Aromatic compounds and organoleptic features of fermented wolfberry wine: Effects of maceration time

ABSTRACT

This study investigated the effect of maceration time on the physicochemical properties and aromatic composition of fermented wolfberry wine. Results showed that middle-time maceration (MM) wine showed higher level on physicochemical features. Esters contributed the most to the overall aroma of wolfberry wine, and its highest level was observed in MM wine. Difference maceration time altered the composition of acids, alcohols, aldehydes, and ketones in wine. Maceration did not alter the level of methionol, whereas MM caused a highest level of 4-vinylguaiacol in wine. Sensory evaluation revealed that MM provided wine with better flavor complexity and fruity notes.

KEYWORDS:

• Wolfberry wine
• Aromatic compounds
• GC–MS
• Maceration
• Organoleptic feature
• Sensory evaluation

Introduction

Wolfberry (Layciumbarbarum L.), also known as goji berry, belongs to solanaceae. It is originated from China and traditionally consumed as a functional food and herbal medicine.^[1] Wolfberry has been now widely cultivated in Asia, Europe, and North America.^[1] Wolfberry fruits have been reported to possess various nutrients constitutes, which provides wolfberry with multiple nutritional and health benefits.^[2] For example, it has been reported that the consumption of wolfberry fruits can improve the function of human liver, kidney, immune system, and circulation system, resulting in the longevity of human.^[3] In China, over about 20,000 tons of fresh wolfberry fruits is annually produced.^[4] Fresh wolfberry fruits are normally processed into dried wolfberry fruits for better shelf life.^[5,6] However, the oxidation and microbial contamination of nutrients can happen in dried wolfberry fruits during storage, reducing their nutritional quality and economic value.^[6] In modern fermentation technology, yeasts are introduced into fruit juice to metabolize sugar to alcohol.^[7] Wolfberry wine after fermentation can prevent the quality loss of wolfberry caused by microbial contamination and increase its commercial profit.

Overall, aroma plays important roles in determining the organoleptic quality of fermented wine, and it thus significantly affects the attraction of wine to customers.^[8,9] Overall, aroma of wine is a result of balancing various volatile compounds in wine.^[10] Basically, volatile compounds in wine mainly consist of esters, fusel alcohols, lactones, aldehydes, C13-norisoprenoids, terpenoids, fatty acids, carbonyls, volatile phenols, and sulfur compounds.^[10] Volatiles derived from fruits are able to be extracted into wine during maceration process, whereas fermented aromatic compounds are mainly synthesized during fermentation process. Wine aging process also results in the formation of aromatic compounds with aging aroma feature.^[11–13] The composition of aromatic compounds is mainly determined by fruit genotype,^[14–16] and fermentation process also affects their evolution and magnitude in wine.^[17,18] In fermentation process, maceration plays an important role in extracting these compounds from fruit skin into wine, and these compounds after maceration process can be further metabolized during fermentation and aging periods.^[10] It has been reported that an appropriate maceration process resulted in a better extraction of aromatic compounds, which significantly enhanced the sensory attributes of wine.^[19] However, excessive maceration has been reported to cause wine with unpleasant flavor.^[20,21] It has been well documented on the effect of maceration time on sensory attributes and aroma feature of wine made of grape.^[19–22] However, such investigations have not been well conducted in fermented wolfberry wine to our best knowledge. Therefore, we selected wolfberry fruits and fermented wolfberry wine under three different maceration time. The physicochemical properties, aromatic composition, and sensory features of these wines were compared. The objective of this study was to investigate if maceration time could affect the aromatic composition and organoleptic quality of wolfberry wine and further to optimize maceration time to produce wolfberry wine with better acceptability.

Materials and methods

Reagents and samples

Dry wolfberry fruits were obtained from Senmiao Corporation (Yinchuan, China). Zym Color Plus Pectinase, ES 488 Saccharomyces cerevisiae, and potassium metabisulfite were obtained from Enartis (Tracete, Italy). Deionized water was purified from a Milli-Q purification system (Millipore, Bedford, MA). Sodium hydroxide, sodium chloride, ammonium sulfate, sodium sulfate, tartaric acid, and ethanol were purchased from Beijing Chemical Works (Beijing, China). Ethyl acetate, isobutyl acetate, ethyl butanoate, isopentyl acetate, ethyl hexanoate, hexyl acetate, ethyl 3-hexenoate, ethyl enanthate, ethyl lactate, caprylic acid methyl ester, ethyl caprylate, isopentyl hexanoate, ethyl nonanoate, ethyl 2-hydroxy-4-methylpentanoate, ethyl caprate, isoamyl caprylate, diethyl succinate, phenethyl acetate, ethyl laurate, acetic acid, butanoic acid, octanoic acid, decanoic acid, 2-methyl-1-propanol, 1-butanol, isopentyl alcohol, 2-heptanol, 1-hexanol, 3-hexen-1-ol, 1-octen-3-ol, 2-ethyl-1-hexanol, 1-octanol, 2,3-butanediol, trans-2-octenol, benzyl alcohol, phenylethyl alcohol, nonanal, furfural, decanal, 6-methyl-5-heptene-2-one, dihydropseudoionone, methionol, naphthalene, mesotol, 4-ethylphenol, and styrene were purchased from Sigma-Aldrich (St. Louis, MO, USA). These commercial standards had a purity above 95%. The internal standard, 4-methyl-2-pentanol, was also a product of Sigma-Aldrich (St. Louis, MO, USA) with a purity of 98%.

