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In winemaking industry, good cleaning and sanitization practices are essential in bottle filling process to preserve quality and avoid subsequent alterations after bottling, when microbes find environment favourable for their development. Devices connected by pipelines, like wine bottling machines, are usually cleaned using Cleaning-in-Place (CIP) method, generally requiring a high consumption of water and the use of chemical cleaning detergents with a negative impact on the environment. Ozone has recently attracted attention due to its efficacy against a broad spectrum of microorganisms and its ability to clean leaving no residues on treated surfaces, protecting the environment and human health. This study aimed to investigate the impact of aqueous (3.5 mg/L for 15 and 30 min of contact time) and gaseous ozone (30 μL/L for 30 and 60 min of contact time) treatments in comparison with usual sanitizing treatment with peracetic acid (1% for 15 min of contact time) on six wine related microorganisms of oenological significance for their potential proliferation in the bottled wine. To this end, an artificially contaminated wine was used to fill rigid and flexible stainless-steel pipes and a bottling machine. The effectiveness of each treatment was evaluated using culture-dependent approach. The microorganisms showed different sensibilities to the treatments, dependent on the sanitization method used. The exposure to aqueous ozone for 30 min was the most effective treatment for pipes cleaning, followed by peracetic acid. On the other hand, when considering the bottling machine, the use of peracetic acid as sanitizing agent led to a complete removal of the cells, while aqueous ozone for a contact of 30 min was able to eliminate all microorganisms except S. cerevisiae.

The use of mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae is gaining attention in recent years due to their ability to modulate the metabolites production of enological interest. In the present study, four of the most popular planted red grape varieties (Cabernet sauvignon, Merlot, Pinot noir and Shiraz) were fermented using the aforementioned species and two different inoculation protocols (inoculation of S. cerevisiae after 24 and 48 h from the Starm. bacillaris inoculation), in order to evaluate their impact on the volatile composition and chromatic characteristics of wines. Analysis from chemical composition showed that titratable acidity and glycerol content exhibited marked differences among wines after fermentation. For volatile compounds, mixed fermented wines using an inoculation delay of 48 h led to reduction of volatile compounds (mainly esters). A shorter 24 h delay produced wines with higher values of color intensity than pure fermented wines. The differences observed between the inoculation protocols can be explained by the growth dynamics of both species during fermentation. These findings suggest that mixed fermentations posed a great potential in reducing metabolites which are considered negative for wine quality (mainly ethyl acetate and volatile fatty acids) and with an improvement of the chromatic profile of the wines.

In this study, we investigated the possible effect of electrolyzed water (EW), aqueous ozone (WO) and gaseous ozone (GO) on Brettanomyces bruxellensis DSM 7001 strain artificially inoculated on the grape surface and on its evolution during the subsequent, inoculated must fermentation. Culture-dependent and -independent techniques were used to evaluate the effectiveness of treatments against B. bruxellensis, as well as its presence during fermentation. Particularly, GO treatment of 24 h decreased its presence by about 2.1 Log, making it possible to reduce significantly the concentration of ethylphenols in the wine in relation to the control wine. EW and WO treatments caused less relevant reductions. The results showed that all the treatments reduced the presence of this yeast on grapes. However, in these experimental conditions it was not possible to achieve a complete removal of this undesirable yeast. Industrial relevance Brettanomyces spp. is considered a wine spoilage yeast due to its ability to produce off-flavors (described as Brett character) and high levels of acetic acid. Broad disinfectant action against microorganisms, eco-friendliness and easiness of on-site application are among the main advantages of the ozone and the electrolyzed water. This study demonstrated the antimicrobial potential of the EW, WO and GO treatments against B. bruxellensis inoculated on post-harvest grapes.

Starmerella bacillaris (synonym Candida zemplinina ) is a non- Saccharomyces yeast that has been proposed as a co-inoculant of selected Saccharomyces cerevisiae strains in mixed culture fermentations to enhance the analytical composition of the wines. In order to acquire further knowledge on the metabolic interactions between these two species, in this study we investigated the impact of oxygen addition and combination of Starm. bacillaris with S. cerevisiae strains on the microbial growth and metabolite production. Fermentations were carried out under two different conditions of oxygen availability. Oxygen availability and strain combination clearly influenced the population dynamics throughout the fermentation. Oxygen concentration increased the survival time of Starm. bacillaris and decreased the growth rate of S. cerevisiae strains in mixed culture fermentations, whereas it did not affect the growth of the latter in pure culture fermentations. This study reveals new knowledge about the influence of oxygen availability on the successional evolution of yeast species during wine fermentation.

