Impact of Sulfur Dioxide and Dimethyl Dicarbonate Treatment on the Quality of White Wines: A Scientific Evaluation

Abstract

The biochemistry and physiology of raw material, the metabolism of microorganisms, and the methods used for processing and storage can affect the stability of wines. Due to the antimicrobial action of sulfur dioxide and dimethyl dicarbonate, the aim of this study is to determine the optimal treatment protocol to maintain the physico–chemical and microbiological stability of white wines with high residual sugar. Thus, the present research focuses on analyzing the influence of both treatments, combined or separate, on 45 wine samples obtained from a blend of Muscat Ottonel and Fetească Regală grape varieties, where different doses of 6% aqueous SO₂ solution (40, 80, and 160 mg/L) and dimethyl bicarbonate (0, 100, and 200 mg/L) were used. In order to assess the ability of dimethyl dicarbonate to suppress microorganisms, varying concentrations of Brettanomyces bruxellensis and Schizosaccharomyces pombe yeasts were inoculated (0, 30, 100 CFU/mL wine). The results indicate that, while sulfur dioxide cannot be entirely substituted in wines, both treatments can effectively lower or inhibit the activity of spoilage microorganisms. For the wines’ physico–chemical and microbiological stability, the treatment that used the synergistic force of sulfur dioxide (160 mg/L) and dimethyl dicarbonate (200 mg/L wine) performed the best.

1. Introduction

The quality of wine is strongly affected by a variety of chemical and biological processes that occur within its complex system [1]. In order to produce a high-quality wine, a series of conditioning and stabilization procedures must be applied to preserve the product’s distinctive color, taste, and flavor [2]. Several chemical, physical, thermal or non-thermal procedures can be applied to assure microbiological stability [3]. To fulfill the demands of modern consumers, winemaking techniques and oenological products have been adapted to improve the physical, chemical, sensory, and microbiological parameters of wine [4]. Throughout history, SO₂ has been used for preserving various food products and beverages (such as fruit juices or wines), due to its anti-oxidazic [5,6,7] and antiseptic roles [8,9,10,11].

SO₂ efficacy is highly dependent on pH value and can benefit the sensory characteristics of food products [12,13,14]. On the other hand, as, lately, sulfur dioxide is linked to consumer allergic reactions, there is an increasing desire to find alternatives for the reduction of SO₂ quantities in wines [14,15]. Thermal and non-thermal processes such as high hydrostatic pressure (HHP), pulsed electric field (PEF), ultraviolet irradiation (UV-C), ultrasound (HPU), or chemical additives such as dimethyl dicarbonate, lysozyme, ascorbic acid, sodium hypochlorite (NaOCl), chitosan, and colloidal silver complex (CSC) have been tested to prevent spoilage caused by microorganisms or faulty biochemical processes during fermentation or storage [3,16]. However, SO₂, at present, cannot be fully removed from the wine-making process as the above-mentioned products and methods may not always be able to fully ensure protection from a microbiological perspective [17].

Dimethyl dicarbonate (C₄H₆O₅) is commonly used as an inhibitor of pathogenic microorganisms in beverages and was initially tested and used for the fruit juice industry [18]. Recent studies have attempted to use this product as defense against microbiological activity in wines [15,19,20] especially in wines with high levels of residual sugar (>5 g/L). The treatment was authorized within viticultural countries of the European Union (Regulation (EC) No. 643/2006) at a maximum dose of 200 mg/L [17]. Dimethyl dicarbonate (DMDC) is recognized as an inhibitor against harmful microorganisms such as Brettanomyces bruxellensis or other spoilage microorganisms when the treatment with sulfur dioxide is not recommended, as the microorganisms may enhance their resistance to treatment [21]. When added to wine, DMDC hydrolyzes to produce methanol and carbon dioxide, hence raising the methanol content [22]. Stafford et al. [23] confirm that 96 mg/L of methanol may be produced when using the maximum amount of dimethyl dicarbonate (200 mg/L).

The International Organisation of Vine and Wine (OIV), the World Health Organization (WHO), the Food and Agriculture Organization (FAO), and the Food Chemical Codex have established legislative guidelines and recommendations that must be followed when using antioxidants and antiseptics such as SO₂ and DMDC in beverage technology. According to Stoeckley et al. [24] and OIV regulations [25] for semidry, demi-sweet and sweet, or special red, white, and rosé wines, the maximum allowed threshold of SO₂ content is 300 mg/L. According to dimethyl dicarbonate suppliers [26], the typical dosage for wine is between 125 and a maximum of 200 mg/L. According to international regulations in force, it can only be utilized only before bottling [27].

Numerous yeast species play a crucial role in winemaking due to their impact on both the chemical and sensory composition of wine, contributing both beneficial and detrimental effects, and thus requiring careful attention [28]. A particular kind of osmophilic yeast called Schizosaccharomyces pombe [29] can thrive in low-water situations, which enables it to survive in beverages with high sugar concentrations [30]. It is helpful in fermentation since its main metabolic function is to convert malic acid into carbon dioxide and alcohol. In wines with residual sugars, the yeast is also known to lead to refermentations, which, in turn, aggravates the quality of the samples [31].

In oenology, Brettanomyces bruxellensis has been considered to be the one of the most harmful yeasts [32], being a significant challenge for winemakers. Numerous investigations have demonstrated the significant phenotypic diversity of Brettanomyces bruxellensis in terms of growth capacity [33], sugars metabolism [34], consumption of nitrogen sources, production of volatile phenols [35,36], sensitivity to pH, temperature, oxygen [37] and sulfur dioxide [38,39]. Certain chemicals, like volatile phenols, are known to be produced by Brettanomyces bruxellensis and are linked to disagreeable smells that are typically referred to as “barnyard” or “horse sweat” [39,40], also medicinal, smoky, spicy, wet wool, phenolic, and/or mousiness taint, which can decrease the sensory quality of the wines [36,41].

The purpose of this study is to monitor the influence of varying doses of SO₂ and DMDC on the quality of white wines. A blend from two local grape varieties (Muscat Ottonel and Fetească regală) with high residual sugar content was used, allowing the study of alternative methods of semi-sweet wine stabilization. The originality of this study stems from the fact that, to the best of the authors’ knowledge, no prior research has examined the application of wines produced from the aforementioned blend in conjunction with DMDC treatments. Consequently, this study advances the understanding of DMDC’s role in wine stability by addressing key factors such as yeast type, yeast concentration, treatment dosage, and the proportional combination of DMDC and SO₂. Additionally, while DMDC is frequently employed in juice production, its application in winemaking remains relatively unmapped. By integrating different SO₂ doses with DMDC under specific experimental conditions—including higher inoculum levels of pathogenic yeasts [14]—this study addresses a significant gap in the literature and explores DMDC potential in maintaining microbiological stability in semi-sweet wines.

The treatments were applied under different experimental conditions: three doses of SO₂ coupled with DMDC and various inoculums of pathogenic yeasts. In Europe, the product can be added to wines that have at least 5 g/L of sugar just before bottling. By demonstrating its practical application in semi-sweet wines with fermentable sugars, this study provides a valuable guideline for winemakers aiming to address microbial instability caused by pathogenic yeasts. Moreover, it highlights an alternative approach to combine SO₂ and DMDC, aligning with consumer preferences and regulatory requirements.

2. Materials and Methods

2.1. Reagents and Standards

In this study, volatile compounds acetaldehyde and methanol were utilized. The solutions were purchased from Merck KGaA (Darmstadt, Germany) and had a concentration of minimum 99.9%. Ethanol was used for the solubilization of compound standards; it was purchased from Merck KGaA (Darmstadt, Germany) and had 99.9% purity. Purified water was produced in house with a Thermo Scientific™ GenPure™UV-TOC system (Thermo Fisher Scientific Inc., Vienna, Austria) and used for the preparation of a 10% (v/v) solution of ethanol in water.