Fermentation

The dry wolfberry fruits (60 kg) were placed in a 200-L fermentation tank and then mixed with 120 L of distilled water. Afterward, 3 g/hL pectinase and 45 mg/L potassium metabisulfite were added to the tank and then, the tank circulation was conducted. Subsequently, the fermentation tank was maintained at 9°C overnight. Sucrose and tartaric acid were added to adjust the sugar content and pH to 220 g/L and 3.6, respectively. Regarding the manufactural recommendation, 20 g/hL of S. cerevisiae was inoculated to initiate fermentation, and the fermentation was conducted at 20°C. The maceration period was determined by the relative density of the free-run juice. Short-time maceration (SM, 48 h), middle-time maceration (MM, 72 h), and long-time maceration (LM, 96 h) were achieved by separating the free-run juice from the pomace at the relative density of 1.072, 1.031, and 1.014, respectively. Afterward, potassium metabisulfite (40 mg/L) was immediately added to the tank when the relative density of the wine remained consistent in three consecutive days. Meanwhile, the tank temperature was reduced to 0°C to terminate the fermentation. After the fermentation, the wolfberry wine was transferred to a 100-L sealed stainless steel tank for further analysis. Each fermentation was carried out in duplicate.

Physicochemical measurement

The physicochemical properties of the wolfberry wines, including ethanol content, residual sugar content, pH, total and free SO₂, and titratable and volatile acidity, were determined according to the Chinese Standard GB 15038-2006.^[23] The polysaccharide content of the wines was measured using the phenol-sulfuric acid method with some modifications.^[24] In briefly, the wine (1 mL) was mixed with 5 mL of ethanol: water (4:1, v/v). The mixture was incubated at room temperature for 12 h, and then centrifuged at 4000 rpm for 10 min to discard the supernatant. Afterward, the resultant pellet was washed with 5 mL of ether, 5 mL acetone, and 5 mL acetone, evaporated to dryness at room temperature, and then dissolved in 10 mL water. Subsequently, the resultant sample (200 µL) was mixed with 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid. The absorbance of the resulting mixture was measured at 490 nm on a UNICO ultraviolet spectrophotometer (Shanghai, China). Glucose was used as the external standard for the quantitation of polysaccharide content in the wolfberry wine.

Volatile compound analyses

A headspace solid-phase micro-extraction was used to extract volatile compounds from the wolfberry wine.^[25] Briefly, the wine sample (5.0 mL) was mixed with 1 g NaCl and 10 µL 1.0038 g/L 4-methyl-2-pentanol (internal standard) in a 15-mL vial. The vial was tightly capped with a PTFE-silicon septum and contained a magnetic stirrer. The sample in the vial was equilibrated at 40°C for 30 min on a heating platform under agitation. Afterward, a carboxen fiber (50/30 µm DVB/Carboxen/PDMS, Supelco, Bellefonte, PA) was inserted into the headspace of the vial and the extraction was conducted for 30 min at 40°C under the continuous agitation. After the extraction, the fiber was immediately desorbed in the GC injector for 8 min and then analyzed using the reported method.^[26] An Agilent 6890 gas chromatogram coupled with an Agilent 5975 mass spectrometry (GC–MS, Agilent Technologies, Santa Clara, CA, USA) was used for the analysis of volatile compounds. A 60 m × 0.25 mm id HP-INNOWAX capillary column with 0.25 μm film thickness (J&W Scientific, Folsom, CA) was used for the volatile compound separation with a carrier gas (helium) flow rate of 1 mL/min in a splitless mode. The temperature in oven was programmed as follows: 50°C held for 1 min and then rose to 220°C at 3°C/min and held at 220°C for 5 min. A 70-eV ionization energy was set under the electron impact mode, and the mass spectrum was recorded at a mass scan of m/z 20–450 under a selective ion mode. A C7–C24 n-alkane series (Supelco, Bellefonte, PA, USA) under the same chromatographic procedure was used to determine the retention indices. Volatile compounds were identified by comparing their mass spectrum with the standard NIST11 library and the reference standard. For the volatile compound without the commercially available standard, their identification was conducted by matching their mass spectrum with NIST11 library and then comparing their retention indices in the NIST standard reference database. Regarding their quantitation, the aromatic standards were dissolved in a synthetic wine model matrix containing 5 g/L tartaric acid, 10.5% (v/v) ethanol, and 24 g/L glucose with the pH of 3.8 adjusted by 5 mol/L NaOH. The mixed standard solution was diluted into 15 successive levels and extracted according the same micro-extraction method for the wolfberry wine. The volatile compound was quantified using their corresponding standard. For the volatile without the commercially available standard, its quantitation was carried out using the volatile standard that had the similar chemical structure or atom numbers. Each measurement was carried out in triplicate.

Sensory evaluation

A professional sensory panel was performed to estimate the sensory differences of the wolfberry wine fermented under different maceration treatments. In this panel, a total of 18 professional tasters, including 9 males and 9 females, were selected. Their age ranged between 21 and 36, and they had completed a 40-h training course of wine sensory evaluation. During the panel, these wolfberry wines were placed in the standard wine-tasting glass at 19 ± 1°C and brought to the taster under a random order in a professional taste panel booth. The tasters graded the sensory attributes of the wine regarding seven descriptors, including flavor complexity, flavor harmony, mouthfeel, fruity note, bitterness, color appearance, and overall acceptability, under a 5-point intensity scale (none: 0; very low: 1; low: 2; medium: 3; high: 4; very high: 5). A 10-min break was given to the taster between the samples.

Odor activity value analysis

Odor activity value (OAV) was used in this study to evaluate the potential contribution of individual volatile compound to the overall aroma of the wolfberry wine. The OAV was calculated using the equation below, and the volatile compound with the OAV value above 1 indicated that this compound significantly contributed to the overall aroma of the wolfberry wine,

Statistical analysis

A one-way analysis of variance (ANOVA) was carried out on SPSS 21.0 (Chicago, IL, USA) to measure the significance of the individual compound concentration induced by different maceration time during the fermentation course under a Duncan’s multiple range tests at a p ≤ 0.05 level.

