Worldwide, beer is a highly consumed beverage. Its production nowadays is carried out at industrial scale, but the consumption of craft beer is also extended (Piacentini, Savi, Olivo, & Scussel, 2015; Schabo, Freire, Sant'Ana, Schaffner, & Magnani, 2021). The beer production process, which consists of malting and brewing, involves the following raw materials: water, barley, hops, adjunct grains (maize, wheat, or rice), sugars or syrups, and yeasts (Pascari, Gil-Samarra, Marín, Ramos, & Sanchis, 2019). In the industrial malting process, after barley is accepted in the malt house, it is cleaned, graded, and stored. The malting process continues with the following stages: steeping, germination, and kilning. During the steeping stage, barley is immersed in aerated water in several steps followed by air-rests (Kunze, 2004; Schwarz, 2017). In this stage, grains take up water until moisture content raises up to approximately 42–47% (w.b.), depending on the malt type to be obtained (Pascari et al., 2019). Temperature will influence the time needed by the grain to reach a moisture content within those values (36–52 h at 20–12 °C) (Kunze, 2004; Wolf-Hall, 2007). Then, steeping water is drained, and grains are allowed to germinate in a high relative humidity atmosphere. The beginning of germination is indicated by the appearance of a small root (“chit”) in the base of each grain. Temperature at which germination takes place varies according to the malt type to be obtained, and may range from 12 to 25 °C (Kunze, 2004; Wolf-Hall, 2007). Germination of the grain leads to the increase of the enzymes needed for the hydrolysis of starch (of barley or adjuncts) into simpler sugars, which are required by yeasts for alcoholic fermentation in the brewing process (Schwarz & Li, 2010). This stage may last 4–5 days and is finished by drying the germinated grains to a moisture content of around 5% (w.b.) in the kilning stage. In the end of the malting process, kilned malt is cooled, rootlets are removed from germinated grains, and malt is stored (Kunze, 2004).

Mould contamination of barley and grains used as adjuncts implies a problem for the malting and brewing industries. Even though moulds belonging to the genera Aspergillus, Penicillium, Alternaria, and Rhizopus can be found in barley (Medina et al., 2006; Piacentini et al., 2015), infection of barley crops by Fusarium species is the most frequent and causes Fusarium head blight (FHB). FHB is produced principally by Fusarium graminearum, although other Fusarium species, such as F. culmorum, F. avenaceum, and F. poae, can cause symptoms. Main consequences of FHB are death of seedlings, loss of germination capacity of grains and the presence of mycotoxins (Lancova et al., 2008; Pascari, Marin, Ramos, & Sanchis, 2022; Piacentini et al., 2019). This will negatively affect the suitability of barley for malting, and also the quality parameters of malt (Schwarz, Horsley, Steffenson, Salas, & Barr, 2006). In addition, mould growth and mycotoxin production is possible during the malting stages of steeping, germination, and kilning, as environmental conditions of those stages are favourable (Jin et al., 2021; Schwarz, 2017). Consequences of Fusarium infection in barley on beer quality are the alteration of beer colour and flavour, and an increased propensity for gushing (Garbe, Schwarz, & Ehmer, 2009; Mastanjević, Mastanjević, & Krstanović, 2017), which is the explosive release of carbon dioxide, foam, and beer after the gentle opening of a bottle or can (Lusk, 2016).

All described problems due to mould occurrence in malting barley evidence the need of strategies for its reduction. Considering the characteristics of the steeping stage in the malting process, treatments with hot water, hydrogen peroxide, and ozone in aqueous phase (or their combination) have been studied for this purpose (Dodd et al., 2011; Kottapalli & Wolf-Hall, 2008; Kottapalli, Wolf-Hall, & Schwarz, 2005). In particular, ozone is an unstable gas with a strong oxidising potential that can directly oxidise compounds and also produce highly reactive free radicals (hydroxyl radicals) (Gottschalk, Libra, & Saupe, 2010). After being produced, ozone has a very short half-life and decomposes into oxygen, leaving no residues on the treated matrix. Antimicrobial action of ozone occurs by oxidative destruction of biomolecules, such as polyunsaturated fatty acids (oxidated to acid peroxides), sulfhydryl groups and amino acids of enzymes, proteins and peptides, which makes cellular membranes sensitive targets (Victorin, 1992). In 2001, ozone applied in gaseous and aqueous phases was approved by the FDA as an antimicrobial agent for the treatment, storage, and processing of food (U.S. Food and Drug Administration, 2001).

The aim of this study was to evaluate the effect of multi-step ozone treatments applied in aqueous phase on the reduction of F. graminearum incidence in artificially contaminated malting barley. Single-step treatments were proposed to select the conditions of multi-step treatments. The treatments were conceived for their application in the steeping step of the malting process, with the novelty of the use of a different proportion of water with respect to the amount of barley used in conventional steeping, and the proposal of shorter steeping periods. Mould growth in treated grains was also determined at temperatures at which germination can take place, and germination parameters (germinative capacity, germinative energy, and water sensitivity), as well as water uptake, were tested to evaluate the effect of ozone treatments on the grains' suitability for malting.