2.2. Grape Varieties and Winemaking

The experimental variants (V1–V45) presented in

Table 1. Experimental samples.

Variant	Yeast	CFU/mL	SO2 mL	DMDC mg/L	Variant	Yeast	CFU/mL	SO2 mL	DMDC mg/L	Variant	Yeast	SO2 mL	CFU/mL	DMDC mg/L
V1	Without yeast	0	40	0	V16	Without yeast	0	80	0	V31	Without yeast	160	0	0
V2				100	V17				100	V32				100
V3				200	V18				200	V33				200
V4	Brettanomyces bruxellensis	30		0	V19	Brettanomyces bruxellensis	30		0	V34	Brettanomyces bruxellensis		30	0
V5				100	V20				100	V35				100
V6				200	V21				200	V36				200
V7		100		0	V22		100		0	V37			100	0
V8				100	V23				100	V38				100
V9				200	V24				200	V39				200
V10	Schizzossacharomyces pombe	30		0	V25	Schizossacharomyces pombe	30		0	V40	Schizossacharomyces pombe		30	0
V11				100	V26				100	V41				100
V12				200	V27				200	V42				200
V13		100		0	V28		100		0	V43			100	0
V14				100	V29				100	V44				100
V15				200	V30				200	V45				200

V1, V2, V3, V16, V17, V18, V31, V32, V33—wine samples, without pathogen yeast inoculation, considered control for this research, DMDC—dimethyl dicarbonate, SO₂—sulfur dioxide 6%, CFU—colony forming units.

were obtained from Muscat Ottonel and Fetească Regală grapes, manually harvested at full ripeness from Iași-Copou vineyard (at 47°10′ north latitudes and 27°35′ east longitudes, Romania) and processed according to a general white wine technology. After harvest, destemming, crushing, and pressing the varieties together, the combined must was transferred to stainless steel tanks for the fermentation phase. The sugar concentration of the must was 264 g/L and titrable acidity was 5.72 g/L tartaric acid. Endozym^® Ice, a liquid enzyme product, was used in concentrations of 4 mL/hL must to maximize the extraction of varietal flavors and to clarify the must. Fermentation was conducted at 10 °C and lasted 14 days, using Saccharomyces yeast from Sodinal^®, as indicated on the package. The resulting wine registered a concentration of 26 g/L of residual sugars. The wine was subsequently inoculated with different concentrations of Brettanomyces bruxellensis strain H531511-2D1 and Schizosaccharomyces pombe strain H104 (30, 100 CFU/mL) supplied by Lanxess^® (Leverkusen, Germany). Following this procedure, the wine was separated into three aliquots and various concentrations of 6% aqueous solution of SO₂ (40, 80, and 160 mg/L) were added.

Wine samples were filtered via sterile filters with 0.2 µm porosity. Various concentrations of dimethyl dicarbonate (100 or 200 mg/L) were added, as can be seen in Table 1. The bottles were kept for 6 months in a temperature-controlled environment at 12 °C.

2.3. Standard Physico–Chemical Parameters

Physico–chemical characterization of wine samples was established in accordance with the analysis techniques indicated by International Organization of Vine and Wine (OIV) [25]. The following parameters were analyzed: alcoholic strength (% vol. alc.); total acidity (g/L tartaric acid), volatile acidity (g/L acetic acid), free and total SO₂ (mg/L) using titrimetric measurements; pH and density (instrumental measurements); residual sugar (g/L) using Luff–Schoorl assay. The wine samples and the analyses were evaluated in triplicate and standard deviations were calculated.

2.4. Microbiological Protocol

The microbiological experiment involved the following steps: preparing the nutrient media, inoculating the yeast strains in/on nutrient media, multiplying the yeast, adapting the yeast to the experiment’s wine concentration, reaching the ideal yeast concentration for inoculation into the wine, and finally performing a microscopic analysis of the wine. A counting chamber Neubauer and a Gerber colony reader (Funke Gerber ColonyStar) were used to determine the yeast concentration (CFU/mL) following standard methodology.

2.4.1. Experimental Protocol for Yeast Strains’ Growth Conditions

In accordance with the experimental protocol of Lanxess^® Deutschland (Berlin, Germany), the yeasts used in the experiments, Schizosaccharomyces pombe and Brettanomyces bruxellensis, were propagated through inoculation on solid media, MEA (malt extract agar) and YMA (yeast malt agar) from Scharlau (Scharlab S.L., Barcelona, Spain), as well as in liquid nutritive media, sterilized must, and malt extract.

Pour plating is required to ascertain the cell titer from the end wine culture. Consequently, we generated serial dilutions from the final colony and inoculated them onto either malt extract agar or nutritional agar. The plates were incubated at 28 °C until they exhibited characteristics indicative of optimal cell growth.

For the final culture, the cell titer must be determined by pour plating. Therefore, we derived serial dilutions from the final culture and, subsequently, inoculated them onto nutrient agar or malt extract agar. Plates were incubated at 28 °C until optimal cellular proliferation was observed [42]. The cell titer of the wine-adapted culture is determined by enumerating the colonies. Simultaneously, the final culture from the preceding phase was chilled to inhibit proliferation until the cells had proliferated on the agar plates. After determining the cell titer of the final culture, the ideal quantity necessary to achieve the requisite final colony titer for dimethyl dicarbonate (DMDC) tests was estimated.

2.4.2. Evaluation of Yeast Concentration

The concentration of yeast cells per milliliter of wine was determined using the dilution factor and a hemocytometer slide. Each species necessitated a yeast concentration of 30 CFU/mL and 100 CFU/mL, which was delivered in three aliquots to the examined wine.

2.5. Quantification of Volatile Compounds

The analysis was performed on a gas chromatograph Agilent 7890B from Agilent Technologies (Santa Clara, CA, USA) equipped with a split/splitless injector and coupled with a flame ionization detector (FID). The injection (1500 μL vapor phase) was performed in split mode (2:1) with injector temperature set at 250 °C using a GC sampler 80 with head-space technology. The carrier gas was hydrogen produced by a generator (Parker–Balston) and the initial hydrogen flow in the column was 1.6 mL/min. The capillary chromatographic column was a high polarity column (nitroterephthalic acid modified polyethylene glycol) with a length of 50 m, an internal diameter of 0.32 mm and a film thickness of 0.50 μm (Phenomenex Zebron FFAP GC). The oven temperature was set at 30 °C and maintained for 5 min, then raised at 2.3 °C/min to 80 °C for 9 min, and then at 25 °C/min to 250 °C and maintained for 3 min. The total run time was 45.5 min. The FID temperature was set to 250 °C. The wine sample (10 mL) was transferred into 20 mL-vial for HS. The vial was then agitated and incubated at 85 °C for 15 min. After incubation, the gas phase was transferred into the inlet of the GC, with a heated syringe at 87 °C as described by Macoviciuc et al. [43] and Cotea et al. [44].

2.6. Chromatic Measurements

Chromatic parameters were analyzed according to the OIV recommended [25] method using attributes of visual perception: chromaticity, tonality, clarity, brightness, and saturation, as was defined by Dumitriu et al. [45].

A SPECORD^® UV-VIS spectrophotometer, SPECORD^® PC 200/205/210/ 250 (Analytik Jena GmbH+Co. KG, Jena, Germany) was used to assess the chromatic properties. The color differences are specified by the CIELab system, which defines a homogeneous 3-dimensional space defined by colorimetric coordinates L*, a*, and b*. The vertical axis denoted by L* was measured from 0—totally opaque to 100—completely transparent. The parameters “+a*” red, “−a*” green, “+b*” yellow, and “−b” blue were registered.

2.7. Sensory Analyses

The experimental wines were subject to a sensory analysis which closely followed the guidelines provided by ISO3591:1997 [46], ISO8589:2010 [47] and the OIV legislation. Aromatic descriptors were generated based on the sensory experiences of the authors and the flavors present in Fetească Regală and Muscat Ottonel varieties during a distinct pre-tasting session that took place a day before the official evaluations.