Results and discussion

Effect of maceration time on physicochemical properties of wolfberry wine

The MM resulted in the wolfberry wine with the highest residual sugar content (Table 1). However, no significant differences were observed in the wines with the SM and the LM. The pH value of the wolfberry wines also showed the statistical differences after different maceration periods. The wine with the MM had the pH value of 4.00, whereas the SM led to the wine with a pH value of 3.72. The wine fermented after the LM showed the lowest pH (3.59). The titratable acidity is a key physicochemical factor that impacts the balance of the flavor, pH condition, and chemical stability of wine.^[27] Its level in the MM and the LM wines was higher than that in the SM wine. Similarly, the MM wine also possessed the highest content of volatile acidity compared to the other wolfberry wines. No significant differences were observed in the free SO₂ level in these wolfberry wines. However, extending the maceration time during the wolfberry fermentation process reduced the total SO₂ level in the wine. Polysaccharides have been considered the important bioactive components in wolfberry, and it has been reported that polysaccharides have the ability of reducing the astringency of wine through inhibiting the interactions between salivary proteins and tannin molecules in wine.^[28] In the present study, the wolfberry wine with the MM showed the highest level of polysaccharides, followed by the LM wine and then the SM wine. Our results were consistent with the previous study where the similar changing trend of polysaccharides was also observed in Tempranillo and Cabernet Sauvignon wines.^[29]

Table 1. Ethanol content, residual sugar content, pH, titratable and volatile acidity, total and free SO₂ content, and polysaccharide content of wolfberry wines fermented under different maceration time.

Physicochemical feature	Maceration time
	SM	MM	LM
Ethanol (%, v/v)	9.92 ± 0.04c	10.94 ± 0.06a	10.56 ± 0.06b
Residual sugar (mg/L)	23.59 ± 0.06b	25.36 ± 0.09a	23.9 ± 0.2b
pH	3.72 ± 0.01b	4.00 ± 0.01a	3.59 ± 0.01c
Titratable acidity (g/L)	14.54 ± 0.09b	15.4 ± 0.2a	15.3 ± 0.1a
Volatile acidity (g/L)	0.71 ± 0.02b	0.88 ± 0.02a	0.74 ± 0.02b
Total SO2 (mg/L)	189 ± 0a	148 ± 6b	127 ± 2c
Free SO2 (mg/L)	11 ± 1a	11 ± 1a	11 ± 1a
Polysaccharides (mg/L)	1176 ± 7b	1464 ± 33a	1306 ± 66ab

Data are mean ± standard deviation of triplicate tests. Different letters in each row represent significant differences at p ≤ 0.05.

SM: Short-time maceration; MM: middle-time maceration; LM: long-time maceration.

Effect of maceration time on volatile composition of wolfberry wine

A total of 93 volatile compounds were identified in the wolfberry wine fermented from different maceration time (Table 2). These volatile compounds included 38 esters, 17 alcohols, 12 aromatic compounds, 7 terpene derivatives, 7 ketones, 6 acids, 5 aldehydes, and 1 sulfur compound regarding their structural feature. Alcohols appeared to be the most abundant volatiles in these wines, followed by esters and acids. Sulfur compound and aromatic compounds also exhibited a moderate level in these wines. However, these wolfberry wines contained a low concentration of aldehydes, ketones, and terpene derivatives.

Table 2. Retention indices and concentration of volatile compounds in wolfberry wine fermented under different maceration time.

