Development and Quality Parameters of Alcoholic Beverages Produced by Mixing Tsipouro and Rose Water

Abstract

Nowadays, there is a global trend of consuming lower-alcohol beverages, while there is a market trend for consuming ready-to-drink products. The present study describes the development of new alcoholic beverages by the simple addition of rose water to the traditional marc spirit tsipouro. New beverages have lower alcohol content (30% v/v) than the mother tsipouro (40% v/v), exhibiting noteworthy antioxidant activity. Tsipouro–rose water beverages exhibited several aroma volatiles which originated from rose water, as determined by SPME GC-MS. Among them were the phenylethyl alcohol, eucalyptol, linalool, citronellol, geraniol, alpha-terpineol, which are known as rose water aroma compounds. The antioxidant activity of tsipouro–rose water beverages as estimated by the Folin, FRAP and DPPH assays appeared to be higher than the diluted tsipouro of the same alcohol content in a dose-dependent manner after mixing and after storage for 30 and 60 days. Preliminary organoleptic evaluation indicated that tsipouro–rose water products exhibit a rose-like aroma and were of acceptable organoleptic quality, especially that with a lower concentration of rose water.

1. Introduction

Distilled spirits have an alcohol content of 30–50% v/v and are produced by distillation from fermented products containing carbohydrates [1]. Grape marc distillates are traditional spirit drinks in all European Mediterranean countries and are recognized as an important part of their national identities [2].

Nowadays, the addition of various aromatic plants or seeds, such as anise, fennel, and saffron, among others, to grape marc distillates has been noticed, and in some cases the products have been commercialized. The main flavoring technique is maceration or infusion which involves soaking ingredients in the spirit to extract their flavor and color, while re-distillation is often used. The enriched products have special organoleptic characteristics and may have pro-health properties [3].

The alcoholic beverage “tsipouro”’ is a traditional, “strong” grape marc distillate produced from residues of the winemaking process, named pomace, either from white or red grapes that abide by the Regulation (EC) No 110/2008. Tsipouro is produced mainly in continental Greece, and its ethanol content range from 40 to 45% v/v. At the European level, four tsipouro distilled spirits have been registered as products of Protected Geographical Indication (PGI). These are the tsipouro of Thessaly, Macedonia, Tyrnavos, and Crete, known as “tsikoudia”. Additionally, at the national level, the tsipouro of Mouzaki, Epirus, and Naousa have also been registered as PGI products. The production process of tsipouro involves the fermentation of the grape marc, followed by two successive distillations and the collection of the second and final distillate. Then, this distillate is diluted with water to a final ethanol content of 40–45% v/v, and the traditional alcoholic drink “tsipouro” is obtained. The order of distillation of the various components depends on their vapor pressure and solubility in ethanol and water. Therefore, the first fraction (“head”) is mainly composed of alcohol-soluble components (e.g., acetaldehyde, ethyl acetate, etc.), which are responsible for strong and intense aromas. The second fraction (“heart”) consists of ethanol, as well as higher alcohols (e.g., propanol, butanol and hexanol). The composition of the last fraction (“tail”) is less volatile compounds, such as 2-phenyl-ethanol and ethyl lactate [4,5,6].

The flowering plant Rosa damascena (Rosaceae) is a significant ornamental and medicinal plant, which is also a source of fragrance. Apart from providing its fragrance, the rose oil shows nutritional, pharmacological, and industrial properties. Rose water is a liquid prepared by the hydrodistillation of fresh rose flowers, and it is also called “hydrosol”. Regardless of the production method, either traditional or industrial, rose water usually contains some rose oil. Rose water is used in cosmetic industries and as a drink and flavoring agent, while its therapeutic properties are also known. It exhibits antinociceptive properties, and it is used to cure eye and oral disorders by exhibiting antimicrobial activities. Its antioxidant and anti-inflammatory activities are helpful for skin care. Rose water is used for flavoring several food and drink products. For such uses, in the European Union it is allowed if some regulations are followed. The main volatiles of rose water are 2-phenylethanol, citronellol, geraniol, linalool, nerol, and other minor compounds such as rose oxides (cis-rose oxide and trans-rose oxide), a- and 2-damascenone, eugenol and methyl eugenol, and some alkanes and alkenes, such as nonadecane, nonadecene, eicosane, henicosane [7,8,9,10,11].

Nowadays, there is a global trend for consuming lower-alcohol beverages, since the main priorities of people are their health and well-being while they like to enjoy their social life [12]. Moreover, there is a market trend for using ready-to-drink products (RTDs), such as cocktails.