The experimental samples were assessed by a professional panel of 20 tasters, 13 men and 7 women. The panel comprised winemakers, lab employees, and researchers. Panel members were trained prior to the sensory analysis stage, to evaluate wines with faults related to refermentations. The panel members classified the intensity (from 0-total absence to 5-overpowering) of aroma descriptors such as honey, white flowers, overripe fruits, peach, and exotic fruits, as was previously decided during the pre-tasting session. The tasting session began in the morning to ensure a better comprehension of the evaluated descriptions, and a wine temperature of 12 °C was used to evaluate the samples. The tasting session was carried out on three different days (15 samples per day) in order to reduce taste fatigue. Similarly, the same team of tasters analyzed the samples under the same conditions.

The samples were coded and stored in transparent white wine glasses. The tasting started with the samples with the lowest doses of SO₂ and DMDC, V1–V15, 40 mg/L SO₂ and 0, 100, 200 mg/L DMDC, followed by V16–V30, 80 mg/L SO₂ and 0, 100, 200 mg/L DMDC and ended with the samples obtained by using the highest treatment doses, 160 mg/L SO₂ and 0, 100, 200 mg/L DMDC. This allowed the tasters to evaluate the samples, unknowingly, from the most affected by the refermentation yeasts to the least spoiled.

The tasters were given information about the applied treatments and that the samples contained alcohol. A tasting sheet was generated using Google Forms. Each taster received a link for each sample. At the end, the data were automatically processed using the answers given by each taster, after which the results were interpreted on a heat map. The mean value of the sensory analysis results is presented as a key value on this study.

2.8. Statistical Analyses

With the help of XLStat (Luminevo, Denver, CO, USA), ANOVA test was conducted. The analysis of variance exposes the presence of significant differences regarding the physico–chemical and chromatic parameters (p < 0.05) between the samples. The analysis includes only a single main factor (variants) for data simplification and does not account for interactions. Additionally, the influence of dimethyl dicarbonate dosage on the main physico–chemical characteristics, chromatic parameters, methanol and acetaldehyde concentrations was made by linear regression analysis. Also, a heatmap was created to help with the sensory characteristics’ better visualization. In that sense, an online platform was used (http://www.heatmapper.ca/expression, accessed on 12 December 2024).

3. Results and Discussion

3.1. Physico–Chemical Parameters

Physico–chemical parameters were analyzed 6 months after bottling to detect the effects of dimethyl dicarbonate and sulfur dioxide treatments on the quality of the samples. The obtained results are presented in

Table 2. Physico–chemical characteristics of variants.