Volatile compound	Retention indices	Standard	Maceration time
			SM	MM	LM
Esters
Acetate esters
Ethyl acetate (mg/L)	764.8	A	70 ± 3b	82.31 ± 2.30a	73.03 ± 0.01ab
Isobutyl acetate (µg/L)	1009.6	A	360 ± 19a	338.4 ± 6.99ab	304.22 ± 0.53b
Isopentyl acetate (mg/L)	1135.9	A	11.8 ± 0.6a	10.22 ± 0.41a	8.34 ± 0.10b
Hexyl acetate (µg/L)	1298.8	A	13.1 ± 1.0b	15.61 ± 0.18a	11.92 ± 0.02b
Heptyl acetate (µg/L)	1377.4	B	0.70 ± 0.05b	1.24 ± 0.08a	0.91 ± 0.08b
2-Ethyl-1-hexanol acetate (µg/L)	1387.2	B	9.0 ± 0.4b	9.3 ± 0.8b	19.2 ± 0.6a
Octyl acetate (µg/L)	1477	B	19 ± 2a	18 ± 1a	19 ± 4a
Trimethylene acetate (µg/L)	1740.3	B	210 ± 83a	290 ± 105a	181 ± 2a
Phenethyl acetate (µg/L)	1826.3	A	585 ± 11a	311 ± 23b	336 ± 15b
Total acetate esters (mg/L)			83 ± 4ab	94 ± 3a	82.2 ± 0.1b
Ethyl esters
Ethyl butanoate (µg/L)	1037.5	A	395 ± 24a	382 ± 7ab	327 ± 4b
Ethyl hexanoate (µg/L)	1244.4	A	618 ± 47a	451 ± 20b	424 ± 13b
Ethyl 3-hexenoate (µg/L)	1307.6	A	2.5 ± 0.2b	3.99 ± 0.08a	1.79 ± 0.05c
Ethyl enanthate (µg/L)	1337.6	A	2.9 ± 0.2b	5.0 ± 0.3a	2.5 ± 0.1b
Ethyl lactate (mg/L)	1346.8	A	2.4 ± 0.1a	3.2 ± 0.1a	3.4 ± 0.4a
Ethyl caprylate (mg/L)	1438.7	A	2.1 ± 0.1a	1.5 ± 0.1b	1.21 ± 0.06b
Ethyl mesitylacetate (µg/L)	1511.8	B	0.53 ± 0.02b	0.62 ± 0.04ab	0.70 ± 0.04a
Ethyl nonanoate (µg/L)	1539.5	A	7.1 ± 0.7b	12 ± 1a	5.56 ± 0.04b
Ethyl 2-hydroxy-4-methylpentanoate (µg/L)	1546.9	A	15.9 ± 0.6a	16.6 ± 0.8a	16 ± 1a
Ethyl caprate (mg/L)	1642.8	A	2.64 ± 0.09a	2.4 ± 0.2a	1.40 ± 0.08b
Diethyl succinate (µg/L)	1680.1	A	180 ± 13ab	210 ± 3a	171 ± 9b
Ethyl 9-decenoate (µg/L)	1694.3	B	49.2 ± 0.2a	14 ± 1c	31.9 ± 0.5b
Mesotol (µg/L)	1824.6	A	2.0 ± 0.5ab	3.0 ± 0.2a	1 ± 0b
Ethyl laurate (µg/L)	1847.5	A	478.0 ± 0.5a	392 ± 43a	274 ± 19b
Ethyl dihydrocinnamate (µg/L)	1895.6	B	1.37 ± 0.07b	1.8 ± 0.1a	1.25 ± 0.05b
Ethyl myristate (µg/L)	2053.2	B	73 ± 6a	57 ± 6ab	36 ± 5b
Ethyl cinnamate (µg/L)	2145.3	B	2.75 ± 0.08b	3.7 ± 0.2a	2.7 ± 0.2b
Ethyl hexadecanoate (µg/L)	2256.1	B	110 ± 3a	101 ± 9a	75 ± 3b
Total ethyl esters (mg/L)			9.1 ± 0.1a	8.7 ± 0.3ab	7.4 ± 0.6b
Other esters
Isobutyl hexanoate (µg/L)	1357.7	B	0.73 ± 0.04a	0.5 ± 0.1a	0.5 ± 0.1a
Caprylic acid methyl ester (µg/L)	1393.8	A	11.5 ± 0.8a	9.9 ± 0.6a	6.3 ± 0.2b
Isopentyl hexanoate (µg/L)	1462.3	A	10.6 ± 0.6a	8.4 ± 0.5b	7.4 ± 0.3b
Isobutyl octanoate (µg/L)	1554.6	B	4.5 ± 0.4a	4.1 ± 0.4a	3.3 ± 0.3a
Methyl caprate (µg/L)	1599	B	58 ± 3a	65 ± 5a	29 ± 1b
Isoamyl caprylate (µg/L)	1662.7	A	25.9 ± 0.5a	18 ± 2b	13.3 ± 0.8b
Benzoic ether (µg/L)	1677.4	B	2.03 ± 0.05b	2.9 ± 0.2a	1.93 ± 0.03b
Methyl 2-(pentyloxy)benzoate (µg/L)	1790.2	B	1.9 ± 0.1b	3.0 ± 0.1a	1.48 ± 0.09b
Isoamyl decanoate (µg/L)	1866.8	B	15.9 ± 0.2a	13 ± 2a	7.6 ± 0.6b
Propanoic acid, 2-methyl-,1-(1,1-dimethylethyl)-2-methyl-1,3-propanediyl ester (µg/L)	1883.2	B	1.4 ± 0.7a	1.4 ± 0.5a	0.97 ± 0.03a
Nonanoic acid, 3-methylbutyl-2 ester (µg/L)	1905.7	B	3.4 ± 0.1a	3.7 ± 0.3a	4.02 ± 0.09a
Total other esters (µg/L)			136 ± 6a	130 ± 11a	76 ± 4b
Total ester (mg/L)			92 ± 4ab	102 ± 3a	89.7 ± 0.7b
Acids
1,2-Dimethyl-2-cyclopentene-1-carboxylic acid (µg/L)	1367.6	B	8.7 ± 0.6b	11.0 ± 0.1a	8.67 ± 0.05b
Acetic acid (µg/L)	1453.8	A	72 ± 7b	206 ± 2a	112 ± 26b
Butanoic acid (mg/L)	1630.4	A	1.25 ± 0.00c	2.2 ± 0.1a	1.74 ± 0.05b
α-Methylbutyric acid (mg/L)	1671.7	B	1.6 ± 0.2b	2.31 ± 0.01a	2.12 ± 0.02a
Octanoic acid (mg/L)	2062.1	A	16 ± 4a	14 ± 2a	12.0 ± 0.7a
Decanoic acid (mg/L)	2271.8	A	22 ± 7a	20 ± 4a	12.7 ± 0.8b
Total acids (mg/L)			41 ± 12a	39 ± 6a	29 ± 2a
Alcohols
2-Methyl-1-propanol (mg/L)	1104	A	36.34 ± 0.06c	39.40 ± 0.04b	42.5 ± 0.8a
1-Butanol (µg/L)	1158.1	A	834 ± 17b	1324 ± 8a	796 ± 18b
