*4.6. Identification of Compounds in Wine* 4.6.1. Phenolics

Identification and quantification of the polyphenol values of the extracts were carried out using an ACQUITY Ultra Performance LC system, equipped with a photodiode array detector with a binary solvent manager (Waters Corporation, Milford, MA, USA) and a mass detector equipped with an electrospray ionization (ESI) source operating in negative and positive modes, as described by Oszmia´nski et al. [51]. Separation of the individual polyphenols was carried out using a UPLC BEH C18 column (1.7 m, 2.1 × 100 mm, Waters Corporation, Milford, MA, USA) at 30 ◦C. The samples (10 L each) were injected, and then the elution was completed in 15 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL/min The mobile phase consisted of solvent A (2.0% formic acid, *v*/*v*) and solvent B (100% acetonitrile). The program began with isocratic elution using 99% of solvent A (0–1 min), then a linear gradient was used until the 12-min point, lowering solvent A to 0%; from 12.5 to 13.5 min, the gradient returned to the initial composition (99% A), then it was maintained at a constant level to re-equilibrate the column. The analysis was carried out using full-scan, data-dependent MS scanning from *m*/*z* 100 to 1500. Leucine enkephalin was used as the reference compound at a concentration of 500 pg/L and at a flow rate of 2 L/min; the [M−H]<sup>−</sup> ion was detected at 554.2615 Da. The [M−H]<sup>−</sup> ion was detected during a 15-min analysis performed within ESI–MS accurate mass experiments, which were permanently introduced via the Lock Spray channel using a Hamilton pump. The lock mass correction was ± 1.00 for the mass window. The mass spectrometer was operated in negative- and positive-ion modes, set to the base peak intensity (BPI) chromatograms, and scaled to 12,400 counts per second (cps) (100%). The optimized MS conditions were as follows: capillary voltage of 2500 V; cone voltage of 30 V, source temperature of 100 ◦C; desolvation temperature of 300 ◦C; desolvation gas (nitrogen) flow rate of 300 L/h. Collision-induced fragmentation experiments were performed using argon as the collision gas, with voltage ramping cycles from 0.3 to 2 V. Characterization of the single components was carried out via the retention time and the accurate molecular masses. Each compound was optimized to its estimated molecular mass [M−H]−/[M+H]<sup>+</sup> in the negative and positive modes before and after fragmentation. The data obtained from UPLC–MS were subsequently entered into the MassLynx 4.0ChromaLynx Application Manager software. On the basis of these data, the software is able to scan different samples for the characterized substances. The runs were monitored at the wavelength for flavonol glycosides of 360 nm. The PDA spectra were measured over the wavelength range of 200–800 nm, in steps of 2 nm. The retention times and spectra were compared to those of the pure standard [51].

### 4.6.2. Glycerol

Analyses were performed using a Shimadzu NEXE-RA XR instrument (Kyoto, Japan) with an RF-20A refractometric detector. Separation was carried out on an Asahipak NH2P-50 250 × 4.6 mm Shodex column (Showa Denko Europe, Munich, Germany). For quantitative measurements, the standard curves prepared for the glycerol standard were used.

#### 4.6.3. Volatile Compound Analysis

Qualitative analysis of the volatile aromas of the two highest-scoring wood-aging wines was carried out using the gas chromatography-mass spectrometer (GC-SPME) technique, using a device equipped with a flame ionization detector (FID) and a DB-WAX capillary column, with helium as a carrier gas. The oven temperature was kept at 40 ◦C for 7 min, followed by an increase to 230 ◦C at a rate of 3 ◦C/min.

#### *4.7. Sensory Evaluation*

The wines were subjected to sensory evaluation. A group comprising 35 tasters evaluated the quality of the wine. Before starting the sensory evaluation of the wines, the testers were trained and informed about the purpose of the assessment. The people who

made the assessment were not professional testers. Wine samples (30 mL) were evaluated in 100 mL wine glasses. Color, aroma, flavor, acidity, and clarity were assessed on a scale of 1 to 10 (with 10 being the best score for a given characteristic). The arithmetic mean for each trait of wine quality was calculated on the basis of individual assessments and a chart was developed.

#### *4.8. Statistical Analysis*

All statistical analyses were performed using the Statistica 12.5 software (StatSoft Polska, Cracow, Poland). The data were then subjected to one-factor variance analysis (ANOVA). Mean comparisons were performed using Tukey's least significant difference (LSD) test; significance was set at *p* < 0.05.

#### **5. Conclusions**

Ionizing radiation effectively eliminated the yeast in the wine. The optimal dose was determined to be 2.5 kGy, resulting in a reduction of yeast population by over 90%, except for one strain. Importantly, the quality of the wine was not significantly reduced. Radiation applied at higher doses worsened the organoleptic and chemical properties of the wine. As the radiation dose increased, the yeast in the wine progressively and rapidly declined. The differences in yeast response are related to the extent of radiation exposure.

Among the yeast breeds studied, only the *S. cerevisiae* ES181 strain exhibited rela-tively higher resistance to radiation doses of 1 and 2.5 kGy. In contrast, the other yeast breeds were subject to reduction, starting from 1 kGy, although to varying degrees. Therefore, it is reasonable to conduct a pretest to determine the sensitivity of the yeasts being used in the different levels of reduction.

In general, it can be said that the breed of yeast used has a very strong influence on the quality of the wine. It is justifiable to use commercial yeast breeds to obtain standardquality wines. However, there are instances where the use of special yeast strains, such as *B. bruxellensis*, can be justified if the aim of vinification is to deliver or produce. *B. bruxellensis* strains rapidly fermented the sugars in the must, resulting in higher alcohol content. This extraction of alcohol contributes to the intensity of the red color in the wine, as it interacts with the anthocyanins.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28124867/s1, Table S1: The content of polyphenols [µg/mL].

**Author Contributions:** Conceptualization, M.B. and I.O.; methodology, M.B., B.J., I.O., S.L.-W., W.M. and U.G.; software, I.O. and M.B.; validation, M.B., I.O., S.L.-W., W.M. and U.G.; formal analysis, M.B. and I.O.; investigation, M.B. and I.O.; resources, M.B. and I.O.; data curation, M.B., I.O., S.L.-W., B.J., W.M. and U.G.; writing—original draft preparation, M.B., I.O., S.L.-W., B.J., W.M. and U.G.; writing—review and editing, M.B., I.O., B.J., S.L.-W.; supervision, I.O.; project administration, I.O.; funding acquisition, I.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by project No. 00020.DDD.6509.00056.2019.16 carried out under the Rural Development Programme—Action WSPÓŁPRACA M16.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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