Variants	Alc. Strength % vol. alc.	Tot. Acidity g/L ac. Tartaric	Vol. Acidity g/L ac. Acetic	Res. Sugars g/L	Density	pH	Free SO2 mg/L	Tot. SO2 mg/L
40 mg/L SO2 With/Without DMDC
V1	14.20 ± 0.23 ab	5.80 ± 0.04 a	0.30 ± 0.13 a	20.70 ± 0.19 ghi	0.9971 ± 0.24 a	3.42 ± 0.07 a	10 ± 0.24 b	40 ± 0.05 b
V2	14.30 ± 0.04 abc	5.80 ± 0.16 a	0.27 ± 0.23 a	21.10 ± 0.13 ijk	0.9969 ± 0.05 a	3.40 ± 0.08 a	10 ± 0.15 b	40 ± 0.12 b
V3	14.30 ± 0.19 abc	5.82 ± 0.13 a	0.21 ± 0.19 a	21.60 ± 0.07 lmno	0.9972 ± 0.04 a	3.26 ± 0.16 a	11 ± 0.18 b	40 ± 0.15 b
V4	14.60 ± 0.07 abcde	6.20 ± 0.16 a	0.19 ± 0.24 a	22.00 ± 0.23 o	0.9975 ± 0.13 a	3.36 ± 0.04 a	10 ± 0.11 b	40 ± 0.18 b
V5	14.70 ± 0.03 bcdef	5.90 ± 0.07 a	0.21 ± 0.19 a	21.00 ± 0.23 jk	0.9972 ± 0.13 a	3.41 ± 0.16 a	10 ± 0.11 b	40 ± 0.05 b
V6	14.80 ± 0.18 cdef	6.10 ± 0.24 a	0.18 ± 0.04 a	20.50 ± 0.19 fgh	0.9970 ± 0.23 a	3.23 ± 0.13 a	11 ± 0.15 b	40 ± 0.19 b
V7	14.50 ± 0.24 abcd	5.90 ± 0.04 a	0.28 ± 0.07 a	21.80 ± 0.13 no	0.9970 ± 0.16 a	3.41 ± 0.23 a	10 ± 0.03 b	39 ± 0.03 a
V8	14.30 ± 0.05 abc	5.90 ± 0.04 a	0.26 ± 0.24 a	19.90 ± 0.16 cde	0.9972 ± 0.05 a	3.41 ± 0.01 a	10 ± 0.03 a	39 ± 0.11 a
V9	14.50 ± 0.12 abcd	5.90 ± 0.04 a	0.19 ± 0.23 a	21.30 ± 0.24 klm	0.9970 ± 0.13 a	3.24 ± 0.16 a	11 ± 0.11 b	40 ± 0.05 b
V10	15.00 ± 0.23 def	6.00 ± 0.13 a	0.21 ± 0.19 a	21.70 ± 0.01 mno	0.9967 ± 0.16 a	3.38 ± 0.24 a	10 ± 0.19 b	39 ± 0.13 a
V11	14.80 ± 0.08 cdef	6.12 ± 0.16 a	0.20 ± 0.13 a	21.20 ± 0.19 jkl	0.9972 ± 0.24 a	3.25 ± 0.04 a	11 ± 0.04 b	40 ± 0.00 b
V12	14.80 ± 0.29 cdef	6.43 ± 0.23 a	0.16 ± 0.13 a	19.20 ± 0.24 b	0.9972 ± 0.08 a	3.26 ± 0.04 a	11 ± 0.22 b	40 ± 0.05 b
V13	14.70 ± 0.16 bcdef	5.90 ± 0.19 a	0.28 ± 0.13 a	20.30 ± 0.04 efg	0.9967 ± 0.16 a	3.37 ± 0.24 a	10 ± 0.03 b	39 ± 0.19 a
V14	14.90 ± 0.04 def	6.12 ± 0.16 a	0.18 ± 0.13 a	21.70 ± 0.19 mno	0.9970 ± 0.23 a	3.21 ± 0.04 a	11 ± 0.05 b	39 ± 0.15 a
V15	14.80 ± 0.26 cdef	6.30 ± 0.16 a	0.18 ± 0.13 a	21.10 ± 0.24 ijk	0.9972 ± 0.04 a	3.23 ± 0.23 a	11 ± 0.11 b	40 ± 0.19 b
p-value (V1–V15)	<0.001 *	0.681	0.681	<0.001 *	1.000	0.550	0.122	1.000
80 mg/L SO2 With/Without DMDC
V16	15.20 ± 0.02 f	6.28 ± 0.09 a	0.17 ± 0.01 a	20.70 ± 0.19 ghi	0.9969 ± 0.08 a	3.25 ± 0.13 a	31 ± 0.24 c	79 ± 0.13 c
V17	15.20 ± 0.13 f	6.12 ± 0.01 a	0.18 ± 0.08 a	21.30 ± 0.02 klm	0.9968 ± 0.03 a	3.21 ± 0.08 a	31 ± 0.05 c	79 ± 0.05 c
V18	15.10 ± 0.01 ef	6.43 ± 0.02 a	0.20 ± 0.01 a	18.50 ± 0.19 a	0.9971 ± 0.11 a	3.26 ± 0.04 a	31 ± 0.07 c	79 ± 0.24 c
V19	15.10 ± 0.08 ef	6.12 ± 0.19 a	0.18 ± 0.07 a	21.00 ± 0.02 ijk	0.9971 ± 0.07 a	3.21 ± 0.15 a	31 ± 0.11 c	79 ± 0.11 c
V20	15.10 ± 0.11 ef	6.43 ± 0.08 a	0.16 ± 0.07 a	19.60 ± 0.01 bc	0.9971 ± 0.02 a	3.27 ± 0.19 a	31 ± 0.03 c	79 ± 0.08 c
V21	15.10 ± 0.21 ef	6.12 ± 0.19 a	0.20 ± 0.13 a	21.40 ± 0.01 klmn	0.9967 ± 0.03 a	3.21 ± 0.08 a	31 ± 0.19 c	79 ± 0.02 c
V22	15.10 ± 0.22 ef	6.12 ± 0.02 a	0.18 ± 0.08 a	21.60 ± 0.07 lmno	0.9973 ± 0.01 a	3.21 ± 0.08 a	31 ± 0.13 c	79 ± 0.15 c
V23	15.00 ± 0.26 def	6.12 ± 0.02 a	0.17 ± 0.03 a	21.20 ± 0.08 jkl	0.9970 ± 0.15 a	3.20 ± 0.07 a	31 ± 0.05 c	79 ± 0.12 c
V24	15.00 ± 0.06 def	6.28 ± 0.13 a	0.18 ± 0.01 a	21.90 ± 0.15 o	0.9975 ± 0.02 a	3.21 ± 0.03 a	31 ± 0.09 c	79 ± 0.03 c
V25	15.00 ± 0.11 def	6.43 ± 0.08 a	0.13 ± 0.02 a	20.10 ± 0.04 def	0.9971 ± 0.13 a	3.27 ± 0.07 a	31 ± 0.00 c	79 ± 0.03 c
V26	15.00 ± 0.17 def	6.12 ± 0.09 a	0.17 ± 0.01 a	20.80 ± 0.08 hij	0.9968 ± 0.04 a	3.22 ± 0.13 a	31 ± 0.05 c	79 ± 0.14 c
V27	15.00 ± 0.27 def	6.12 ± 0.13 a	0.13 ± 0.09 a	19.70 ± 0.03 cd	0.9965 ± 0.13 a	3.22 ± 0.01 a	31 ± 0.03 c	79 ± 0.05 c
V28	14.90 ± 0.12 def	6.12 ± 0.13 a	0.18 ± 0.02 a	21.30 ± 0.08 klm	0.9972 ± 0.07 a	3.22 ± 0.09 a	31 ± 0.19 c	79 ± 0.01 c
V29	14.90 ± 0.14 def	6.28 ± 0.08 a	0.17 ± 0.01 a	20.70 ± 0.13 ghi	0.9966 ± 0.04 a	3.22 ± 0.09 a	31 ± 0.15 c	79 ± 0.02 c
V30	14.80 ± 0.21 cdef	6.12 ± 0.13 a	0.19 ± 0.01 a	21.40 ± 0.19 klmn	0.9968 ± 0.08 a	3.21 ± 0.07 a	31 ± 0.11 c	79 ± 0.09 c
p-value (V16–V30)	0.972	0.129	0.129	<0.001 *	1.000	0.999	1.000	0.972
160 mg/L SO2 With/Without DMDC
V31	14.20 ± 0.13 ab	6.20 ± 0.01 a	0.27 ± 0.19 a	21.30 ± 0.02 klm	0.9953 ± 0.03 a	3.19 ± 0.08 a	95 ± 0.09 d	159 ± 0.28 d
V32	14.30 ± 0.02 abc	6.28 ± 0.01 a	0.27 ± 0.08 a	21.20 ± 0.09 jkl	0.9950 ± 0.04 a	3.19 ± 0.07 a	95 ± 0.23 d	159 ± 0.16 d
V33	14.20 ± 0.19 ab	6.20 ± 0.19 a	0.26 ± 0.03 a	21.30 ± 0.08 klm	0.9953 ± 0.13 a	3.18 ± 0.15 a	95 ± 0.01 d	159 ± 0.24 d
V34	14.20 ± 0.07 ab	6.40 ± 0.13 a	0.25 ± 0.01 a	21.30 ± 0.08 klm	0.9953 ± 0.01 a	3.18 ± 0.19 a	95 ± 0.00 d	159 ± 0.02 d
V35	14.20 ± 0.01 ab	6.28 ± 0.09 a	0.24 ± 0.08 a	21.30 ± 0.09 klm	0.9953 ± 0.07 a	3.18 ± 0.13 a	95 ± 0.28 d	159 ± 0.28 d
V36	14.20 ± 0.03 ab	6.43 ± 0.08 a	0.24 ± 0.02 a	21.30 ± 0.15 klm	0.9953 ± 0.09 a	3.22 ± 0.19 a	95 ± 0.23 d	159 ± 0.07 d
V37	14.10 ± 0.07 a	6.30 ± 0.13 a	0.24 ± 0.04 a	21.40 ± 0.02 klmn	0.9954 ± 0.09 a	3.19 ± 0.08 a	95 ± 0.09 d	159 ± 0.16 d
V38	14.20 ± 0.21 ab	6.20 ± 0.08 a	0.18 ± 0.04 a	21.30 ± 0.07 klm	0.9953 ± 0.13 a	3.18 ± 0.09 a	95 ± 0.03 d	159 ± 0.01 d
V39	14.20 ± 0.15 ab	6.28 ± 0.13 a	0.17 ± 0.09 a	21.30 ± 0.15 klm	0.9953 ± 0.08 a	3.18 ± 0.07 a	95 ± 0.24 d	159 ± 0.28 d
V40	14.20 ± 0.02 ab	6.28 ± 0.13 a	0.17 ± 0.08 a	21.30 ± 0.07 klm	0.9953 ± 0.03 a	3.20 ± 0.09 a	95 ± 0.02 d	159 ± 0.05 d
V41	14.10 ± 0.13 a	6.28 ± 0.15 a	0.16 ± 0.13 a	21.40 ± 0.11 klmn	0.9954 ± 0.19 a	3.17 ± 0.08 a	95 ± 0.07 d	159 ± 0.09 d
V42	14.10 ± 0.21 a	6.28 ± 0.07 a	0.16 ± 0.13 a	21.40 ± 0.15 klmn	0.9954 ± 0.08 a	3.17 ± 0.19 a	95 ± 0.02 d	159 ± 0.24 d
V43	14.10 ± 0.15 a	6.12 ± 0.08 a	0.16 ± 0.07 a	21.40 ± 0.02 klmn	0.9954 ± 0.04 a	3.18 ± 0.13 a	95 ± 0.03 d	159 ± 0.33 d
V44	14.10 ± 0.13 a	6.12 ± 0.04 a	0.16 ± 0.13 a	21.40 ± 0.07 klmn	0.9954 ± 0.15 a	3.18 ± 0.02 a	95 ± 0.07 d	159 ± 0.11 d
V45	14.10 ± 0.03 a	6.12 ± 0.19 a	0.16 ± 0.11 a	21.40 ± 0.09 klmn	0.9954 ± 0.01 a	3.18 ± 0.19 a	95 ± 0.16 d	159 ± 0.00 d
p-value (V31–V45)	0.722	<0.001 *	0.055	0.268	1.000	1.000	1.000	0.987
p-value (V1–V45)	<0.001 *	<0.001 *	0.006 *	<0.001 *	1.000	0.367	<0.001 *	0.722

The data were obtained in triplicate and represent the arithmetic mean of the results, including the standard deviation. The * highlights a statistically significant difference in the analyzed parameters, depending on the applied treatments. The superscript letters highlight the homogeneous groups for each individual physico–chemical parameter.

. When all samples were analyzed, significant differences (p < 0.05) were observed for the majority of parameters, except for density and pH. It is obvious that different doses of sulfur dioxide and dimethyl dicarbonate significantly affect the physical–chemical quality of the analyzed samples due to complementary actions manifested on wines (antioxidant effects, enzyme inhibition, and antimicrobial action).