Background and Aims The aim of this work was to assess and compare the chemical composition and colour characteristics of Barbera red wines obtained after partial alcohol reduction using three promising methodologies for implementation at the industrial level. Methods and Results Alcohol reduction was achieved by: (i) pre-fermentation addition of liquid derived from grape must (reverse osmosis by-product); (ii) mixed fermentations with strains of Starmerella bacillaris and Saccharomyces cerevisiae; and (iii) dealcoholisation of wine post-fermentation with a polypropylene membrane. The microbiological approach enabled the production of wines with a slightly lower alcohol concentration (−0.2 to −0.3% v/v), while facilitating the release of anthocyanin and some esters of fatty acids (ethyl hexanoate and ethyl dodecanoate) that could contribute positively to wine aroma with pleasant nuances. The low impact of the partial replacement of grape juice on the chemical composition and chromatic characteristics of Barbera wines makes this technique a good option for reducing the ethanol concentration by up to 1.0–2.0% v/v. In contrast, the use of a polypropylene membrane influenced negatively the composition of red wines by reducing significantly the concentration of esters (−60%) and anthocyanin (−17%), independently of the dealcoholisation level (up to −2% v/v). Conclusions The alcohol reduction strategies can greatly influence the volatile and phenolic composition of the wine. The choice of either a technological or microbiological approach in the wine industry is dependent on the alcohol reduction required. Significance of the Study This is the first comparative study of three strategies to reduce the alcohol concentration on the same batch of must/wine.

Recently the use of ozone as sanitizing agent has been proposed on winegrapes in order to control mycobiota after harvest. The aim of this work was to investigate possible indirect physico-chemical effects of ozone treatment on berry skin phenolic composition and extractability. Vitis vinifera L. cv Nebbiolo and Barbera, chosen for their different anthocyanin profiles, were post-harvest treated for 24 and 72 h with gaseous ozone (30 μL/L). Skin anthocyanin and flavanol extractability was assessed during maceration (6, 24, 48, 96, 168 and 240 h) using a wine-like solution. In our experimental conditions, ozone did not affect significantly the final extraction yield of anthocyanins (TA), proanthocyanidins (PRO), and flavanols reactive to vanillin (FRV) in Barbera, although TA and FRV extractabilities were higher in control samples than in ozone-treated samples during the first stages of maceration. In Nebbiolo, the final TA extraction yield was positively influenced by the ozone treatment (68.6, 64.2, and 59.9% for 24 h ozone-treated berries, 72 h ozone-treated berries and control samples, respectively). Final PRO and FRV extractability also increased in both ozone-treated samples compared to the control (+ 8.6–9.1% for PRO and + 7.3–11.7% for FRV). No significant differences were found among treatments for individual anthocyanins in both cultivars at the end of maceration. Therefore, the use of ozone as sanitizing agent in red varieties prior to winemaking process can be considered because it did not negatively affect the extractability of skin anthocyanins and flavanols.

Starmerella bacillaris has been proposed as a non-Saccharomyces species candidate to be used in mixed fermentations with Saccharomyces cerevisiae for the production of wine. Among the prospective applications, reductions in ethanol content and the acetic acid produced from high sugar musts have attracted particular attention. In this study, we described the fermentation behavior of six strains of S. bacillaris in grape must with initial sugar concentration ranging from 200 to 330 g/L. Time (days of fermentation) was a second variable that was monitored for its influence on fermentation. Response surface methodology was employed to model the behavior of the strains. The six strains generally behaved uniformly. Residual sugar concentration and production of ethanol, glycerol, and acetic acid mainly depended on time. Initial sugar concentration in the must was positively associated with residual glucose in musts with higher initial sugar concentration. Similarly, malic acid consumption showed a dependence on time and sugar concentration and was inhibited in musts with a higher sugar content. The behavior of S. bacillaris strains can be considered compatible with enological practices that may involve mixed fermentation with S. cerevisiae.

Decreasing the ethanol content in wine is a current challenge, mainly due to the global climate change and to the consumer preference for wines from grapes with increased maturity. In this study, a central composite design (CCD) and response surface methodology (RSM) approach was used to investigate the potential application of Starmerella bacillaris (synonym Candida zemplinina) in combination with Saccharomyces cerevisiae, in mixed (co-inoculated and sequential) cultures, to understand better the mechanism of co-habitation and achieve the objective of reducing the ethanol in wines. Laboratory scale fermentations demonstrated a decrease up to 0.7 % (v/v) of ethanol and an increase of about 4.2 g/L of glycerol when S. cerevisiae was inoculated with a delay of 48 h with respect to the inoculation of S. bacillaris. Pilot-scale fermentations, carried out in winemaking conditions, confirmed the laboratory results. This study demonstrates that the combination of strains and inoculation protocol could help to reduce the ethanol content in wines.

