Chitosan in wine industry: identification of origin by a multidisciplinary approach and application in oenology

Aim: Chitosan is a promising antimicrobial agent, capable of providing control of a wide range of spoilage microorganisms. To guarantee the fugal origin of chitosan, the only authorized for oenological practices, a multidisciplinary approach based on the measurement of the stable isotope ratios, Fourier transform infrared spectrometry and thermogravimetric analysis was proposed. In addition, the activity of chitosan against food related microorganisms was evaluated by different experiments aimed to discriminate between chemical and physical action of chitosan vs. the microorganisms. Method: The measurement of the stable isotope ratios (SIR) of carbon δ13C, nitrogen δ15N, oxygen δ18O and hydrogen δ2H of 35 samples of chitosan and the data of maximum degradation temperatures (obtained by TGA) combined with those of the peak areas of amide I and NH2/Amide II (obtained by FTIR) were employed to discriminate chitosan different sources (fungal grown on different substrates vs crustacean). The antimicrobial activity was tested in static and stirred conditions, in a synthetic media, using type strains of most common technological or spoilage microorganism. Viability was evaluated by Petri plate counts. The activity of the soluble portion of chitosan was checked by inoculating microorganisms in the media after chitosan removal. Results: The Kruskal-Wallis test showed that δ13C and δ18O were the most significant parameters able to classified chitosan into three different groups (from fungus grown on C3 photosynthetic cycle plant substrate, from fungus on C4 substrate and from crustacean). HCA and PCA analysis based on TGA, FTIR and SIR data successfully distributed the tested samples into informative clusters. Tests of chitosan antimicrobial activity highlighted the different sensitivity of microorganisms to chitosans, allowing selective control of spoilage agents. However, yeast and bacteria involved in fermentation were damaged by chitosan, and the synthetic media treated with this molecule showed a less fermentative aptitude. Conclusion: A robust analytical strategy for the correct identification of chitosan samples from crustaceans or fungi was presented, based on the observation that diverse biosynthetic pathways during the formation of the chitin influenced the isotopic composition of chitosan. Results of toxicity tests suggest that chitosan is a promising tool in fermented beverage production, but an in-depth study of the biochemical interaction between chitosan and food microorganisms is necessary.