The total acidity of grapes normally ranges from 5 to 16 g/L, while lower values of about 5 to 7 g/L of tartaric acid are observed in wine [48]. There are several factors that can influence this parameter, such as grape variety, ripeness, technological, storage conditions, and climatic conditions [49]. The total acidity of the analyzed samples ranged from 4.74 to 6.43 g/L of tartaric acid across the variants, following the limits established by the current legislation. In Figure 1, it can be observed that, for the samples treated with the lowest concentration of SO₂ (40 mg/L), the results are statistically significant. The total acidity fluctuated between 5.80 and 6.43 g/L, which might relate to its role in stabilizing wine. It can be concluded that the small concentrations of SO₂ are not enough to prevent chemical changes in semi-sweet wines. These variations can affect the wine’s perceived acidity. Volatile acidity refers to the total content of volatile acids removed from wine by steam distillation. Acetic acid accounts for 95–99% of the total volatile acidity, with the remainder consisting of small amounts of lactic acid, propionic acid, butyric acid, and formic acid. Volatile acidity is an important parameter in assessing the quality and health of wine [50]. The values recorded in this study ranged from 0.13 to 0.30 g/L acetic acid and were also within the legal limit of 1.08 g/L acetic acid in wine [51]. Yeast activity plays a key role in controlling volatile acidity in wine, while sulfur dioxide levels influence yeast performance and help regulate the production of volatile acids [52].

pH is an important parameter in winemaking and is closely related to wine stability. The ideal pH level in wines is between 3.2 and 3.6. In general, if the wine has a high acidity level, it will have a low pH. Wines with high acidity and low pH are considered stable; therefore, the growth of bacteria and other microorganisms is inhibited in this type of environment. SO₂ affects the concentration of volatile and fixed acids, which are important for the pH and overall quality of red and white wines [53]. According to Kodur, 2011 [54], wine quality is negatively affected by high pH (>3.8), which is associated with decreased color stability and undesirable flavors. On the contrary, in this study, the analyzed samples showed pH values ranging from 3.17 (V4, V42) to 3.47 (V15), all below 3.8. From a chemical perspective, this indicates that the wines are stable.

Alcoholic strength is considered one of winemaking’s most important parameters, as it represents the result of the fermentation of sugars in must, which can cause significant changes in wine quality [50]. The final alcohol content is affected by factors such as the initial sugar concentration, the yeast strain used, the fermentation temperature, and the yeast’s tolerance to alcohol [55]. The analyzed variants showed variations in values ranging from 14.3% vol. alc. to 15.2% vol. alc. in V1-V30, consistent with the initial sugar content of the raw materials used (264 g/L sugars in must). Yeast activity during fermentation is responsible for the variations in alcoholic strength in semi-sweet wines, especially when considering that the increasing sugar content of grape must be a result of climatic change [56]. Samples V1–V30 experienced fluctuations of alcoholic strength, likely due to the activity of yeast in semi-sweet wines. It seems that the treatment schemes administered (with 40, 80 mg/L SO₂ with or without DMDC) were not sufficient to ensure physico–chemical stability of the analyzed samples. In contrast, the variants V31-V45 were the most stable, the alcohol concentration values being similar 14.10–14.30% vol. alc. The interaction between SO₂ and dimethyl dicarbonate in this case favored the physico–chemical stability of wines. Also, it has been demonstrated by Nieto-Rojo et al., 2015 [56], that adding DMDC and SO₂ treatments enhances wine’s sensory profile while keeping alcohol levels within acceptable limits. According to Figure 1, SO₂ manifests a significantly negative influence on wine’s alcoholic strength, while dimethyl dicarbonate has no significant impact on the dependent variable.

DMDC and SO₂ both have an impact on fermentation depending on when they are added. SO₂ is frequently employed throughout the whole process, but in contrast, DMDC is usually applied at wine bottling [57]. Thus, the values obtained vary according to the treatments mentioned, the different doses administered, the chemical composition of the wines, and the activity of inoculated yeasts. The analyzed samples show a sugar concentration above 18.50 g/L (V18) in samples where the SO₂ and DMDC treatment was deficient and of 22.00 g/L (V4) in variants where the combined behavior of the used products led to the inactivation of yeasts. The biological instability of wines is closely related to the higher content of sugars that could lead to refermentations [49]. As observed in Figure 1d, SO₂ treatment has a significant effect on microbiological stability of wines with residual sugar, a pattern also observed in other research [40,42]. Dimethyl dicarbonate, despite its chemical use in beverages stabilization, does not show the same influence over residual sugar in this case. The same effect of the combined treatments is confirmed by the volatile acidity in Figure 1c, as the acetic acid concentration is kept under control and there is no microbial spoilage.

Free SO₂ can be consumed by bacteria, fungi, or wild yeasts, which decreases its effectiveness. By metabolizing SO₂ and transforming it into bound forms, microorganisms can lower the amount of active SO₂. Including both free and bound forms, this is the overall amount of SO₂ in the wine. It is measured in order to evaluate the wine’s total sulfur dioxide level and its potential for preservative action. The key balance in winemaking is to ensure there is enough free SO₂ to maintain the wine’s stability without excessive levels that could result in off flavors or undesirable effects in the wine. Total SO₂ can be higher in some wines, but if the free SO₂ is low, the wine may be more susceptible to spoilage and oxidation [28]. The SO₂ concentrations registered differences between the analyzed groups. For the group V1–V15 (40 mg/L SO₂), values of 10 or 11 mg/L of free SO₂ and 39 or 40 mg/L of total SO₂ were registered. For the group of samples treated with 80 mg/L SO₂ and dimethyl dicarbonate (V16–V30) the values were 31 mg/L for free SO₂ and 79 mg/L of total SO₂. For the third group, V31-V45, treated with 160 mg/L SO₂ the values were 95 mg/L of free SO₂ and 159 mg/L of total SO₂. It can be seen that there were minor fluctuations in the values for the first group (V1–V15), while no variations were observed in the other two groups, indicating that the samples were physico–chemically balanced. This aspect emphasizes that there were no major differences between the initial doses applied in the experiment and the values obtained from the analysis. According to Gallego et al. [58], Delfini et al. [59], and Lisanti et al. [60], it can be observed that the action of dimethyl dicarbonate and sulfur dioxide on chemical composition of wines offer stability and conservation.

In this study, the application of both treatments, sulfur dioxide and dimethyl dicarbonate was effective in physico–chemical wine stability. By contrast, the variants without dimethyl dicarbonate showed slight physico–chemical instability. The results indicate that 160 mg/L SO₂ in combination with dimethyl dicarbonate (100 and 200 mL) contribute to maintaining wine stability results found also by Costa et al. [61] and Divol et al. [62].

3.2. Chromatic Measurements

Wine color parameters are key factors in assessing wine quality being influenced by factors such as grape variety, pH, temperature, different oenological treatments applied pre- or post-fermentation, certain physico–chemical processes, and applied winemaking technology, etc. The ANOVA test results for the selected variables (L*, a*, b*) show significant differences (p < 0.05) between samples. It can be observed that SO₂, dimethyl dicarbonate concentrations and inoculated yeasts have a major impact on wine’s chromatic parameters.

From

Table 3. Colorimetric parameters of wine samples.

