PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Tiziana Civera

Curriculum vitae

Curriculum Vitae

Phd thesis

Project title: Agro-industrial by-products with bioactive effects: an eco-friendly strategy to improve food safety and quality

Background:

In the last years, the interest in bioactive molecules derived from food waste has remarkably increased [1]. In fact, the presence of biologically active compounds in fruit and vegetable by-products could represent a relevant opportunity to control the microbial contamination by spoilage bacteria and foodborne pathogens, in order to extend the shelf-life and improve the safety of food products [2,3].

 

Aims: 

The PhD project aims to:

- test in vitro the antibacterial and antibiofilm activities of extracts derived from local agricultural by-products (hazelnut pericarp and apple pomace);

- estimate in vivo the effect of hazelnut pericarp and apple pomace extracts on prolonging the shelf-life of meat preparations, using culture-dependent and culture-independent approaches, along with the impact on sensory and technological characteristics;

- examine the effect of the extract showing the best performance against selected foodborne pathogens (S. enterica, L. monocytogenes) using challenge tests. 

 

Materials and Methods:

During the first year, a detailed characterization of hazelnut pericarp (HP) extract obtained with subcritical water technology was conducted. Subsequently, in vitro and in vivo tests were performed following the protocols previously described.

During the second year, two apple pomace (AP) extracts, A and B, were collected by means of a pilot system from SepareCo s.r.l., in supercritical carbon dioxide conditions (extraction temperature = 60°C, final pressure = 250 bar). Extract A was treated with a solution of ethanol and water, whereas B was re-hydrated before being processed in microwave. The AP extracts were characterized through the identification of both the volatile fraction and the water soluble components, using respectively gas and liquid chromatography coupled with mass spectrometry. Additionally, AP extracts underwent a quantification of key phytochemicals.  

The antimicrobial activity of AP extracts was assayed in vitro, against reference and wild strains (in general more resistant of previous ones) of spoilage bacteria and foodborne pathogens. The screening of antibacterial properties was achieved following the method described by Clinical and Laboratory Standards Institute (CLSI) for the minimum inhibitory concentration (MIC) determination, with some modifications [4]. Serial dilutions of AP extracts (2.5% - 5% - 10% - 20%) were tested.

In parallel, the assessment of antibiofilm properties of the AP extracts was established by the determination of the minimum biofilm eradication concentration (MBEC) following Innovotech guidelines [5].

In vitro results revealed that the best performance was obtained with a 10% of extract B, so in vivo tests were carried out with it. Two batches of beef burger were prepared and treated with the extract and following tested at three storage times (0, 3 and 6 days). Microbiological analyses (Total Viable Count, Enterobacteriaceae and Pseudomonas spp. determination) and colour evaluations (L*, a*, b* attributes) were performed at each time, on each batch, in triplicate (n = 18). Control samples (untreated beef burger) were included in each experiment (n = 36). Moreover, DNA extraction was carried out from each sample (n = 36) using DNeasy PowerSoil Pro Kit (QIAGEN) in order to characterize the microbiota using the 16S metabarcoding. 

 

Results:

With regard to hazelnut pericarp (HP) extract in vitro tests, a different response on the tested bacterial species to different extract concentrations was revealed (ranging from 2.5 mg/mL to 20 mg/mL). In addition, a positive antimicrobial effect was observed almost exclusively using high concentrations of extract. Furthermore, the inclusion of HP extract in beef burgers negatively influenced the colour and involved a significant cooking weight loss.

About AP extracts, phytochemical analysis revealed that they contain a substantial amount of ursolic and oleanolic acid, which along with phenolic compounds contribute to the observed bioactivity of the extracts [6,7].

The AP extracts showed evidence of antimicrobial activity at concentrations ranging between 10%-20%. Nevertheless, the wild strain of B. cereus was never inhibited. 

Concerning MBEC determination, biofilms were eradicated at AP extracts concentration of 5%, although none of the tested concentrations eradicated the biofilm of S. enterica.

About in vivo tests, a noticeable difference in the microbial growth was observed between control and treated burgers at 3 and 6 days of refrigerated storage for each analyzed item, showing an antimicrobial effect of extract B. As concern colorimetric and spectro-colorimetric evaluations, the inclusion of the extract did not appear to influence the colour of the meat preparations. Data analysis of the 16S metabarcoding sequencing results are in progress.

 

Future developments:

An additional 50 kg of apple pomace was extracted in supercritical carbon dioxide conditions and evaluation of the in vitro antimicrobial activity against selected foodborne pathogens will be carried out to test the new batch. Then, the effect of the AP extract of new collection on short-ripened sausages will be estimated during my abroad period through the application of culture-dependent and culture-independent approaches. Sensory and technological characteristics as colour, pH, Aw and texture will be evaluated as well. Moreover, specific challenge tests will be performed to study the potential antimicrobial activity of AP extract towards L. monocytogenes and S. enterica in short-ripened sausages. 

 

References:

[1] Roila R., Primavilla S., Ranucci D., Galarini R., Codini M., Giusepponi D., Altissimi C., Valiani A., Casagrande-Proietti P., Branciari R.,<< Measuring the antimicrobial activity of natural extracts against food spoilage bacteria to enhance food hygiene: preliminary in vitro results >>, Acta IMEKO, vol.13, no.2, pp. 1-5, 2024.

[2] Gyawali R., Ibrahim S. A.,<< Natural products as antimicrobial agents >>, Food Control, 46, pp. 412- 429, 2014.

[3] Negi P. S.,<< Plant extracts for the control of bacterial growth: efficacy, stability and safety issues for food application >>, International Journal of Food Microbiology, 156, pp. 7-17, 2012.

[4] Clinical and Laboratory Standards Institute, <<Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Ninth Edition >>, 2012.

[5] Innovotech, << MBEC Assay: For High-Throughput Antimicrobial Susceptibility Testing of Biofilms >>, 2015.

[6] Cargnin S. T. , Gnoatto S. B., << Ursolic acid from apple pomace and traditional plants: a valuable triterpenoid with functional properties >>, Food Chemistry, 220, pp. 477-489, 2017.

[7] Jesus J. A., Lago J. H. G., Laurenti M. D., Yamamoto E. S., Passero L. F. D., << Antimicrobial activity of oleanolic and ursolic acids: an update >>, Evidence-Based Complementary and Alternative Medicine, 2015.

Research activities

Co supervisor

Pierluigi Aldo Di Ciccio 

Congress abstract, with active partecipation: 

- Muratore G., Di Ciccio P. A., Morra P., Forte C., Mannino G., Civera T. (2024). Control of spoilage and pathogenic bacteria with antimicrobial compounds deriving from food industry waste and investigation of potential applications in meat products. In proceeding of the 77th SISVET Congress, Parma (Italy); 12-14/06/2024.

- Muratore G., Di Ciccio P. A., Morra P., Mannino G., Berta C. M., Destefano R., Civera T. (2024). Apple pomace (AP) extract as a promising antimicrobial agent. In proceeding of the InnovAntiBiofilm Congress, Porto (Portugal); 09/12/2024. 

 

Congress abstract (as co-author): 

Pattono D., Muratore G., Catania A. M., Ala U., Dalmasso A., Rossetto P., Bottero M. T. (2023). Evaluation of bacterial translocation in routine and emergency slaughtering; in proceeding of the 76th SISVET Congress, Bari (Italy); 21-23/06/2023.