The aim of the present study was the development of new alcoholic beverages by the simple mixing of tsipouro and rose water with an alcohol content of 30% v/v exhibiting the volatiles and aroma characteristics of rose water and noteworthy antioxidant activity.

2. Materials and Methods

2.1. Materials

2.1.1. Samples

The distillate tsipouro was kindly donated by Zoinos winery, Zitsa, Ioannina, Greece (vintage 2021) and it came from the local grape cultivar Vitis vinifera var. Debina. It was packaged in transparent glass bottles of 200 mL. The rose water was kindly donated by Kitsos essential oils, Tzoumerka-Ioannina, Greece, and it came from the rose cultivar Rosa Damascena. It was packaged in non-transparent plastic bottles of 150 mL.

2.1.2. Reagents

Gallic acid (anhydrous) 97.5–102.5%, Methanol (MeOH), Folin–Ciocalteu’s Phenol Reagent 2N, H₂O₂ 30%, and K₂HPO₄. 3H₂O 99% were purchased from Merck (Darmstadt, Germany). Sulfuric acid H₂SO₄ 95–97%, Na₂CO₃, 2,2-Diphenyl-1-Picrylhydrazyl (DPPH), HCl puriss ≥ 37%, FeCl₃ 97%, 1,10-Phenanthroline monohydrate > 99.5%, and 4-Methyl-2-pentanol 99% were purchased from Sigma-Aldrich (St. Louis, MO, USA). KOH pellets and NaOH pellets 97% were from Mallinckrodt (Chemical Works, St. Louis, MO, USA). Ethanol (EtOH) 99,8% denaturated with IPA, MEK and Bitrex were purchased from PanReac AppliChem (Darmstadt, Germany). A total of 0.05 mol L⁻¹ I₂ Fixanal was from Honeywell Fluka (Charlotte, NC, USA). 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) ≥99.0% FW = 312.33 g/mol was from Fluka Analytical, part of Sigma Aldrich Co. LLC (Gillingham, UK). FeSO₄.7H₂O pure and Phenol red p.a. indicator were purchased from POCh S.A.Gliwice (Gliwice, Slaskie, Poland). The starch soluble analytical reagent was from Riedel-de Haen (Seelze, Germany). In all experimental procedures, the solvents used were of analytical grade.

2.2. Methodology

2.2.1. Procedure for Preparation of Tsipouro–Rose Water Products

For the preparation of 100 mL of tsipouro–rose water products of a lower alcoholic strength (30% v/v), the following procedures were followed. Diluted tsipouro with water (TW): 75 mL of tsipouro 40% v/v + 25 mL of deionized water were put in a flask followed by a simple shaken process. Tsipouro–Rose Water 1 (TR1): 75 mL of tsipouro 40% v/v + 10 mL of rose water + 15 mL of deionized water were put in a flask followed by a simple shaken process. Tsipouro–Rose Water 2 (TR2): 75 mL of tsipouro 40% v/v + 20 mL of rose water + 5 mL of deionized water were put in a flask followed by a simple shaken process. The procedure was repeated on demand.

In each case, 45 mL were put in transparent glass bottles of 50 mL capacity with special plastic cups that offer leakproof protection (Figure S1). Samples were analyzed promptly (t = 0 days), while bottles were kept in a temperature-controlled chamber (Velp Scientifica, Usmate Velate, Italy) at 20 °C and analyzed after 30 (t = 30 days) and 60 (t = 60 days) days. The ratios of tsipouro and rose water (the concentration of rose water used) were selected after sensory evaluation, while the antioxidant activity was also evaluated.

2.2.2. Methods of Analysis

Gross Composition of Tsipouro, Rose Water and Tsipouro–Rose Water Products

For the determination of alcohol strength by volume, the method described by the International Organization of Vine and Wine (OIV) was followed (Method OIV-MA-AS312-01B:R2019, 1, 2021) using an alcohol meter with a range of 0–100% v/v after distillation [13].

For the determination of both free and total sulfur dioxide, the method described by the International Organization of Vine and Wine [14] was followed (Method OIV-MA-AS323-04B) with some modifications. For free SO₂ determination, 25 mL of the sample and 2.5 mL of 3.18 mol L⁻¹ H₂SO₄ solution were placed in a 250 mL conical flask and titrated with I₂ 0.01 mol L⁻¹ using 1 mL of starch indicator, until the color of the solution became blue. The free sulfur was calculated as follows: free SO₂ (mg/L) = 12.8 x V, where V = the volume of 0.01 mol L⁻¹ I₂ solution consumed in mL. Correspondingly, for determining the total SO₂, 25 mL of the sample and 12.5 mL of 1 mol L⁻¹ KOH solution were placed in a 250 mL conical flask, the solution was left to stand for 15 min, and then, it was acidified with 5 mL of 3.18 mol L⁻ H₂SO₄ solution. It was titrated with I₂ 0.01 mol L⁻¹ using a starch indicator until the color of the solution turned blue, and the calculation of the total SO₂ content was performed as described above (OIV).