Samples	Luminosity L 0–100	Colorimetric Coordinates
		a Red (+)/Green (−)	b Yellow (+)/Blue (−)
40 mg SO2
V1	55.3 ± 0.03 a	8.86 ± 0.19 a	31.29 ± 0.01 zd
V2	58.5 ± 0.11 a	8.52 ± 0.07 a	31.81 ± 0.05 zf
V3	54.7 ± 0.13 a	9.15 ± 0.19 a	32.61 ± 0.09 zm
V4	63.5 ± 0.09 a	8.44 ± 0.18 a	32.40 ± 0.11 zj
V5	60.9 ± 0.20 a	8.74 ± 0.90 a	32.46 ± 0.01 zl
V6	64.2 ± 0.05 a	8.38 ± 0.19 a	31.98 ± 0.03 zh
V7	56.4 ± 0.16 a	9.27 ± 0.09 a	32.41 ± 0.11 zk
V8	54.8 ± 0.07 a	10.09 ± 0.20 a	33.34 ± 0.01 zo
V9	55.5 ± 0.18 a	9.28 ± 0.03 a	31.86 ± 0.07 zg
V10	59.8 ± 0.19 a	9.00 ± 0.11 a	32.82 ± 0.05 zn
V11	67.4 ± 0.01 a	7.95 ± 0.90 a	32.15 ± 0.18 zi
V12	60.2 ± 0.20 a	9.33 ± 0.19 a	30.65 ± 0.01 zb
V13	65.9 ± 0.22 a	7.47 ± 0.03 a	31.13 ± 0.07 zc
V14	61.9 ± 0.11 a	8.91 ± 0.20 a	31.38 ± 0.18 ze
V15	60.2 ± 0.03 a	8.93 ± 0.19 a	32.15 ± 0.09 zi
p-value (V1–V15)	0.000 *	0.000 *	0.000 *
80 mg SO2
V16	96.6 ± 0.25 b	1.17 ± 0.03 a	7.39 ± 0.18 w
V17	96.8 ± 0.09 b	1.08 ± 0.05 a	7.31 ± 0.20 u
V18	96.7 ± 0.11 b	1.12 ± 0.19 a	7.29 ± 0.07 s
V19	96.5 ± 0.07 b	1.29 ± 0.01 a	7.48 ± 0.09 za
V20	96.7 ± 0.03 b	1.09 ± 0.05 a	7.37 ± 0.18 v
V21	96.7 ± 0.22 b	1.13 ± 0.09 a	7.29 ± 0.20 s
V22	97.1 ± 0.16 b	0.65 ± 0.11 a	7.12 ± 0.07 n
V23	96.9 ± 0.19 b	0.88 ± 0.20 a	7.20 ± 0.01 p
V24	96.8 ± 0.05 b	0.99 ± 0.03 a	7.25 ± 0.18 q
V25	96.9 ± 0.01 b	0.96 ± 0.07 a	7.17 ± 0.11 o
V26	96.7 ± 0.18 b	1.15 ± 0.09 a	7.41 ± 0.19 y
V27	96.7 ± 0.11 b	1.15 ± 0.05 a	7.40 ± 0.16 x
V28	96.4 ± 0.09 b	1.00 ± 0.03 a	7.26 ± 0.18 r
V29	96.8 ± 0.20 b	0.97 ± 0.11 a	7.30 ± 0.01 t
V30	96.7 ± 0.03 b	0.98 ± 0.07 a	7.37 ± 0.16 v
p-value (V16–V30)	0.000 *	0.000 *	0.000 *
160 mg SO2
V31	98.2 ± 0.11 b	−0.25 ± 0.20 a	5.67 ± 0.01 g
V32	98.2 ± 0.19 b	−0.25 ± 0.18 a	5.64 ± 0.16 f
V33	98.2 ± 0.01 b	−0.24 ± 0.09 a	5.67 ± 0.03 g
V34	98.2 ± 0.20 b	−0.36 ± 0.11 a	5.79 ± 0.13 h
V35	98.2 ± 0.07 b	−0.34 ± 0.19 a	5.80 ± 0.01 i
V36	98.1 ± 0.09 b	−0.28 ± 0.18 a	5.85 ± 0.22 j
V37	98.4 ± 0.20 b	−0.40 ± 0.09 a	5.52 ± 0.03 b
V38	98.4 ± 0.19 b	−0.38 ± 0.05 a	5.56 ± 0.01 d
V39	98.3 ± 0.03 b	−0.22 ± 0.11 a	5.62 ± 0.16 e
V40	98.4 ± 0.20 b	−0.31 ± 0.07 a	5.54 ± 0.09 c
V41	98.2 ± 0.01 b	−0.29 ± 0.05 a	5.61 ± 0.01 a
V42	98.3 ± 0.09 b	−0.31 ± 0.03 a	5.51 ± 0.18 k
V43	98.2 ± 0.18 b	−0.22 ± 0.07 a	5.92 ± 0.01 m
V44	98.1 ± 0.19 b	−0.17 ± 0.09 a	5.97 ± 0.20 l
V45	98.1 ± 0.11 b	−0.16 ± 0.16 a	5.96 ± 0.05 l
p-value (V31–V45)	0.000 *	0.000 *	0.000 *
p-value (V1–V45)	0.000 *	0.000 *	0.000 *

The data were obtained in triplicate and represent the arithmetic mean of the results, including the standard deviation. The * highlights a statistically significant difference in the analyzed parameters, depending on the applied treatments. The superscript letters highlight the homogeneous groups for each individual chromatic parameter.

, it is observed that samples V1-V15 have a high degree of oxidation, with wine colors showing brownish shades, indicating that the treatment with 40 mg/L SO₂ was insufficient for long-term antioxidative protection. For the chromatic indicator L*, there is a considerable increase in value with higher concentrations of sulfur dioxide and dimethyl dicarbonate (from 60 with 40 mg/L SO₂ to 99 with 160 mg/L SO₂). In the case of the a* indicator, the values were much lower in samples treated with higher amounts of SO₂ (from 9.00 in the first group to −0.50 in the 160 mg/L SO₂ group) indicating good stability provided by SO₂ and dimethyl dicarbonate treatments. For the b* parameter, the average values decreased from 33.00 in the 40 mg/L SO₂ group to 5.00 in the 160 mg/L SO₂ group. Others samples showed high clarity values, such as 96.50 (V19) and 97.10 (V22) for samples treated with 80 mg/L SO₂ with and without dimethyl dicarbonate. Samples treated with 160 mg/L SO₂ with and without dimethyl dicarbonate, had similar values ranging from 98.00 to 98.60.

The results show a significant chromatic difference between samples treated with 40/80 mg/L SO₂ and dimethyl dicarbonate and those treated with 160 mg/L SO₂ and dimethyl dicarbonate. This supports the hypothesis that the administered treatments can contribute to wine color stabilization, as also confirmed by the findings of Santos et al. [17]. The sterilizing action of DMDC can be beneficial in preventing microbial spoilage, which could otherwise lead to wines color degradation. For example, wild yeast spoilage or bacterial infections could cause a wine to become cloudy or loose color intensity [63]. Thus, to prevent these microbial problems, treatment with DMDC helps preserve the clarity and aroma freshness of wine, indirectly supporting color stability, results that are also confirmed in this study.

The color parameters indicated that the highest dose of SO₂ (160 mg/L) and dimethyl dicarbonate provided the white wines with stability and antioxidative protection. Comparative findings were presented by Santos et al. [17] and Delfini et al. [59]. So, this treatment scheme is a good alternative for producing quality wines. From Figure 2, SO₂ has a strong and statistically significant effect on luminosity. These results suggest that higher levels of sulfur dioxide are associated with brighter wine samples. This could be due to its antioxidative effects, which may impact the optical properties of the wine.

Dimethyl dicarbonate, on the other hand, does not significantly influence luminosity, implying its role in the wine samples is unrelated to this characteristic. The results from linear regression suggest that higher levels of sulfur dioxide reduce the values of the red component of the CIELab system. This could be due to its role in preventing oxidation. Dimethyl dicarbonate does not have a significant influence on the red-green axis of wine color, indicating that its role does not affect this specific color parameter.

3.3. Microbiological Results

From a technological standpoint, the inactivation of microbes by treatments is a significant topic for oenologists in wine production and has been the focus of various studies. This study utilized Schizosaccharomyces pombe and Brettanomyces bruxellensis yeasts to demonstrate strain-specific resistance based on the treatments administered to the wines. In the samples treated with 40 mg SO₂/L, both with and without dimethyl dicarbonate (samples V1–V15), Schizosaccharomyces pombe and Brettanomyces bruxellensis were identified, irrespective of the dimethyl dicarbonate dosage applied.

