Use of chitin and its derivatives obtained from sustainable sources as an alternative to the use of chemical additives in the food production

Winemaking is a complex process in which the management of microbiological control plays a crucial role in determining the quality of the final product. Traditionally, the addition of sulphur dioxide (SO2) has been the main method for ensuring the microbiological stabilisation of wine, due to its broad spectrum of action against unwanted microorganisms and its ability to prevent oxidative phenomena. However, in recent years, interest toward tools as alternatives to SO2 has grown significantly due to concerns over its negative health effects on consumers and its potential organoleptic impact on wine. Scientific research has focused its efforts on finding alternative methods to reduce or eliminate the use of SO2, while maintaining the microbiological stability of the wine. Among the potential solutions, the use of chitosan, derived from sustainable sources such as insects, could represent a promising innovation. This type of chitosan not only might assure the microbiological stabilization of the wine, but, as a consequence of its source, has also a reduced environmental impact, meeting the needs of an increasingly sustainability-conscious wine industry. This Industrial PhD thesis sets out to explore chemical methods for the microbiological control of winemaking, analysing both traditional and more innovative treatments, with a focus on chitosan derived from alternative sources, such as insects. Through a critical review of the available evidence, the efficacy, limitations and effects of these alternatives on wine quality are discussed, offering an integrated view of future prospects for more sustainable winemaking. This project was financed by the Basilicata Region (Italy), the activities were carried out in collaboration with the industrial partner of the project ‘Cantina di Venosa’ located in Venosa (Potenza, Italy), University Institute of Vine and Wine Jules Guyot - University of Burgundy, Dijon (France) and the Department of Biosciences, Biotechnology and Environment - University of Bari ‘Aldo Moro’ (Italy). The activities were carried out following a scale-up approach, which allowed the transition from laboratory experiments to industrial-scale studies. The preliminary study of the literature was essential in order to plan the research activities to follow during the project. Indeed, Chapter 1 provides an overview of the chemical methods used for microbiological control in winemaking. The traditional use of sulphur dioxide (SO2) as the main antimicrobial agent to prevent microbial spoilage and maintain wine quality is emphasised. However, the growing interest in alternatives to SO2 is also discussed, as it may have negative health effects. Alternative chemical compounds, such as sorbic acid, dimethyl dicarbonate (DMDC), lysozyme and chitosan are examined, focusing on the latter and alternative, sustainable sources for its supply, such as insects. It is concluded that although these treatments are effective, there is still no single compound that can completely replace SO2. A combination of methods might be the best solution to reduce the sulphite content in wine. The first laboratory activities, reported in Chapter 2, involved the selection and study of several non-Saccharomyces yeast strains, potentially present during wine fermentation, with a focus on their resistance to commercial and insect-based chitosan. The strains were tested under controlled conditions to determine the resistance level to chitosan, in comparison to SO2 tolerance. This characteristic was assayed by using different approaches, which were the test on liquid and agarized media, added with different concentrations of antimicrobials, and the influence of SO2 and chitosan addition during laboratory-scale fermentations, using different combinations of the antimicrobials. These activities showed that many non-Saccharomyces yeast strains have medium/high resistance to chitosan; good results were obtained by the combined use of chitosan and low SO2 levels (20 mg/L) during inoculated fermentations with Saccharomyces cerevisiae. This approach might be a promising tool for production of low-sulphite wines. Another research step was addressed to evaluate the antimicrobial activity of chitosan against Brettanomyces bruxellensis, a yeast known to cause sensory defects in wines. Furthermore, the use of fungal chitosan is the current approved application for the control of these wine-contaminating yeasts during barrel aging. The study reported in Chapter 3 was conducted in a synthetic medium, and the effectiveness of three types of chitosan (from crustacean, insect and fungi) was compared. It was shown that chitosan from crustaceans and chitosan from insects significantly reduced the microbial population, showing greater efficacy than the fungal chitosan permitted in oenology. The evaluation of the effect of different chitosan types by using the flow cytometry technique on B. bruxellensis cells, in comparison to S. cerevisiae, confirmed the effectiveness of this compound only against Brettanomyces yeasts. Furthermore, insect-based chitosan showed better antimicrobial activity compared to the fungal chitosan currently permitted in oenology. Finally, after confirming the effectiveness of chitosan at laboratory scale, the scale-up approach included experiments at higher volumes to test the effectiveness under conditions closer to real production. Chapter 4 reports the results obtained by transferring the lab-scale tests to real vinification conditions. These activities concerned the artificial contamination with a B. bruxellensis strain of wine produced by the ‘Cantina di Venosa’ cellar and added with the different types of chitosan tested in the previous studies. The scale-up approach adopted in this study allowed to test the effectiveness of chitosan as a natural antimicrobial in winemaking and wine ageing. The results suggest that chitosan, especially those obtained from sustainable sources such as insects, could be a promising alternative to SO₂ for both the production of low-sulphite wines and the protection of wine from contaminating yeasts during storage and ageing.