Isopentyl alcohol (mg/L)	1216	A	220 ± 6a	213 ± 3a	213 ± 5a
Pentyl alcohol (µg/L)	1255.5	B	14.2 ± 0.6b	22.2 ± 0.5a	14.8 ± 0.2b
2-Heptanol (µg/L)	1320	A	6.4 ± 0.4a	7 ± 1a	7.7 ± 0.4a
1-Hexanol (µg/L)	1352.5	A	40 ± 3c	77 ± 2a	63 ± 1b
Dowanol peat (µg/L)	1376.9	B	20 ± 3b	66.7 ± 0.1a	28 ± 6b
3-Hexen-1-ol (µg/L)	1384.7	A	22.4 ± 0.7b	137 ± 13a	15 ± 3b
1-Octen-3-ol (µg/L)	1448.6	A	3.2 ± 0.2b	7.6 ± 0.2a	3.54 ± 0.01b
2-Ethyl-1-hexanol (µg/L)	1488.8	A	1.9 ± 0.3b	3.3 ± 0.2a	4.1 ± 0.2a
2,3-Butanediol, [R-(R*,R*)]– (mg/L)	1538.9	B	324 ± 140a	948 ± 343a	458 ± 8a
1-Octanol (µg/L)	1557	A	17.37 ± 0.08a	18 ± 1a	14.1 ± 0.5b
2,3-Butanediol, [S-(R*,R*)]– (mg/L)	1574.9	A	121 ± 52a	247 ± 110a	119 ± 31a
trans-2-Octenol (µg/L)	1616	A	2.57 ± 0.08a	2.63 ± 0.08a	2.44 ± 0.03a
E-5-Decen-1-ol (µg/L)	1795.2	B	2.4 ± 1.0a	1.7 ± 0.2a	1.7 ± 0.1a
Benzyl alcohol (µg/L)	1883.4	A	198 ± 23b	560 ± 51a	311 ± 8b
Phenylethyl alcohol (mg/L)	1920.2	A	36 ± 7a	29 ± 4a	43 ± 1a
4-Tolylcarbinol (µg/L)	1981.6	B	3.1 ± 0.2a	2.5 ± 0.2a	1.8 ± 0.2b
Total alcohols (mg/L)			739 ± 194a	1478 ± 460a	876 ± 32a
Aldehydes
Nonanal (µg/L)	1399.2	A	0.7 ± 0.2ab	1.27 ± 0.01a	0.5 ± 0.2b
Furfural (µg/L)	1471.8	A	149 ± 13a	185 ± 16a	211 ± 24a
Decanal (µg/L)	1504.4	A	3 ± 1a	4.0 ± 0.9a	2.9 ± 0.5a
Safranal (µg/L)	1656.8	B	6.75 ± 0.07b	11.8 ± 0.7a	8.6 ± 0.5b
2-Butyl-2-octenal (µg/L)	1675.4	B	0.02 ± 0.00c	0.03 ± 0.00a	0.02 ± 0.00b
Total aldehydes (µg/L)			160 ± 12a	202 ± 17a	223 ± 24a
Ketones
2,2,6-Trimethylcyclohexanone (µg/L)	1325	B	nd	1.8 ± 0.7a	nd
6-Methyl-5-heptene-2-one (µg/L)	1344.7	A	4 ± 1b	12 ± 2a	6 ± 1ab
2-Undecanone (µg/L)	1603.7	B	1.5 ± 0.1ab	1.29 ± 0.08b	1.90 ± 0.06a
β-Cyclocitral (µg/L)	1630.9	B	4.4 ± 0.4c	8.0 ± 0.2a	5.8 ± 0.1b
Dihydropseudoionone (µg/L)	1861.4	A	1.03 ± 0.09b	2.9 ± 0.3a	4.1 ± 0.5a
2-Acetylpyrrole (µg/L)	1981.6	B	92 ± 21b	213 ± 36a	125 ± 5ab
(2,6,6-Trimethyl-2-hydroxycyclohexylidene) acetic acid lactone (µg/L)	2372.3	B	7 ± 1a	13 ± 4a	5.7 ± 0.5a
Total ketones (µg/L)			110 ± 23b	252 ± 43a	148 ± 5ab
Sulfur compounds
Methionol (mg/L)	1720.6	A	2.4 ± 0.6a	1.6 ± 0.3a	3.1 ± 0.5a
Aromatic compounds
Isodurene (µg/L)	1498.2	B	2.8 ± 0.2a	2.90 ± 0.08a	3.14 ± 0.06a
1,1,4,6-Tetramethylindane (µg/L)	1677.5	B	13.3 ± 0.5a	16 ± 1a	14.11 ± 0.01a
2,5-Diisopropyl-p-xylene (µg/L)	1694.1	B	53 ± 3a	40 ± 2b	49.9 ± 0.5a
Naphthalene (µg/L)	1756	A	3.40 ± 0.03a	3.6 ± 0.4a	3.30 ± 0.07a
2,5,8-Trimethyl-1,2-dihydronaphthalene (µg/L)	1758.3	B	13 ± 2a	10.9 ± 0.9a	12.6 ± 0.3a
β-Methylnaphthalene (µg/L)	1870	B	9.82 ± 0.06a	11.7 ± 1.18a	11.6 ± 0.2a
α-Methylnaphthalene (µg/L)	1906.9	B	6.59 ± 0.03a	7.6 ± 0.8a	8.2 ± 0.3a
α-Calacorene (µg/L)	1931.9	B	11.8 ± 0.3ab	13 ± 1a	9.8 ± 0.3b
4-Ethylphenol (µg/L)	2182.8	A	9 ± 3a	6.4 ± 1.0a	4.8 ± 0.4a
4-Vinylguaiacol (µg/L)	2208.5	B	581 ± 95b	1207 ± 184a	465 ± 16b
2,4-Di-tert-butylphenol (µg/L)	2308.7	B	43 ± 2a	39 ± 2a	40 ± 2a
Coumaran (µg/L)	2403.1	B	509 ± 138a	852 ± 249a	346 ± 27a
Total aromatic compounds (mg/L)			1.3 ± 0.2ab	2.2 ± 0.4a	0.97 ± 0.05b
Terpernoids and norisoprenoids
Styrene (µg/L)	1269.8	A	117 ± 13b	161 ± 6a	147 ± 5ab
Ionene (µg/L)	1489.7	B	13.9 ± 0.4a	15.8 ± 1.2a	14.6 ± 0.5a
1-Oxaspiro[4.5]dec-6-ene, 2,6,10,10-tetr (µg/L)	1549.5	B	2.6 ± 0.2b	4.4 ± 0.3a	3.1 ± 0.1b
α-Cedrene (µg/L)	1625.9	B	0.39 ± 0.06a	0.34 ± 0.03a	0.38 ± 0.04a
β-Ionone (µg/L)	1951.7	B	3.13 ± 0.03b	8.6 ± 0.6a	6.8 ± 0.5a
2,6-Dimethyl-2,6-undecadien-10-ol (µg/L)	1955.5	B	0.18 ± 0.00c	0.83 ± 0.05a	0.53 ± 0.04b
Cedrol (µg/L)	2137.5	B	0.07 ± 0.01a	0.07 ± 0.00a	0.06 ± 0.00a
Total terpernoids and norisoprenoids (µg/L)			137 ± 12b	191 ± 8a	172 ± 6ab