For the determination of total, fixed and volatile acidity, the methods described by the International Methods of Analysis of Spirituous Beverages of Vitivinicultural Origin were used (Method OIV-MA-BS-12: R2009) [15].

UV Spectrum Acquisition of Tsipouro and Rose Water

An amount of 1 mL of each sample was transferred into a quartz cuvette with an optical path of 1 cm using an automatic pipette, and the spectrum was taken. Zeroing of the instrument was carried out with ethanol 40% v/v or deionized DI water for tsipouro and rose water, respectively. For UV spectrum acquisition, a UV—1280 spectrophotometer (Shimatzu, Kyoto, Japan) was used.

Volatiles of Rose Water and Tsipouro–Rose Water Products

An amount of 980 μL of each sample was placed into a screw-capped glass vial of 4 mL capacity with a Teflon-rubber septum. All samples were kept at −20 °C until their analysis, when they defrosted at room temperature. Then, 20 μL of the internal standard solution (4-methyl-2-pentanol, 75 g L⁻¹) was added at a final concentration of 150 mg L⁻¹. Then, each sample was left for acclimatization at 40 °C for 10 min under magnetic stirring at 500 rpm. Then, a constant length of the fiber was exposed to the headspace for 20 min. For the extraction procedure, a Divinylbenzene/Carboxen/PDMS 50/30 µm (Supelco, Bellefonte, PA, USA) fiber was used. Before use, the fiber was submitted to a cleaning process. Moreover, the whole system of fiber and chromatogram was checked by running the program of analysis but without the sample, and the system was ready for analysis when a pure baseline was taken. A Gas Chromtograph Mass Spectrometer GCMS-QP2010 Ultra (Shimatzu, Kyoto, Japan) was used for the determination of volatiles.

Following extraction, the fiber transferred to the injection port of the chromatograph and the desorption of volatiles took place at 240 °C for 15 min using a 0.75 mm i.d. liner (Supelco, Bellefonte, PA, USA). The MS was operated in electron impact mode with the electron energy set at 70 eV. Mass range applied was 35–450 m/z. Source and transfer line temperatures were set at 200 and 260 °C, respectively. An Innowax fused-silica column (30 m × 0.32 mm, 0.5 µm film thickness) was used. The carrier gas was helium with a flow rate of 1.5 mL/min and an average velocity of 44.2 cm/s. The oven temperature was programmed from 40 °C for 6.5 min and then increased to 60, 155, 210 and 240 °C at rates of 2.0, 4.0, 6.0 and 20 °C/min, respectively. It was held at 240 °C for 5.5 min. Solvent delay was adjusted at 2.5–4.5/3.3 min.

Peak identification was performed by comparing mass spectra with those obtained from the Wiley 275 and NIST 98 libraries, and peaks with >80% similarity were retained. Semiquantitative data were expressed in mg/L (area of compound/area of internal standard) x concentration of internal standard). Peaks exhibiting an area less than 100,000 were not considered.

Antioxidant Activity of Tsipouro, Rose Water and Tsipouro–Rose Water Products

Antioxidant activity was determined using the Folin, FRAP and DPPH assays.

The assessment of antioxidant activity by the Folin–Ciocalteu assay was based on the method developed by Folin and Ciocalteu for proteins and used for total phenolics and other antioxidants [16,17,18]. The procedure used is as follows. In a 1.5 mL Eppendorf tube, 700 μL of deionized water, 100 μL of the sample and 50 μL of Folin–Ciocalteu reagent were added and vortexed. After exactly 1 min, 150 μL of aqueous 20% w/v sodium carbonate was added; the mixture was vortexed and allowed to stand at room temperature in the dark for 60 min. The absorbance was measured at 750 nm using an LLG-uniSPEC UV/VIS spectrophotometer (Meckenheim, Germany). In parallel, a blank was prepared using 100 μL of 40% v/v ethanol (or 30% v/v ethanol or deionized water) instead of the sample. Zeroing of the spectrophotometer was performed using the blank. Calibration curves were made using gallic acid as a standard in the suitable solvent (EtOH 40% for distillate tsipouro, EtOH 30% for products of tsipouro and deionized water for rose water). Results were expressed as mg gallic acid per liter. C = (A − 0.0141)/0.0104, R² = 0.9995 (EtOH 40% v/v); C = (A − 0.0073)/0.0105, R² = 0.9997 (EtOH 30% v/v); C = (A − 0.0067)/0.0104, R² = 0.9997 (deionized water).