In Figure 3 some microscopic aspects and measurements of the studied yeasts are presented.

Bacterial species from the lactic acid bacteria group exhibiting spherical (cocci) and cylindrical (bacilli) morphological features were identified. The wine displayed sediment at the bottle’s base, color alterations (oxidation), and noticeable CO₂ production due to pathogenic refermentation.

Yeast species introduced as inoculum were found in samples treated with 80 mg/L SO₂, both with 100 mg/L dimethyl dicarbonate and without it (samples V16–V30). The wine was transparent, although the presence of CO₂ was apparent. This outcome can be associated with the inoculum of Schizosaccharomyces and Brettanomyces yeasts, as the lower treatment levels are inadequate to guarantee the wines’ optimal shelf-life.

Conversely, in samples subjected to the maximum quantity of sulfur dioxide (160 mg/L) and any of the two doses of dimethyl dicarbonate, no yeasts or other microbes were detected. The wine had the characteristic hue of its originating types, manifesting as a transparent, straw-yellow liquid, with no evidence of refermentation observed. This resulted from the synergistic interaction between sulfur dioxide and dimethyl dicarbonate, validating their capacity to suppress microorganisms as indicated by Ough [18] and Divol et al. [62]. In samples with 160 mg/L SO₂ and no DMDC, some colonies of yeasts were identified in Petri plates. In this case, treatments with DMDC proved important for assuring microbiological stabilization even if sulfur dioxide was administered in high concentrations.

The data indicate that sulfur dioxide and dimethyl dicarbonate function synergistically and merit additional research to enhance the microbiological and antioxidative stability of wines (Figure 4), corroborating results reported by Costa et al. [61].

Nonetheless, it was shown that samples exposed to the lowest quantities of SO₂ (40 and 80 mg/L) failed to provide enough protection, as indicated by sedimentation, turbidity, and refermentation. In contrast, elevating the content of dimethyl dicarbonate enhanced the wine’s color and clarity, removing sediment and preventing refermentation. One of the main advantages of DMDC is its quick reactivity, which enables it to effectively inactivate yeast, thereby preventing interference with a possible refermentation process. DMDC is particularly effective when applied exactly before bottling, serving as a conservation agent for wine. Comparable findings were also reported by Costa et al. [61]. In his research, the optimal dose of dimethyl dicarbonate to prevent the growth of 500 CFU/mL inocula of Schizosaccharomyces pombe, Dekkera bruxellensis, Saccharomyces cerevisiae, and Pichia guilliermondii was determined to be 100 mg/L without SO₂ treatment.

Dimethyl dicarbonate is an efficient antimicrobial against a broad spectrum of microorganisms, particularly yeasts, although its efficacy is diminished against bacteria responsible for wine contamination, as corroborated by Divol et al. [62], Zhang et al., [63] and Renouf et al. [64]. To enhance its efficacy and reduce the necessity for elevated concentrations, the chemical is often utilized with other preservation techniques, such as sulfur dioxide (SO₂).

3.4. Volatile Compounds

It has been demonstrated that combining SO₂ with DMDC is beneficial to the wine volatile profile [20]. It is important to highlight whether the applied treatment schemes may or may not lead to appearance of undesirable aroma components and their various concentrations. In that sense, the main volatile compounds of interest for this study were acetaldehyde and methanol represented in Figure 5 and Figure 6.

Ethanal, or acetaldehyde, is a volatile carbonyl molecule that can be produced by wine oxidation or yeast activity during biological processes [65]. Increased levels of ethanal are also correlated with an increase in ethanol content, as observed in the case of samples V16–V30. Ethanol can be oxidized by acetic acid bacteria and yeasts to produce acetaldehyde. It is thought to be a by-product of alcoholic fermentation, even the amount produced by different yeasts species. Yeasts’ production of acetaldehyde can be affected by oxygen and SO₂. Acetaldehyde and SO₂ can react to create a reversible mixture. Acetaldehyde is usually reduced or removed as a result of this process, avoiding accumulation at undesired levels [66].

According to Osborne et al. [67], it plays a big part in the flavor, color, and stability of wine. In accordance with Liu et al. [68], the substance is unstable and can react with amino acids to form unwanted taste compounds. Smaller concentrations of acetaldehyde can result in fruity or nutty flavors and herbaceous (green grass) notes in freshly fermented wine [67]. However, excess acetaldehyde at concentrations above 125 mg/L causes changes, irritation, and unpleasant smells in wines [69,70].

Samples treated with 40 mg/L SO₂ and without dimethyl dicarbonate had acetaldehyde values ranging from 4.58 mg/L (V4) to 6.6 mg/L (V13). When treated with 40 mg/L SO₂ and 100 mg/L dimethyl dicarbonate, levels ranged from 4.37 mg/L (V2) to 12.27 mg/L (V14). Those preserved with 40 mg/L SO₂ and 200 mg/L dimethyl dicarbonate had values from 4.53 mg/L (V6) to 6.12 mg/L (V15).

Sulfur dioxide (SO₂) plays a vital role in preserving the freshness and flavor balance of wine by reducing acetaldehyde levels. However, excessive amounts of SO₂ can inhibit fermentation or lead to the appearance of undesirable flavors impacting the wine’s aromatic profile. By preventing the oxidation of phenolic compounds, SO₂ also helps maintain wine color, particularly in white wines, indirectly influencing acetaldehyde levels by inhibiting oxidative processes responsible for its formation [71]. While DMDC does not directly react with acetaldehyde, it can indirectly reduce its production by preventing microbial contamination. For example, also wild yeasts can increase acetaldehyde formation through oxidative reactions, potentially compromising wine quality. DMDC can effectively inhibit microbial development and thus spoilage, thereby reducing the conditions that promote acetaldehyde growth, as observed in the results [72].

Variants with 80 mg/L SO₂ and no dimethyl dicarbonate presented 18.49 mg/L (V25)–27.44 mg/L (V28) of acetaldehyde. When 100 mg/L of dimethyl dicarbonate were added, a range between 14.63 mg/L (V20) and 27.63 mg/L (V23) was observed. Samples treated with 200 mg/L dimethyl dicarbonate presented 13.01 mg/L (V21)–25.21 mg/L (V30) of acetaldehyde. Wines obtained after the addition of 160 mg/L SO₂ showed 7.48 mg/L (V34)–11.29 mg/L (V40) levels of the mentioned compound.

Wines treated with the same amount of SO₂ and also 100 mg/L of dimethyl dicarbonate recorded values ranging from 6.25 mg/L (V38) to 11.11 mg/L (V35) of ethanal. Those treated with the same amount of SO₂ but with 200 mg/L dimethyl dicarbonate had values ranging from 7.18 mg/L (V44) to 13.23 mg/L (V45). The samples treated with different concentrations of SO₂ (40, 80, or 160 mg/L) with or without dimethyl dicarbonate, showed the lowest acetaldehyde levels.

Preliminary results have been published by our research team from previous experiments using these treatments under the same conditions [73,74]. This indicates that the treatments did not significantly impact the sensory profiles of the wines.

Methyl pectinesterases, which naturally occur in fruit, break down pectins to produce methanol before and during alcoholic fermentation. Methanol production can increase during maceration, especially in red wines compared to rosé or white wines. The International Organization of Vine and Wine (OIV) sets strict limits on methanol levels in wines: <400 mg/L for red wines and <250 mg/L for white or rosé wines [25]. The amounts of methanol in wine depend on several factors, such as grape variety [6] and their health aspects, fermentation temperature, maceration conditions, and different treatments applied in the winemaking process [75]. In the analyzed samples, increased methanol content can be attributed to the hydrolysis of dimethyl dicarbonate (DMDC) into methanol and carbon dioxide. These findings were also presented by Ough et al. [18] and Stafford et al. [23].

