(620c) On the Quantitative Investigation of the Antimicrobial Efficacy of Grape Seed Extracts Against Food-Related Bacterial Pathogens.

Over the last years, the trends in food consumption have changed significantly. Consumers are demanding products that are minimally processed, without the use of chemical preservatives or antibiotics. In addition, there is a growing concern about the role of antimicrobial resistance (AMR) in disease outbreaks. Antimicrobial resistance (AMR) is driven by the extensive-use of antibiotics and preventive measures need to be taken against their excessive use¹. Moreover, due to climate change, environmental awareness is more present as compared to the past, urging the food industry follow a more sustainable approach on the production design. Traditional processing methods such as pasteurization or sterilization cannot meet all the above demands and novel processing control strategies should, therefore, be developed².

The possibility to use natural antimicrobials to replace chemical preservatives and the extensive use of antibiotics has been one of the major goals of the food industry. Fruit and vegetable by-products are particularly interesting as sources of natural antimicrobials. Valorising waste of a food processing line which otherwise would have been disposed is an added value for researching their antimicrobial activity, as it contributes to the reduction of food waste³. Grape by-products are derived from grape processing to produce wine and juice. They represent 20% of the total weight of the fruit of the grape, making their disposal a challenging problem for wineries and for the grape juice industry. Grape by-products are composed by the skins, the seeds and in some cases the stems of the fruit. However, the seeds contain higher concentrations of antioxidant and antimicrobial compounds, such as polyphenols, in comparison with other grape by-products. Most studies conducted to date, are focused on the extraction of polyphenols from locally produced grape pomaces and test whether these extracts have a microbial inhibitory effect, without specific systematic monitoring of the microbial dynamics/kinetics⁴. The limited studies that have quantified the inhibition following treatment with extracts from grape by-products used specific food products, deeming the results relevant only for those specific food products⁵.

Overall, in order to use grape by-products effectively as a natural antimicrobial strategy in a larger scale, it is important to study the detailed microbial dynamics of, Gram-negative and Gram-positive, foodborne pathogens robustly controlled systems (2D or 3D food models) instead of food products, as different physicochemical and rheological properties can be better monitored. Such fundamental studies will enable a better understanding of the exact concentration ranges at which grape pomace extracts are bacteriostatic and bactericidal, their mode of action and how they could be utilized, individually or in combination with other novel technologies (ultrasound, cold atmospheric plasma, pulsed electric fields) to attain sustainable food processing. The aim of our work is therefore to perform fundamental systematic studies of the impact of grape seed extracts on the microbial dynamics of food related pathogens in model systems.

METHODS

Grape seed extracts (GSE) were purchased from Bulk powders. The inactivation effect of GSE was tested against L. monocytogenes and E. coli inoculated in TSBYE supplemented with 1% and 4% GSE. Additionally, two levels of initial microbial population were tested, i.e., 10⁵ and 10⁸ CFU/ml. The effect of 1% GSE against mid-exponential or early stationary phase L. monocytogenes was also studied, to enable an understanding of the impact of the growth phase on the potential antimicrobial resistance. After the inoculation of L. monocytogenes in the nutrient broth, microbial dynamics were monitored for 24h. Furthermore, potential morphological changes of the cells treated with GSE, were observed using scanning electron microscopy (SEM). To identify the percentage of sub-lethally injured cells and dead cells, flow cytometry methods were carried out, staining with propidium iodide (PI) and bis-(1,3-dibutylbarbituric acid) trimethaine oxanol (BOX), respectively.

RESULTS

Treatment with GSE showed great antimicrobial activity against Gram-positive L. monocytogenes and Gram-negative E. coli,especially for lower initial inoculum levels (10⁵ CFU/ml) and for mid-exponential cultures phase cells. The microbial inactivation was substantially enhanced for higher GSE concentrations ,i.e., 4%. Analysis of the treated samples using flow cytometry showed significant differences on the ratio of dead and sub-lethally injured cells when the initial inoculum concentration, the GSE concentration and/or growth phase were changed, in alignment with the microbial inactivation kinetics.

CONCLUSIONS

This work shows that GSE have good potential as a natural antimicrobial strategy against food related bacterial pathogens. This insight to the microbial dynamics is essential for the development of novel and robust green/sustainable processing control strategies, i.e., application of GSE individually or coupled with other novel technologies in a hurdle approach.

Acknowledgements:

This work was supported by the Department of Chemical and Process Engineering of the University of Surrey, the Doctoral College of the University of Surrey and R-processing Limited. E.V. is grateful to the Royal Academy of Engineering, for an industrial Fellowship.

References:

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