The assessment of reducing (antioxidant) power by the FRAP assay was based on the method developed by Benzie and Strain [19] and adopted on food samples [20,21]. The procedure used is as follows. The sample (50 μL) was mixed with 50 μL of FeCl₃ solution (3 mmol L⁻¹ in 0.05 mol L⁻¹ HCl) in a 1.5 mL Eppendorf tube and incubated for 30 min in a water bath at 37 °C. Then, 900 μL of TPTZ solution (1 mmol L⁻¹ in 0.05 mol L⁻¹ HCl) was added, and the mixture was vortexed. After exactly 10 min, the absorbance was recorded at 620 nm using an LLG-uniSPEC UV/VIS spectrophotometer (Meckenheim, Germany). To measure the absorbance of each sample, the instrument was zeroed with the corresponding blank. The blank was prepared for each sample in the same way, but 0.05 mol L⁻¹ HCl solution was added instead of the FeCl₃ solution. The antioxidant activity by the Ferric Reducing Antioxidant Power was calculated from the calibration curves using gallic acid as a standard in the suitable solvent (EtOH 40% v/v for distillate tsipouro, EtOH 30% v/v for products of tsipouro and deionized water for rose water). Results were expressed as mg gallic acid per liter. C = (A + 0.0010)/0.0323, R² = 0.9991 (EtOH 40% v/v); C = (A − 0.0026)/0.0338, R² = 0.9990 (EtOH 30% v/v); C = (A − 0.0018)/0.0337, R² = 0.9991 (deionized water). In parallel, a control was prepared using 50 μL of 40% v/v ethanol (or 30% v/v ethanol or deionized water) instead of each sample or gallic acid. Values for each control were subtracted from the values of the corresponding samples.

The assessment of scavenging (antioxidant) activity is based on the method developed by Blois [22] and adopted on food samples [20,21]. The procedure used is as follows: 0.5 mL of DPPH solution (90 mg L⁻¹ in MeOH) and 0.8 mL of the sample were transferred into a 1 cm glass cuvette. The kinetics of the reaction were monitored at 515 nm for 20 min, starting at the moment of addition of the sample (t = 0). For the measurements, a UV—1280 spectrophotometer (Shimatzu, Kyoto, Japan) was used. The spectrophotometer was zeroed with a solution of 0.5 mL of methanol and 0.8 mL of ethanol 40% v/v (or 30% v/v or deionized water, respectively). For the control, 0.8 mL of 40% v/v ethanol (or 30% v/v ethanol or deionized water) was added to 0.5 mL of methanolic DPPH solution, instead of the sample. The % inhibition of the DPPH radical was calculated using the formula: % inhibition = [(Ac − As)/Ac] × 100, where Ac was the absorbance of the control sample and As was the absorbance of the sample at a certain time. The % initial scavenging rate of the DPPH radical was calculated by considering the absorbance at 1 min, while the absorbance at 18 min were used to calculate the % total scavenging.

2.2.3. Organoleptic Evaluation of Tsipouro–Rose Water Products

Preliminary organoleptic evaluation was carried out by two persons familiar with the organoleptic properties of tsipouro. In a well-ventilated area, 10 mL of each sample (TW, TR1, TR2) were transferred into a transparent glass and evaluated for aroma and taste. All samples of each trial, TW, TR1, TR2 of 0, 30, and 60 days-old, were evaluated.

2.2.4. Statistical Analysis

The whole experiment including the products’ preparation, their analyses and organoleptic evaluation was repeated three times (three independent batches).

Values reported are means of the three trials with standard deviations. The results were analyzed statistically with the use of SPSS Statistics version 28.0.1.0 (142) of the IBM Company. Differences between means were examined by ANOVA using Duncan’s test at a significant level of p < 0.05. When two groups were examined, the paired test was employed.

3. Results and Discussion

3.1. Basic Chemical Composition of Tsipouro, Rose Water and Tsipouro–Rose Water Products

Gross composition of the mother tsipouro used was as follows: alcoholic strength (% v/v): 40 ± 0, free sulfur dioxide (mg L⁻¹): 5.5 ± 0.8, total sulfur dioxide (mg L⁻¹): 57.0 ± 1.0, total acidity (mg L⁻¹ acetic acid): 160 ± 7, fixed acidity (mg L⁻¹ acetic acid): 20 ± 7, and volatile acidity (mg L⁻¹ acetic acid): 140 ± 7. Gross composition of the rose water was as follows: alcoholic strength (% v/v): 0 ± 0, free sulfur dioxide (mg L⁻¹): 0.0 ± 0.0, total sulfur dioxide (mg L⁻¹): 0.0 ± 0.0, total acidity (mg L⁻¹ acetic acid): 92 ± 7, fixed acidity (mg L⁻¹ acetic acid): 64 ± 7, and volatile acidity (mg L⁻¹ acetic acid): 22 ± 7.

