

The bioactive extracts and/or fractions as well as the resulting formulations (micro/nanoemulsions, microcapsules & loaded diatom frustules) will be provided to PPC for further steps.

  1. Two individual approaches will be tested at lab scale (i) adding dried microcapsules and diatoms during extrusion process in core or skin layer and (ii) surface coating with micro/nanoemulsions of the plastic films by using bar-coating technique after extrusion. To optimize coating grammage, different size of k-bar (k-hand coater) will be used.
  2. For extrusion approach, ratio of microcapsules and diatom frustules, temperature, time and pressure will be investigated in coordination with PPC. For extrusion approach, frustules and microcapsules will be mixed with polymer particles and will be extruded to form packaging.
  3. If the release rate of AM/AO agents will be too low to achieve antimicrobial/antioxidant effect as stated in “critical risks for implementation” table, then a secondary technique (using a semipermeable patch) will be applied as stated in the mitigations for critical risks table.

To compare and improve the release of antimicrobial agents, a separate semipermeable patch of loaded frustules and microcapsules can be prepared which will be attached to the surface of original packaging. The patch material will have the same polymer with the packaging (PE, PA or BOPP). The semipermeable patch will be obtained from the commercial patches that are already used in food packaging. The semipermeable patch will prevent the escape of diatom frustules and microcapsules while allowing water transfer and release of AM/AO agents. The patch that is loaded with frustules and microcapsules will be attached to the surface of the packaging with hot press.

The use of micro/nanoemulsions, diatom frustules and microcapsules with potential combinations with nanopatterned films will be investigated. NIL will be performed either directly on the packaging film consisting of microcapsules, micro/nano-emulsions or frustules, either directly or on a sacrificial coating applied to the package film, using at the lowest temperature possible. The option of patch configuration is particularly attractive to address risk of NIL process affecting the encapsulated AM/AO agents by separating the NIL process from and integration of microcapsules or frustules.

The antimicrobial activity of the combined plastic active packagings will be evaluated.
  1. The release rate of AM/AO agents, thermal stability (TGA, DSC), microstructural, mechanical, barrier and physiochemical characterization tests (standard flexible film tests, ASTM F1249, ASTM D3985, ASTM D1003, ASTI D2578, ASTM D1204, ASTM D882, ASTM D1894 and PPC internal methods) of the final packaging by the industrial partners PPC and ÜB.
  2. The objective of this work package is to reach around 80% release of these AM/AO agents to the headspace of package or in contact with food during the entire shelf-life of the product. Newly developed food packaging systems will be sent to the accredited laboratories to be tested for overall migration and specific migration according to Commission regulation (EU) No 10/2011 on plastic materials and articles intended to come into contact with food.

WP5: Validation in-situ, WP Lead: UCBL

  1. Final validation of the optimum packaging films will be done with challenge tests in real food against target microorganisms specific for the application.
  2. The optimum package films from WP3 and 4 will be reproduced and quality-tested for the purpose. Sterilized (irradiated) food samples will be surface-inoculated with each one of the relevant target microorganisms for the food application and stored under normal retail conditions.
  3. The preservative effect of the novel packaging films will be evaluated by determination of cell counts with classic microbiological methods and use of selective media at regular intervals during storage.
  4. The preservative effect of packaging film in low water activity cakes will be evaluated in cake recipes lacking any preservative against fungi.
  5. Accelerated shelf-life studies will be conducted by placing samples into humidity- and temperature- controlled chambers able to apply elevated stress conditions.
  6. Samples will be analysed by visual inspection as well as chemical and textural analyses.
  7. Texture profile analyses to determine the changes in hardness, gumminess, chewiness, cohesiveness and springiness will be performed.
  8. Hunterlab color measurements will be performed to monitor the maintenance of color during shelf life.
  9. Wet chemistry analyses like moisture, pH, peroxide value and free fatty acid content will also be performed to evaluate the oxidative stability.

WP6: Regulatory and environmental considerations, WP Lead: ÜB

To identify and quantify ingredients of the bioactive extracts and/or enriched fractions selected in WP2 will be analyzed with gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and/or NMR spectroscopy.

The compatibility of the selected bioactive extracts, emulsions, diatom frustules and biomimetic approaches will progressively be evaluated to conform to the relevant regulations. In this regard, Turkish Food Codex Regulation on materials and articles intended to come into contact with foodstuffs which is drafted in accordance with Regulation (EC) no 1935/2004 of the European Parliament and of the Council will be followed.
  1. Sustainability assessment of the developed packaging system will rely on standardized methodology for the life cycle assessment (ISO 14040 and 14044). The assessment will rely on the recent product environmental footprint (PEF) guidelines for packaging of various foods and on the Environmental footprint 3.0 (EF 3.0) life cycle assessment method. The LCAs performed in this project will address many environmental impacts, using state-of-the-art methodologies, and considering the assessment scope from cradle to grave.
  2. Special attention will be devoted to defining the variations on the end-of-life options defining the circularity potential for the developed packaging. Antimicrobial functions of packaging will be given extra consideration and will require relevant adaptation of methodologies and functional units. This task will also consider the potential rebound effects i.e., when seeking to avoid one environmental problem others can arise, causing an unwanted shifting of environmental burdens on the local, regional or global scale. Uncertainties are unavoidable in LCA, arising e.g., from methodological choices, limitations and data gaps. We will handle uncertainties by gathering statistically sound data from partners and industry, by performing scenario, sensitivity and uncertainty analyses, and in communication of results highlight the most critical assumptions.
  3. Conclusions on the level of environmental impact of packaging and antimicrobial functions of the packaging will be made in comparative aspects to more conventional packaging options. Achievement of TRL 4 and 5 allows for the collection of more industrially relevant data, which can be effectively used for the LCA. Relying on the low TRL data for the LCA (classically relying on industrial data) requires the development of specifically tailored approaches for the upscaling modelling.

Developed packaging system will be evaluated by PPC according to recycling regulations of the packaging material.