Data are mean ± standard deviation of triplicate tests. Different letters in each row represent significant differences at p ≤ 0.05. “nd” indicated “not detected.” Retention indices are based on HP-innowax column. “A” and “B” in standard column indicate that compound is identified with and without reference standard, respectively.

The OAV is an important indictor to reflect the contribution of individual volatile compound to the overall aroma of wine.^[30–32]. It has been accepted that volatile compound with its concentration above its odor threshold (OAV above 1) could significantly contribute its flavor notes to the overall aroma of wine.^[30–32] In the present study, a total of 18 volatile compounds, including 5 esters, 3 acids, 4 alcohols, 1 sulfur compound, and 1 aromatic compound, showed their OAV value above 1 in at least 1 of the 3 wolfberry wines. This indicated that these compounds played important roles in the overall aroma of the wolfberry wines (Table 3).

Table 3. Odor threshold, and flavor note and OAVs of major volatile compounds in wolfberry wine fermented from different maceration time.

Compounds
Esters	Short-time maceration	Middle-time maceration	Long-time maceration	Odor threshold (μg/L)	Ref.^a	Descriptor	Ref.^a
Ethyl acetate	5.8 ± 0.4b	6.9 ± 0.3a	6.09 ± 0.00ab	7500	^[Citation1]	Pineapple, fruity, varnish	^[Citation6]
Isobutyl acetate	0.22 ± 0.01a	0.21 ± 0ab	0.19 ± 0.00b	1600	^[Citation2]	Banana, fruity, sweet	^[Citation7]
Ethyl butanoate	0.99 ± 0.06a	0.96 ± 0.02ab	0.82 ± 0.01b	400	^[Citation3]	Banana, pineapple, strawberry	^[Citation4]
Isopentyl acetate	74 ± 4a	64 ± 3a	52.1 ± 0.6b	160	^[Citation4]	Sweety, fruity	^[Citation3]
Ethyl hexanoate	7.7 ± 0.6a	5.6 ± 0.3b	5.3 ± 0.2b	154,636	^[Citation3]	Fruity, buttery	^[Citation3]
Ethyl caprylate	3.7 ± 0.2a	2.6 ± 0.2b	2.1 ± 0.1b	580	^[Citation3]	Sweet, floral, fruity, banana, pear	^[Citation3]
Ethyl caprate	13.2 ± 0.5a	12 ± 1a	7.0 ± 0.4b	200	^[Citation3]	Fruity, fatty	^[Citation3]
Phenethyl acetate	0.33 ± 0.01a	0.17 ± 0.01b	0.19 ± 0.01b	1800	^[Citation3]	Fruity, rose	^[Citation3]
Ethyl laurate	0.32 ± 0.00a	0.26 ± 0.03a	0.18 ± 0.01b	1500	^[Citation3]	Oily, fatty, fruity	^[Citation3]
Acids
Butanoic acid	7.22 ± 0.02c	12.8 ± 0.6a	10.0 ± 0.3b	173	^[Citation5]	Butterlike, cheesy, stinky, floral	^[Citation9]
Octanoic acid	31 ± 8a	28 ± 4a	24 ± 1a	500	^[Citation3]	Rancid, cheese, fatty acid	^[Citation3]
Decanoic acid	22 ± 7a	20 ± 4a	12.7 ± 0.8a	1000	^[Citation5]	Fatty, rancid	^[Citation3]
Alcohols
Isopentyl alcohol	3.7 ± 0.1a	3.54 ± 0.04a	3.54 ± 0.08a	60,000	^[Citation4]	Solvent, sweet, alcohol, nail polish	^[Citation3]
2,3-Butanediol, [R-(R*,R*)]–	2.2 ± 0.9a	6 ± 2a	3.05 ± 0.06a	150,000	^[Citation3]	Fruity, sweet, butter	^[Citation3]
2,3-Butanediol, [S-(R*,R*)]–	0.8 ± 0.4a	1.6 ± 0.7a	0.8 ± 0.2a	150,000	^[Citation6]	Fruity, sweet, butter	^[Citation3]
Phenylethyl alcohol	2.6 ± 0.5a	2.1 ± 0.3a	3.1 ± 0.1a	14,000	^[Citation3]	Roses, honey	^[Citation3]
Sulfur compounds
Methionol	2.4 ± 0.6a	1.6 ± 0.3a	3.1 ± 0.5a	1000	^[Citation3]	Cabbage, cooked potato, garlic	^[Citation3]
Aromatic compounds
4-Vinylguaiacol	0.53 ± 0.09b	1.1 ± 0.2a	0.42 ± 0.01b	1100	^[Citation5]	Phenolic, bitter, pharmaceutic spicy	^[Citation7]

Data are mean ± standard deviation of triplicate tests. Different letters in each row represent significant differences at p ≤ 0.05.

^aReference: ^[1]Guth (1997), ^[2]Tao and Li (2010), ^[3]Cai, Zhu, Wang, Lu, Lan, Reeves, et al. (2014), ^[4]Zhu, Zhu, Li, Zhang, Zhang, and Fan (2016), ^[5]Ferreira, López, and Cacho (2000), ^[6]Wu, Zhu, Cui, Duan, and Pan (2011), ^[7]Coelho, Vilanova, Genisheva, Oliveira, Teixeira, and Domingues (2015), ^[8]Shu, Chang, Teng, Zhen, Shu, Hong, et al. (2013).