In the UV spectrum of mother tsipouro (Figure 1), peaks were observed at approximately 280 nm, while in the UV spectrum of rose water (Figure 2), peaks were observed at approximately 280 nm and at 255–260 nm. Since it is known that at 280 nm the phenolic ring is absorbed and at about 255 nm the benzoic acids are absorbed [23,24], UV spectra indicate that the mother tsipouro and rose water that were used contained some phenolics.

Total, fixed and volatile acidity of the tsipouro–rose water products are presented in

Table 1. Acidity of tsipouro–rose water products.

	ΤW	ΤR1	ΤR2
Total acidity (mg L−1 acetic acid)	124 a ± 7	140 b ± 7	136 ab ± 7
Fixed acidity (mg L−1 acetic acid)	24 a ± 0	28 a ± 7	24 a ± 0
Volatile acidity (mg L−1 acetic acid)	100 a ± 7	116 b ± 7	112 ab ± 7

TW: diluted tsipouro, TR1: product of tsipouro with low concentration of rose water, TR2: product of tsipouro with high concentration of rose water. The results presented in the table are the means (n = 3) ± S.D. Superscripts a, b show the comparison among TW, TR1, TR2 for any acidity. Means that do not have a common superscript differ significantly.

.

The tsipouro–rose water with a high concentration of rose water exhibited statistically similar acidities with diluted tsipouro. The two tsipouro–rose water products also exhibited statistically similar acidities. The tsipouro–rose water product with a low concentration of rose water exhibited higher total acidity and volatile acidity while exhibiting a similar fixed acidity in comparison to the diluted tsipouro. All the above differences and similarities can be attributed to the acidities of the rose water along with the fact that deionized water quickly becomes slightly acidic since it absorbs atmospheric carbon dioxide. It should be noted that both tsipouro–rose water products exhibited statistically lower total and volatile acidities than the mother tsipouro, while demonstrating statistically similar fixed acidities, indicating the acceptance of tsipouro–rose water products regarding their acidities.

3.2. Volatiles of Rose Water and Tsipouro–Rose Water Products

The rose water and tsipouro–rose water products were analyzed by SPME-GC/MS, and the concentrations of their main volatiles are presented in

Table 2. Main volatiles of rose water and tsipouro–rose water products.

Volatile	RW	TW	TR1	TR2
Acetaldehyde	0.504 ± 0.078	1.1 a ± 0.5	1.4 a ± 0.17	1.6 a ± 0.18
Ethyl Acetate	0.139 ± 0.016	75 a ± 5	70 a ± 13	79 a ± 2
alpha-Pinene	n.d.	1.6 a ± 1.5	0.65 a ± 1.13	1.1 a ± 0.9
Isobutanol	n.d.	24.6 a ± 2.1	24.4 a ± 0.6	22.9 a ± 1.4
Isoamyl acetate	n.d.	10.5 a ± 1.3	10.4 a ± 1.2	13.0 a ± 4.9
beta.-Myrcene	10.552 ± 0.797	n.d.	n.d.	n.d.
Eucalyptol	45.238 ± 5.114	n.d.	6.2 b ± 1.6	11.6 c ± 1.8
Isoamyl alcohol	n.d.	386 a ± 4	378 a ± 19	372 a ± 6
Hexanoic acid, ethyl ester	n.d.	23 a ± 3	29 a ± 3	27 a ± 4
β-cis-ocimene	5.272 ± 0.712	n.d.	n.d.	n.d.
cis Rose oxide	12.283 ± 2.554	n.d.	n.d.	2.7 b ± 0.2
1-Hexanol	2.675 ± 0.577	18.9 a ± 0.5	20.4 a ± 1.8	18.7 a ± 2.0
Octanoic acid, ethyl ester	n.d.	98.2 a ± 1.7	105.4 a ± 10.2	92.6 a ± 2.2
Furfural	n.d.	9.2 a ± 1.1	10.3 a ± 0.8	8.8 a ± 2.4
Nerol oxide	1.712 ± 0.687	n.d.	n.d.	n.d.
Benzaldehyde	3.443 ± 0.297	n.d.	n.d.	n.d.
Linalool	433.287 ± 2.627	1.14 a ± 0.26	40.4 b ± 2.1	75 c ± 16
Terpinen-4-ol	18.437 ± 6.318	n.d.	1.8 b ± 0.7	3.5 c ± 1.0
Decanoic acid, ethyl ester	n.d.	32.5 a ± 2.1	33.7 a ± 2.7	29.0 a ± 1.1
Diethyl succinate	n.d.	2.8 a ± 0.3	2.8 a ± 1.0	2.5 a ± 0.4
alpha-terpineol	62.302 ± 6.703	n.d.	6.3 b ± 0.7	12.0 c ± 2.7
Citral	8.602 ± 2.557	n.d.	n.d.	n.d.
1, 1, 5-Trimethyl-1, 2-dihydronaphthalene	n.d.	1.6 a ± 1.7	2.11 a ± 0.27	1.0 a ± 1.8
α-citronellol	2.063 ± 1.412	n.d.	17.5 b ± 2.2	35 c ± 5
Nerol	2.554 ± 0.571	0.3 a ± 0.5	3.1 b ± 0.5	6.7 c ± 0.9
Dodecanoic acid, ethyl ester	0.784 ± 0.075	1.8 a ± 0.8	3.1 ab ± 1.1	3.9 b ± 1.0
Geraniol	140.686 ± 14.213	n.d.	6.6 b ± 0.9	14.2 c ± 2.3
Phenylethyl alcohol	80.997 ± 8.436	2.1 a ± 0.7	12.4 b ± 1.3	25.2 c ± 2.8
Methyleugenol	20.416 ± 3.026	n.d.	n.d.	0.9 b ± 0.1
Nerolidol	4.487 ± 1.152	n.d.	n.d.	n.d.
Eugenol	2.285 ± 0.249	n.d.	0.4 a ± 0.4	1.1 a ± 1.0
Hexadecanoic acid, ethyl ester	1.052 ± 0.216	2.4 a ± 0.5	3.2 a ± 1.1	3.86 a ± 0.25

