A Bacillus-based biofungicide agent prevents ochratoxins occurrence in grapes and impacts the volatile profile throughout the Chardonnay winemaking stages

Abstract

Bacillus-based biocontrol agents have emerged as a strategy to eliminate or reduce the use of synthetic fungicides that are detrimental to health and the environment. In vineyards, a special concern arises from the control of Aspergillus carbonarius, a fungus known for its potential to produce ochratoxins. Ochratoxin A (OTA) is the most toxic form among ochratoxins and its maximum limit in wine has been established in Europe and Brazil as 2 μg/kg. Wine quality, especially the volatile profile, may be influenced by the antifungal strategies, since fungicide residues are transferred from grapes to must during winemaking. The objective of this study was to evaluate, for the first time, the impact of a biocontrol strategy containing Bacillus velezensis P1 on the volatile profile and occurrence of ochratoxins when grapes infected with A. carbonarius were used in winemaking. The evaluation of ochratoxins was carried out by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS), and volatile compounds were analyzed using comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry (GC × GC/qMS). Six ochratoxins were identified in must prepared with Chardonnay grapes inoculated with A. carbonarius (ochratoxin α, ochratoxin β, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide and OTA). Although winemaking causes a decrease in the levels of all forms of ochratoxins, the co-occurrence of these mycotoxins was verified in wine made with grapes containing A. carbonarius. B. velezensis P1 prevented the occurrence of ochratoxins in must, ensuring the safety of wines. Regarding the volatile profile, a predominant presence of terpenic compounds was verified in samples treated with B. velezensis when compared with those not treated with the biocontrol strategy, whereas the presence of A. carbonarius resulted in a higher concentration of volatile compounds with an odor described as fatty/waxy, possibly compromising wine quality. Therefore, B. velezensis P1 is a new biofungicide possibility to produce ochratoxin-free grapes and high-quality wines.

Keywords: Aspergillus carbonarius; Bacillus velezensis; Biological control; GC × GC/MS; LC-QToF-MS; Ochratoxin A.