Optimization of “Zaoheibao” wine fermentation process and analysis of aroma substances

Abstract

The “Zaoheibao” fruit with rich rose-scented grapes was used as the main material to optimize the fermentation process of “Zaoheibao” wine. Its composition in volatile aroma compounds was determined by headspace solid phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC–MS) analysis, indicating that “Zaoheibao” wine has strong ester, fruity and floral characteristics. The addition of 30 mg/L SO₂ and 200 mg/L yeast, and a fermentation time of 6 days were indicated as the optimal fermentation conditions, where 35 volatile aroma substances were detected and a relative ester content of 45.59% was achieved. Among the identified ester compounds, isoamyl ester, ethyl octanoate, ethyl acetate, 2-methyltetrahydrothiophen-3-one, 4-hydroxy-2-butanone, linalool and 2,4-dimethyl-1-heptene contributed mainly to the aroma characteristics of “Zaoheibao” wine. The obtained results will allow the production of higher quality “Zaoheibao” wine and serve as a guide to improve the characteristics of other wine varieties.

Keywords:

• “Zaoheibao” wine
• process optimization
• aroma substances

Introduction

The “Zaoheibao” grape is a new variety that has been bred at the Fruit Tree Research Institute of the Shanxi Academy of Agricultural Sciences after multiple mutations of the “Guibao” and “Zaomeigui” diploids using colchicine [1]. It is an Eurasian variety with large bunches of 430 g, with a conical shape and shoulders, and average length and width of 16.7 and 14.5 cm, respectively. The berries, with dense fruit kernels, are large, short oval with an average longitudinal diameter of 2.4 cm and a horizontal diameter of 2.3 cm. It should be noted that although the “Zaoheibao” grape has been promoted and planted in many places, its growth performance retains the original variety characteristics.

Wine is generally prepared by fermentation with yeast under various conditions [2,3], which can affect its quality, taste and volatile aroma. Especially the fermentation aroma generated by the yeast activity can significantly influence the aroma of the produced wine [4] In addition, the amount of yeast added plays an important role in the fermentation process, as it affects the metabolism of the yeast itself and the growth, reproduction, and conversion of alcohols. Low yeast amounts result in slow fermentation speed, while high yeast amounts lead to a cloudy wine body [5], which negatively affects its color. Thus, to select the right type of yeast, strains with strong alcohol production capacity should be first considered, followed by a strong ester production capacity and alcohol tolerance. Carrau et al. [6] analyzed the effect of the inoculation amount of Saccharomyces cerevisiae on the fermentation aroma, and concluded that a concentration of 10⁵ CFU/L increased the content of esters and monoterpenes and reduced the content of higher alcohols and medium-chain fatty acids.

The duration of the fermentation cycle can also affect the extent of wine fermentation. If it is too short, the fermentation will not be completed, while a very long cycle can increase the production cost. Liu et al. [7] studied the dynamics of volatile compounds during the traditional solid-state fermentation of Gutian Hong Qu glutinous rice wine, and found that although the extension of the fermentation cycle can increase the total amount of aroma substances of alkyd esters, the content of single aroma substances such as ɑ-terpineol, cedarol and ethyl isobutyrate is significantly reduced. Furthermore, during the wine fermentation process, the addition of SO₂ will change the pH value of the fermentation environment, which will ultimately affect the fermentation quality by affecting the activity of Saccharomyces cerevisiae and non-Saccharomyces cerevisiae. The addition of an appropriate amount of SO₂ can inhibit the formation and growth of harmful microorganisms and prevent the deterioration of wine. However, excessive use of SO₂ may alter its taste and flavor, while it is harmful for human health [8]. Sun et al. [9] found that during the brewing process, the concentration of isobutanol in the volatile aroma content increased with increasing SO₂, while the concentration of n-propanol, isoamyl acetate and ethyl lactate decreased, indicating that a SO₂ excess can negatively affect the wine quality.

To date, studies on the composition of volatile aroma substances in wine have focused mainly on classic varieties such as Chardonnay and Merlot [10,11]. It has been reported that among the volatile compounds, esters contribute significantly to the aroma and odor of wine, while they can also enhance its complexity [12], which is an important indicator of the quality of fruit wine and vinegar [13]. Moreover, for the analysis and classification of volatile aroma substances, various techniques, such as sensory evaluation analysis, principal component analysis (PCA) and cluster analysis, can be used [14]. However, no studies have been published on the analysis of volatile aroma components and the brewing process of rich “Zaoheibao” wine.

Thus, in this study, the fruit of “Zaoheibao” grape was used as the test material. An orthogonal experiment design was applied without adding external sugar to determine the optimal SO₂ and yeast amounts and optimize the fermentation cycle. Aiming to develop a fermentation process that would afford a wine with rich ester and fruity floral characteristics, headspace solid phase microextraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC–MS) was used to detect and analyze the composition of volatile aroma substances in “Zaoheibao” wine and to determine the optimal brewing conditions.