n.d.= not detected. Values, mg L⁻¹, as 4-methyl-2-pentanol, are the means (n = 3) ± S.D. RW: rose water; TW: diluted tsipouro; TR1: product of tsipouro with low concentration of rose water; TR2: product of tsipouro with high concentration of rose water. Superscripts a, b, c show the comparison among TW, TR1, TR2 for any volatile. Means that do not have a common superscript differ significantly.

.

Rose water exhibited many/several volatile compounds. Among them, the main volatiles reported in the literature [7,8,9] for rose water includes phenylethyl ethanol, geraniol, citronellol, linalool, alpha-terpineol, and eucalyptol.

The volatiles of diluted tsipouro (TW) represent the volatiles of tsipouro but at lower concentrations due to its dilution. Among the volatiles of TW were phenylethanol, acetaldehyde, ethyl acetate, hexanoic and octanoic ethyl esters, and linalool. The main volatile compounds of tsipouro are higher alcohols (like 2-phenylethanol and amyl alcohols), esters (such as ethyl acetate), aldehydes (like acetaldehyde), and terpenes that contribute to tsipouro’s characteristic aroma and flavor [2,5,6].

TR1 and TR2 products exhibited volatiles detected only or mainly in TW, such as acetaldehyde, ethyl acetate, isoamyl acetate, isoamyl alcohol, and hexanoic and octanoic ethyl esters. Moreover, TR1 and TR2 products exhibited volatiles detected only or mainly in rose water such as phenylethanol, linalool, geraniol, citronellol, a-terpineol and eucalyptol. Based on that, it is logical to expect that the above volatiles of rose water origin would/will contribute to tsipouro–rose water products. These substances are mainly responsible for the fact that rose water products generally acquire an aromatic and pleasant character [7,8,9,25].

3.3. Determination of the Antioxidant Activity

The antioxidant activity of the mother tsipouro, rose water, and tsipouro–rose water products were evaluated using the Folin, FRAP-reducing power and DPPH-scavenging capacity assays. The antioxidant activity of the mother tsipouro and rose water as estimated by the Folin assay was 12.3 ± 0.1 and 18.8 ± 0.2 mg L⁻¹ as gallic acid, respectively.

The antioxidant activity of tsipouro–rose water products after mixing and after storage for 30 and 60 days is presented in

Table 3. Antioxidant activity—Folin–Ciocalteu method of tsipouro products, expressed as mg L⁻¹ gallic acid, during their storage at 20 °C.

Storage Time, Days	ΤW	ΤR1	TR2
0 days	9.9 aC ± 0.3	12.2 bC ± 0.2	15.7 cC ± 0.3
30 days	8.5 aB ± 0.4	11.2 bB ± 0.5	14.8 cB ± 0.3
60 days	7.3 aA ± 0.3	9.2 bA ± 0.3	12.7 cA ± 0.2

TW: diluted tsipouro, TR1: product of tsipouro with low concentration of rose water, TR2: product of tsipouro with high concentration of rose water. The results presented in the table are the means (n = 3) ± S.D. Superscripts a, b, c show the horizontal comparison, while the A, B, C express the vertical comparison. Means that do not have a common superscript differ significantly.