Alcohols

Fusel alcohols (also known as higher alcohols), as the main secondary product of yeast metabolism, are synthesized from amino acids at the early stage of alcoholic fermentation.^[33] These fusel alcohols normally bring pungent and strong odor to wine.^[32] Their contribution to the wine overall aroma mainly relies on their concentration in wine.^[34] It has been reported that these compounds could provide the wine aroma with satisfactory complexity when their level in wine was below 300 mg/L. However, a negative effect on the wine aroma was observed when wine contained the concentration of these alcohols above 400 mg/L.^[34]

In the present study, the concentration of the total fusel alcohols was higher than 400 mg/L in all the wolfberry wines (Table 2). It has been reported that maceration treatment during fermentation process resulted in an increase on the level of fusel alcohols in grape wines.^[35,36] However, different maceration time did not significantly alter their accumulation in the wolfberry wine. This might be attributed to the differences on the chemical components in grape and wolfberry. Regarding the individual fusel alcohols, isopentyl alcohol, 2,3-butanediol (R,R), and phenylethyl alcohol possessed their concentration above their threshold in all the wolfberry wine. Meanwhile, the 2,3-butanediol (S,R) concentration in the MM wine was above its odor threshold (Table 3). Among these fusel alcohols, isopentyl alcohol is formed from deamination and decarboxylation of isoleucine during fermentation.^[37,38]. This volatile exhibits sweet note, and it can bring herbaceous, cacao, and vinous aroma to wine.^[37,38] 2,3-Butanediol (S,R) and 2,3-butanediol (R,R) provide the fruity, sweet, and butter flavor notes.^[32] Phenylethyl alcohol contributed the rose and honey notes to the overall aroma of wine.^[39] Different maceration time during the wolfberry wine fermentation did not induce the content differences on these major fusel alcohols in the wine. However, different maceration time significantly affected the accumulation of some fusel alcohols in the wine (Table 2). For example, the concentration of 2-methyl-1-propanol increased in the wolfberry wine with the increase of the maceration period during the wine fermentation. The similar observation was also found in a published report.^[10] Although their concentration was not above its threshold, the difference on these fusel alcohols could induce the sensory differences of the wolfberry wines due to their potential synergistic effect. For example, 1-hexanol has been reported to enhance the overall aroma of wine when its level was higher than 20 mg/L but lower than 100 mg/L in wine.^[40,41] The wolfberry wine fermented from the MM exhibited much higher concentration of 1-butanol, pentyl alcohol, 1-hexanol, dowanol peat, 3-hexen-1-ol, 1-octen-3-ol, and benzyl alcohol (Table 2). Therefore, it was expected that the MM could improve the flavor notes of the wolfberry wine due to the enhanced level of these fusel alcohols.

Esters

Esters are able to be synthesized in wine via the conjugation of alcohols and acids under the activity of yeasts during alcoholic fermentation.^[34] These esters can be grouped into acetate esters, ethyl esters, and other esters regarding their structural nature and synthesis path. For example, acetate esters are the major products of fusel alcohols via the acetyl–CoA reaction, whereas ethanolysis of acyl–CoA can result in the formation of ethyl esters in wine.^[34,39] Esters play the essential roles in contributing to wine with the floral and fruity notes.^[42]

In this study, the concentration of the total esters in the wine was significantly affected by different maceration time (Table 2). The MM wine contained the highest ester content, followed by the wine fermented from the SM and then the LM. Additionally, the wolfberry wine fermented from the MM had higher level of the total acetate ester content and the total other ester content compared with the LM wine. Extending the maceration period during fermentation might induce the hydrolysis of esters, which resulted in a concentration decrease of esters in wine.^[10,43] However, the total content of ethyl esters was higher in the SM wine although these esters were not dominant in the wine.

Among acetate esters in the wine, ethyl acetate and isopentyl acetate were the major acetate esters that possessed their OAV above 1 (Table 3). Ethyl acetate has been described as the pineapple, fruity, and varnish notes.^[31] This volatile showed higher concentration in the MM and LM wines compared to the SM wine. Besides, isopentyl acetate has been confirmed to be the representative volatile compound in wolfberry wine and it displays the sweet, banana, and fruity aroma.^[44] This volatile appeared to be higher in the wine with the MM and the SM. The SM wine had higher level of phenethyl acetate and isobutyl acetates. However, the aroma contribution of these two acetate esters was limited since their concentration was below their threshold.

Ethyl caprate, ethyl laurate, ethyl lactate, ethyl caprylate, ethyl hexanoate, and ethyl butanoate were the major ethyl esters present in the wolfberry wine. It has been reported that these esters provided wine with the fruity, fatty, banana, pear, and green apple notes.^[32,45] Extending the maceration time during the wolfberry wine fermentation decreased their accumulation in the wine except for ethyl lactate (Table 2). Our results were consistent with the previous reports.^[46,47] Such a decrease on the concentration of these ethyl esters might result from their nonenzymatic hydrolysis during the fermentation.^[10] There were 12 esters that did not belong to ethyl ester or acetate ester (Table 2). Although maceration time showed the effect on the accumulation of these esters in the wolfberry wine, their relative low concentration might not significantly result in a difference on the total aroma contribution of the wolfberry wines.