The effects of continuous exposure to ozone gas (O3, 30 μL/L, 24 h) and immersion in electrolyzed water (EW, 400 mg/L free chlorine, 10 min) on skin hardness of Italia and Muscat Hamburg table grapes, and Merlot and Barbera wine grapes were investigated and compared with those of control treatments (air and deionized water). Skin hardness was instrumentally evaluated via measurements of skin break energy using a puncture test. For all cultivars and density classes, ozone treatment was more effective in increasing skin hardness than EW treatment, although the differences were not always significant. For the same treatment, skin hardening was independent of berry density, and the magnitude of this effect was cultivar-dependent. This skin hardening might positively affect the postharvest life of table grapes. In wine grapes, increases in skin break energy values were related to higher yields and slow extraction kinetics of phenolic compounds during maceration. Practical Applications The purpose and the originality of this work were to determine the real impact of continuous exposure to ozone gas and immersion in electrolyzed water on skin hardness in table grape and wine grape varieties. Differences in the treatment effect due to the grape maturity level were studied in berries sorted according to density. For all cultivars and density classes evaluated, ozone treatment was more effective for increasing skin hardness than electrolyzed water treatment although the differences were not always significant. Therefore, the use of ozone gas as alternative sanitizing agent could extend the postharvest life of table grapes and facilitate the extraction of phenolic compounds from wine grapes during maceration according to increased skin hardness.

Mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae affect the chemical composition of wines, by modulating various metabolites of oenological interest. The current study was carried out to elucidate the effect of sequential inoculation of the above mentioned species on the production of white wines, especially on the chemical and aromatic characteristics of Chardonnay, Muscat, Riesling and Sauvignon blanc wines. Titratable acidity and glycerol content exhibited evident differences among the wines after fermentation. For volatile compounds, mixed fermentations led to a reduction of the total esters, including ethyl acetate, which is a compound responsible for wine deterioration. However, Sauvignon blanc wines fermented by mixed cultures contained significantly higher levels of esters and thiols, both associated with positive sensory attributes. These findings suggest that sequential inoculations possess great potential in affecting and modulating the chemical and aromatic profile of white wines, especially those produced from Sauvignon blanc grapes.

Ozone represents a potent antimicrobial compound that is already proposed as a possible sanitizing agent, especially for surface decontamination of fruits and vegetables. The main objective of this study was to evaluate the effect of ozone, either in aqueous or gaseous form, on wine grape mycobiota and its impact during spontaneous and inoculated fermentations. Gaseous (32±1μL/L, 12 and 24h) and aqueous (5±0.25mg/L, 6 and 12min) ozone were tested as sanitizing treatments. A multiphasic approach was used employing culture-dependent (traditional plate counts) and -independent techniques, based on DNA and RNA amplification (PCR-denaturing gradient gel electrophoresis [DGGE] and reverse transcription PCR [RT-PCR]-DGGE), respectively. Microbiological analysis data highlighted a reduction of >0.5LogCFU/mL of the total yeasts present on grape berry surfaces after ozone treatments, mainly due to the reduction of apiculate yeasts. The chemical analysis of the wines, produced from the treated grapes, showed higher acetic acid content in the untreated spontaneous fermentations (0.52g/L) compared to the treated (ranged from 0.16 to 0.38g/L), while all fermentation-inoculated wines contained higher amounts of pleasant volatile compounds.

Electrolyzed water (EW) has recently attracted much attention due to its efficacy against a broad spectrum of microorganisms. In this study, we investigated the impact of two EW treatments (40 and 400 mg/L free chlorine) on grape mycobiota using culture-dependent and -independent approaches. Moreover, the effect of yeast inoculation on treated and non-treated grapes was also considered. At the end of the fermentation, the wines produced were subjected to chemical and aroma analyses. The results revealed a decrease of about 0.5 log CFU/mL of the total yeast population on grapes surface independently of the dose of EW applied. Yeast inoculation and EW treatments shortened the time needed by Saccharomyces cerevisiae to dominate apiculate yeasts, particularly, 2 days for inoculated and 7 days for spontaneous fermentations. A decrease of acetic acid (about 55%) was also observed compared to untreated spontaneous fermentation. In addition, aroma analysis highlighted a positive contribution of inoculated yeast on the wine aromas, since they had approximately 50 % higher pleasant esters compared to spontaneous fermented wines. Industrial Relevance: Sulfur dioxide is widely used in crushed grapes prior to fermentation due to its antimicrobial and antioxidant activity. However, legislative rules, health risks and negative consumer perception related to its presence and use have resulted in a need to find new sanitizers able to reduce its use. The effectiveness of EW to reduce yeast species able to produce high levels of undesirable compounds was demonstrated. This research introduced an innovative antimicrobial agent, which could assist in the first step of wine production to reduce the use of SO2.