. At any time of testing, the tsipouro–rose water products exhibited higher antioxidant activity than the control of diluted tsipouro. Moreover, the antioxidant activity increased proportional to the concentration of the added rose water.

During storage, antioxidant activity decreased in both the control and the products of tsipouro–rose water. However, in all cases the order was the tsipouro–rose water product with a high concentration of rose water (TR2) > the tsipouro–water product with a low concentration of rose water > diluted tsipouro with water (TW).

Moreover, it should be noticed that after mixing the TR2 tsipouro–rose water product, it exhibited higher antioxidant activity than the mother tsipouro (40% v/v), 15.7 ± 0.3 versus 12.3 ± 0.1, while the TR1 tsipouro–rose water product was similar.

The reducing power of tsipouro and rose water as estimated by the FRAP assay was 6.9 ± 0.1 and 10.5 ± 0.3 mg L⁻¹ as gallic acid, respectively. In

Table 4. Antioxidant activity—FRAP Method of tsipouro products, expressed as mg L⁻¹ gallic acid, during their storage at 20 °C.

Storage Time, Days	ΤW	ΤR1	TR2
0 days	5.3 aC ± 0.2	6.3 bC ± 0.2	7.1 cB ± 0.1
30 days	4.8 aB ± 0.1	5.5 bB ± 0.0	6.9 cB ± 0.0
60 days	3.9 aA ± 0.3	4.7 bA ± 0.1	5.5 cA ± 0.0

TW: diluted tsipouro, TR1: product of tsipouro with low concentration of rose water, TR2: product of tsipouro with high concentration of rose water. The results are the means (n = 3) ± S.D. Superscripts a, b, c show the horizontal comparison, while the A, B, C express the vertical comparison. Means that do not have a common superscript differ significantly.

, the reducing power of the tsipouro–rose water products as estimated by the FRAP assay are presented. At any time of testing, the tsipouro–rose water products exhibited higher antioxidant activity than the control of diluted tsipouro. Moreover, the antioxidant activity increased proportional to the concentration of the added rose water.

During storage, antioxidant activity decreased in both the control and the product of tsipouro–rose water with a low concentration of rose water. However, the tsipouro–rose water product with a high concentration of rose water exhibited stable antioxidant activity after 30 days of storage. At any sampling time, the antioxidant activity followed the order of the tsipouro–water product with a high concentration of rose water (TR2) > the tsipouro–water product with a low concentration of rose water > the diluted tsipouro with water (TW). On the other hand, after mixing (t = 0), both of the tsipouro–rose water products exhibited similar antioxidant activity to that of the mother tsipouro (40% vol.).

The kinetics of the DPPH radical scavenging by tsipouro, rose water, and tsipouro–rose water products were followed. By looking at the curves, two times were selected to calculate the % scavenging the DPPH radical. Absorbances at 1 min (before the plateau) are used to calculate the % initial rate, and the absorbances at 18 min (in the plateau) are used to calculate the % total scavenging.

The kinetics of the DPPH radical scavenging by the tsipouro and rose water are given in Figures S1 and S2. Thus, for tsipouro 40% v/v, the % initial scavenging rate at 1 min was calculated to be 16.4 ± 0.8%, while the % total scavenging at 18 min was 21.2 ± 1.6%. The corresponding values for the rose water were 33.8 ± 1.4 and 47.6 ± 1.5%, respectively.

The kinetics of the DPPH radical scavenging by the tsipouro–rose water products after mixing (t- = 0 days) are given in Figure S3.

Table 5. Scavenging of the DPPH free radical by tsipouro–rose water products after mixing (t- = 0 days) and after storage for 30 and 60 days.

	Storage Time, Days	ΤW	ΤR1	TR2
% Initial rate of DPPH scavenging (1 min)	0 days	13.0 aC ± 0.6	24.0 bC ± 1.0	26.9 cC ± 0.9
	30 days	8.6 aB ± 1.0	20.7 bB ± 1.1	24.6 cB ± 1.3
	60 days	6.1 aA ± 0.6	16.2 bA ± 0.6	21.4 cA ± 0.7
% Total DPPH scavenging (18 min)	0 days	14.7 aB ± 0.4	34.6 bB ± 1.8	43.2 cA ± 1.8
	30 days	12.3 aA ± 1.3	31.6 bA ± 1.7	42.8 cA ± 2.1
	60 days	11.3 aA ± 1.5	28.7 bA ± 0.4	41.4 cA ± 0.4

TW: diluted tsipouro, TR1: product of tsipouro with low concentration of rose water, TR2: product of tsipouro with high concentration of rose water. The results presented in the table are the means (n = 3) ± S.D. Superscripts a, b, c show the horizontal comparison, while the A, B, C express the vertical comparison. Means that do not have a common superscript differ significantly.

presents the % initial rate and % total scavenging of the DPPH radical by the tsipouro–rose water products after mixing (t- = 0 days) and after storage for 30 and 60 days.