Samples treated with 40 mg/L SO₂ and without dimethyl dicarbonate recorded the minimum methanol content (52.11 mg/L) in V7, and a maximum value (76.97 mg/L) in V13. On the other hand, samples treated with 100 mg/L dimethyl dicarbonate had the lowest value of 93.44 mg/L (V20) and the highest value of 121.09 mg/L (V14). Those treated with 200 mg/L dimethyl dicarbonate presented between 115.87 mg/L (V12) and 160.2 mg/L (V40) of methanol. For the treatment with 80 mg/L SO₂ and without dimethyl dicarbonate, the samples contained from 51.07 mg/L (V25) to 64.52 mg/L (V28) of methanol. The variants with the addition of 100 mg/L dimethyl dicarbonate contained values between 92.43 mg/L (V20) and 102.02 mg/L (V23).

Wines treated with 40 mg/L SO₂ and 200 mg/L dimethyl dicarbonate had methanol content ranging from 95.93 mg/L (V21) to 155.71 mg/L (V30). The administration of 160 mg/L SO₂ and 100 mg/L dimethyl dicarbonate produced 69.64 mg/L methanol (V44) and 100.95 mg/L (V41). In contrast, adding 200 mg/L dimethyl dicarbonate led to an increase in methanol content from 119.75 mg/L (V36) to 148.82 mg/L methanol (V39). An increasing trend in methanol concentrations due to the hydrolysis of dimethyl dicarbonate into methanol and carbon dioxide was also observed by other authors (Santos et al. [17] and Lisanti et al. [60]). However, the resulting concentrations are within the legislative limit for white wines, which is 250 mg/L methanol [25]. Therefore, the analyzed samples are balanced and not hazardous to consumer health. The additional methanol resulting from DMDC as a food additive used within regulations does not represent a significantly higher risk than the hazard from the methanol naturally produced and from its normal occurrence in foods [76,77].

To evaluate the impact of SO₂ and dimethyl dicarbonate doses, linear regression was applied (Figure 7). The results highlighted that 82% of the variability of the methanol and only 1% of the variability of the acetaldehyde (dependent variables) can be explained by SO₂ and DMDC doses (explanatory variables). As shown in Figure 7, the larger absolute values of standardized coefficients indicate a stronger effect of DMDC dose on methanol concentrations (r = 0.900). Acetaldehyde is not significantly affected by these treatments. This figure displays the standardized coefficient from a linear regression model predicting methanol and acetaldehyde concentrations in the analyzed wines. The coefficient indicates the change in these compound levels (in standard deviations) for a one standard deviation change in each predictor variable (SO₂ and dimethyl dicarbonate doses, and their interaction). Larger absolute values represent stronger effects, with positive coefficients indicating a direct relationship and negative coefficients indicating an inverse relationship. As shown in Figure 7a, dimethyl dicarbonate is a key variable that significantly influences the increasing concentration of the dependent variable (methanol). SO₂ has little to no effect, and its contribution may not be statistically meaningful. On the other hand, SO₂ levels manifest important impact on acetaldehyde formation (Figure 7b).

3.5. Sensory Analyses

The character of wine is primarily determined by its acidity, bitterness, or sweetness. These qualities are produced by non-volatile substances that dissolve in water or an alcohol blend. Sulfur dioxide is known to enhance the sensory quality of wine by maintaining the freshness and primary grape aromas while preventing wine “fatigue” caused by intense oxidation and aeration [78]. These changes are generally negative when the concentration is higher, leading to sensory characteristics that are perceived as less desirable by wine consumers. This effect has been evidenced in study realized by Salton and colleagues in 2000 [79] highlighting the potential drawbacks of SO₂ usage in some cases. However, wines with improved sensory qualities are produced when DMDC and SO₂ concentrations are combined [55]. Wines treated with the lowest amount of SO₂ (40 mg/L) with or without dimethyl dicarbonate, exhibited the most pronounced oxidative-related flavors such as over-ripe fruits, cooked fruits, jam, with the highest values of oxidation and refermentation parameters. The heatmap presented in Figure 8 illustrates the existing relationship between the various wine samples and the analyzed sensory descriptors, following a gradient color scheme. Wines within the same cluster manifest similar patterns of sensory attributes, highlighting how different treatments influence the wine styles. A higher intensity is observed for the descriptors (e.g., D6-grapefruit, D10-wild flowers) compared to the others. This suggests that these characteristics are representative of the majority of analyzed samples. Descriptors such as refermentation (D20) and oxidation (D21) show low values in most samples, indicating that these characteristics are either rare or undesirable in this type of wine.

The high intensity of descriptors D20 (refermentation) and D21 (oxidation) in a few samples (e.g., wines 41–45) could indicate microbiological stability issues in the production or storage process. The main characteristic that distinguishes Schizosaccharomyces pombe from other fermentative yeasts is its ability to metabolize malic acid into ethanol and CO₂, explaining the occurrence of refermentation in samples with this type of yeast, a result also confirmed by other authors [17], and by international regulations in force [27].

As the treatment concentrations increased to 80 mg/L SO₂ and dimethyl dicarbonate, the resulting wines were more balanced, with reduced oxidation and refermentation faults due to the stabilization action of the doses administered. The floral and fruity notes (tropical fruits, ripe fruits, honey, and wildflowers) of the dimethyl dicarbonate—treated samples were more expressive compared to the SO₂-only treated variants. The samples treated with the highest amount of SO₂ (160 mg/L) and dimethyl dicarbonate were characterized by a ‘clean’, elegant, balanced profile with good stability [59] confirm that dimethyl dicarbonate can partially replace SO₂ and is an effective conservation method against pathogenic microorganisms that are responsible for undesirable flavors in wines. Threlfall et al. [14] also confirmed that using dimethyl dicarbonate prevents the action of microorganisms and unpleasant odors or flavors in wine, especially when using the maximum dose of 200 mg/L, authorized by the International Wine and Vine Organization, a result also confirmed in this study. The results demonstrate dimethyl dicarbonate potential to improve and preserve wine quality, inhibit the growth of pathogenic microorganisms, and maintain the wine’s aroma profile, as was also demonstrated by Lisanti et al. [60].

4. Conclusions

In samples where sulfur dioxide was used without the added synergistic effect of dimethyl dicarbonate (DMDC), Schizosaccharomyces pombe and Brettanomyces bruxellensis produce significant changes in the chemical, microbiological, and sensory characteristics of the wines. However, treatments combining sulfur dioxide and DMDC at concentrations above 40 mg/L resulted in optimal stabilization. Methanol and acetaldehyde quantification confirmed that wines treated with DMDC meet the required standards for consumption. The interaction between sulfur dioxide and DMDC also aid in preserving sensory characteristics specific to the grape varieties, while balanced, refined, and elegant aromas are highlighted. This study presents a method for adjusting winemaking techniques to produce high-quality demi-sweet and sweet wines using various DMDC dosages. The results demonstrate that the combined treatment of sulfur dioxide and DMDC significantly increases both microbiological and oxidative stability, offering valuable insights for future oenological practices.

These preliminary findings offer valuable insights for ever-evolving winemaking technologies aimed at obtaining high quality wines with residual sugars. The study provides the wine industry with critical data for dealing with microbial development and the arising issues for ensuring wine quality, while the used methods aid in developing preventive strategies against pathogenic yeast species in winemaking. However, factors such as inconsistency in raw material, grape quality and structure (e.g., grape variety, ripeness degree, and climatic settings), as well as winemaking conditions (e.g., temperature during fermentation), influence the results. Future research should focus on evaluating the inhibitory efficacy of DMDC on pathogenic yeasts using different grape varieties and treatment doses, as well as assessing its impact on chemical composition and sensory aspect of wines. Additionally, regular microbiological stability during storage, such as periodic analyses every three months, should be integrated into future studies.