Acids

Fatty acids are mainly formed via enzymatic reactions during wine fermentation, and they play important roles in affecting the overall aroma of wine.^[39] It has been reported that fatty acids possess the cheese, fruity, rancid, and fatty notes.^[39] No significant differences on the concentration of the total acids were observed among the different maceration wines (Table 2). However, the individual fatty acids showed the content differences in the wines treated with different maceration time. For example, octanoic acid, decanoic acid, and butanoic acid appeared to show the obvious contribution to the wolfberry wine aroma due to their high concentration (Tables 2 and 3). Butanoic acid showed the highest concentration in the MM wine, whereas extending maceration time resulted in a content decrease of decanoic acid. Decanoic acid could be used as an energy source for yeasts during maceration and fermentation process, and its consumption by yeasts might cause the decrease on its level in wine.^[10] The octanoic acid concentration in the wolfberry wine was not affected by the maceration time. In addition, the MM wine contained higher content of 1, 2-dimetyl-2-cyclopentene-1-carboxylic acid and acetic acid. However, their effect on the overall aroma might be limited since their concentration was not above their threshold. It has been proposed that fatty acids normally induce unpleasant odors to wine,^[39] and different maceration time did not alter too much on the fatty acid composition or concentration.

Aromatic compounds

Aromatic compounds are basically derived from the metabolisms of phenolic acids during fermentation, and their composition is mainly determined by fruit genotype.^[48,49] The total aromatic compound content was observed to be the highest in the MM wine, followed by the wine fermented from the SM (Table 2). However, extending the maceration time during the wolfberry wine fermentation resulted in a low content of the total aromatic compounds. Regarding individual aromatic compounds, only 4-vinylguaiacol and coumaran appeared to contain high level in the wine (Table 2), and the MM wine showed the highest level of 4-vinylguaiacol. However, the concentration of 4-vinylguaiacol in the MM wine was only higher than its threshold (Table 3). The other aromatic compounds existed in the wine with a low level. Normally, 4-vinylguaiacol displays a bitter and pharmaceutic-spicy flavor to the wine aroma.^[48]

Aldehydes and ketones

Aldehydes and ketones belong to carbonyl compounds. These compounds are synthesized from the metabolism of unsaturated fatty acids under the lipoxygenase activity during fermentation process.^[39] The MM caused a higher content of the total ketones in the wolfberry wine, whereas the total aldehyde content of the wine was not altered by different maceration time (Table 2). Furfural (caramel odor) and 2-acetylpyrrole (burnt) were the dominant individual aldehyde and ketone in the wine, respectively.^[39,50] 2-Acetylpyrrole showed the highest concentration in the wine fermented from the MM. Similarly, the maceration time also induced the difference on the level of the other carbonyl compounds in the wine. However, their low concentration might bring little effect on the overall wine aroma contribution (Table 2).

Other volatile compounds

Methionol was the only sulfur compound detected in the wolfberry wines, and it has the cabbage, cooked potato, and garlic odor.^[32] Its concentration in the wine was higher than its threshold (Table 3). However, the different maceration time did not change its concentration in the wine (Table 2). The content of the total terpene derivatives was observed to be higher in the MM wine, which mainly resulted from higher level of styrene in this wine (Table 2). However, its concentration in the wine was not higher than its odor threshold, limiting its aroma contribution.

Sensory evaluation

The sensory attributes of these different maceration treated wines were compared regarding their flavor complexity, flavor harmony, mouthfeel, wolfberry fruity notes, bitterness, color appearance, and overall acceptability (Fig. 1). Among these attributes, flavor complexity, flavor harmony, and wolfberry fruity notes are directly determined by the volatile composition and concentration in wine, whereas phenolic composition mainly affects its color appearance.^[51] Bitterness, mouthfeel, and overall acceptability of wine are a combined result of physicochemical properties (such as polysaccharide content), aromatic profile, and phenolic composition.^{[8,9,27,51–53]} In the present study, the flavor complexity and wolfberry fruity notes in the wine fermented from the MM were given higher score (Fig. 1). These indicated that the accumulation of the volatile compounds related to these aroma features was enhanced in the wine. Acetate esters and ethyl esters displayed its concentration higher than their threshold in the wine, and these volatiles contributed to the fruity notes to the wine aroma. Therefore, it was speculated that MM benefited the accumulation of acetate esters and ethyl esters in the wine, which significantly improved the fruity aroma of the wine. Additionally, a lower score was given to the MM wine regarding its color appearance (Fig. 1). Since phenolic compounds are the critical components that affect the color feature of wine, we speculated that the color distinction might result from the alteration of phenolic compound composition in the wolfberry wine processed with the MM .^[51] A further study regarding the maceration effect on composition of phenolic compounds in wolfberry wine should be carried out. The mouthfeel, bitterness, and overall acceptability of these wines were given the similar scores by the tasters, indicating that these organoleptic features in wine were not altered by different maceration time. It has been reported that grape wine with LM treatment was received a high score on the bitterness and mouthfeel.^[29] These differences might be attributed to the fruit differences between grapes and wolfberry.

Figure 1. Organoleptic features of wolfberry wine fermented from different maceration time. Organoleptic attributes were evaluated in terms of flavor complexity, flavor harmony, mouthfeel, wolfberry fruity notes, bitterness, color appearance, and overall acceptability. Different letters in each row represent significant differences at p ≤ 0.05.

Conclusion

The effect of different maceration time on wolfberry wine fermentation was investigated and the wine fermented from the MM showed the high concentration of esters, especially isopentyl acetate, ethyl caprate, ethyl acetate, and ethyl hexanoate. The MM wine also contained higher level of ketones, 4-vinylguaiacol, and terpene derivatives. Esters appeared to be the primary volatile compounds that significantly contributed to the overall aroma of the wolfberry wine due to their level higher than their threshold. Sensory evaluation revealed that the wolfberry wine with the MM exhibited better organoleptic features on the flavor complexity and wolfberry fruity notes.

Acknowledgment

This study was finally supported by the Fundamental Research Funds for the Central Universities (Grant Numbers 2015ZCQ-SW-04 and YX2015-15).