At any time of testing, the tsipouro–rose water products exhibited a higher scavenging capacity, both the % initial rate and % total scavenging, of DPPH radical than the control of diluted tsipouro. Moreover, the scavenging capacity increased proportional to the concentration of the added rose water.

During storage, % initial rate of scavenging decreased in both the control and the products of tsipouro–rose water. On the other hand, % total scavenging appeared to be stable in some cases of TW and TR1, while it was stable at any time of sampling in the case of TR2. At any sampling time, the scavenging capacity, % initial rate and % total scavenging, followed the order of the tsipouro–water product with a high concentration of rose water (TR2) > the tsipouro–water product with a low concentration of rose water > the diluted tsipouro (TW). Moreover, it should be noticed that after mixing both tsipouro–rose water products, they exhibited a higher % initial rate and % total scavenging than those of the mother tsipouro (40% vol.).

The Folin assay is often referred to as a method for determination of total phenolics, the FRAP assay for determination of reducing capacity, and the DPPH assay for determination of the scavenging capacity of the stable DPPH free radical. However, Folin, FRAP and DPPH assays are methods for the assessment of antioxidant activity [26]. The tsipouro (mother tsipouro 40% vol) exhibited some antioxidant activity as estimated by the above three methods. Limited antioxidant activity of tsipouro has been reported by others [22]. Differences in antioxidant activity among various tsipouro can be attributed to the antioxidant potential of grape varieties used and to the technology applied, especially the application of an aging period in wood barrels [4]. It has been reported that various distilled spirits (cognac, bourbon whisky, vodka, gin) exhibited high or zero antioxidant activity correlated with their phenol concentrations. Wood aging is recognized to be the main source of phenols leading to high antioxidant activities of the spirits [27]. The rose water exhibited some antioxidant activity as estimated by the above three assays. The antioxidant activity of rose water has been reported by others [9,10,28].

Results obtained by these three assays indicate that tsipouro–rose water products of 30% vol. alcohol exhibit a higher antioxidant activity than the control diluted tsipouro of the same alcohol content in a dose-dependent manner. Moreover, tsipouro–rose water products exhibited higher antioxidant activity during storage than the control diluted tsipouro. All of the above show the positive effect of rose water on antioxidant activity.

In addition, the tsipouro–rose water products exhibited similar or higher antioxidant activity compared to the mother tsipouro (40% v/v). This may be attributed to an effect of rose water and/or to alcohol–phenolics interactions that may modify antioxidant activity [29]. It is noticed that in all cases (diluted tsipouro and both tsipouro–rose water products), antioxidant activity decreased during storage time. This, under the experimental conditions used, may be attributed to oxidation of non-volatiles such as phenolics and volatiles such as terpenes. Oxidation phenomena and a decrease in antioxidant activity during the storage of similar products under similar conditions have been reported by others [30,31].

3.4. Organoleptic Evaluation

At t = 0 days, the day of fortification, the control diluted tsipouro was rated as a typical commercial tsipouro with a sense of milder intensity in both taste and aroma. The tsipouro–rose water with a low concentration of rose water exhibited a strong but pleasant rose aroma, with a slightly sweet taste. The tsipouro–rose water with a high concentration of rose water presented a rather strong rose flavor. The tsipouro–rose water with a low concentration of rose water was more acceptable by both testers.

At t = 30 days and t = 60 days, testers express descriptions similar with those at t = 0. It should be noted that a more thorough organoleptic examination is required. However, from the preliminary examination, it becomes clear that the addition of a certain amount of rose water to tsipouro contributes to the production of a more aromatic and pleasant beverage.

4. Conclusions

The present study describes the development of new alcoholic beverages by the simple addition of rose water to the traditional marc spirit tsipouro. New beverages have lower alcohol content (30% v/v) than the mother beverage (40% v/v), exhibit noteworthy antioxidant activity and several volatiles of rose water origin, and are of acceptable organoleptic quality. The present work can be extended by studying the stability of the new product during long-term preservation and by providing more information on their acceptability by consumers.