Materials and methods

Materials and reagents

The fruits of the “Zaoheibao” grape were obtained from the Shanxi Agricultural University Fruit Research Institute (Taigu, Shanxi) and the yeast starter (Saccharomyces cerevisiae) was purchased from Angel Yeast Co., Ltd. (Yichang, Hubei). Na₂SO₃ was used as the SO₂ source and was obtained from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.

Fermentation process

The fermentation process for the production of “Zaoheibao” wine is shown in Figure 1. In particular, fully ripe and undecayed “Zaoheibao” fruit was collected in August and the peel and juice were retained. After squeezing the juice, the grape juice (without the addition of external sugar) was poured into the fermentation tank. The required SO₂ amount for each treatment was then added in the form of Na₂SO₃ and the mixture was maintained at room temperature for 12 h. Afterwards, the appropriate amount of yeast, specific for fruit wine, which was previously activated and stirred at 35–40 °C in warm water until dissolved, was added to each test sample. The alcohol fermentation was performed at 28 °C. Samples of 20–30 mL were collected at different fermentation time intervals and were stored in an ultra-low temperature (-80 °C) refrigerator (Qingdao Haier Special Electric Co., Ltd.) for about one month. In October–December, they were dissolved in ice, frozen at ultra-low temperature, and centrifuged (Eppendorf) at 8228 g (8000 r/min) for 10 min. The supernatant was collected, placed (5 mL) in a headspace vial, and analyzed by HS-SPME and GC–MS at the Experimental Center of Shanxi Agricultural University.

Figure 1. Fermentation process from the “Zaoheibao” grape fruits to wine.

Determination of optimal yeast amount

The “Zaoheibao” grape juice was divided into four equal parts and 30 mg/L SO₂ was added in each sample. Then, 50, 100, 150 and 200 mg/L Angel fruit wine yeast were added to each sample, respectively, followed by fermentation at 25–28 °C for 6 days. The relative contents of ester volatile aroma substances detected for each sample were then compared to identify the optimal yeast amount for high quality “Zaoheibao” wine.

Determination of optimal SO₂ amount

The “Zaoheibao” grape juice was separated into four equal parts and 150 mg/L Angel fruit wine yeast was added in each sample. Then, 20, 30, 40 and 50 mg/L SO₂ were added to each sample, respectively, followed by fermentation at 25–28 °C for 6 days. In order to identify the optimal SO₂ amount, the relative content of ester volatile aroma substances detected for each sample was compared.

Determination of optimal fermentation time

The “Zaoheibao” grape juice was divided into three equal parts and 150 mg/L Angel fruit wine yeast and 30 mg/L SO₂ were added. The fermentation was performed at 25–28 °C and samples from the fermentation broth were collected after 2, 6 and 10 days to compare the relative content of ester volatile aroma substances.

Orthogonal optimization experiment

Based on the single-factor test, an orthogonal test was also performed to determine the optimal conditions for the fermentation of “Zaoheibao” wine. The factor levels are presented in Table 1 and the treatment tests are shown in Table 2. The relative content of esters in Table 3 is presented as an optimized value.

Table 1. Level table of the orthogonal test factors.

Level	SO2 (mg/L)	Yeast (mg/L)	Fermentation time (days)
1	20	100	2
2	30	150	6
3	40	200	10

Table 2. Table of orthogonal test analysis.

Treatment No.	A (SO2)	B (Yeast)	C (Fermentation time)
1	1	1	1
2	1	2	2
3	1	3	3
4	2	1	3
5	2	2	1
6	2	3	2
7	3	1	2
8	3	2	3
9	3	3	1

Table 3. Orthogonal test results for “Zaoheibao” wine.

Treatment No.	A(SO2)	B (Yeast)	C (Fermentation time)	Blank	Relative content of esters (%)
1	1	1	1	2	22.42
2	1	2	2	2	26.23
3	1	3	3	2	34.18
4	2	1	3	3	23.69
5	2	2	1	3	31.57
6	2	3	2	1	45.59
7	3	1	2	1	20.20
8	3	2	3	1	22.13
9	3	3	1	3	27.26
mean1	27.61	22.1	27.08	29.31
mean2	33.62	26	30.67	27.61
mean3	23.2	35.68	26.67	27.50
R	10.42	13.58	4	1.81

Extraction of HS-SPME

In brief, 5 mL of wine were placed in the headspace bottle and the stirrer was started. Equilibration was done in a 40 °C water bath for 15 min. The DVB/CAR/PDMS (American Thermo Fisher Scientific) extraction fiber head was inserted. Absorption was carried out at 40 °C for 30 min, and desorption of the extraction head at the GC inlet for 5 min, followed by automatic sample injection for GC-MS analysis.

GC–MS analysis

The GC–MS analysis of the volatile aroma compounds was performed on a Thermo Fisher (American Thermo Fisher Scientific) instrument using a HP-5MS chromatographic column and an inlet temperature of 280 °C. An initial temperature of 40 °C was applied, which was maintained for 3 min and then increased to 180 °C at a rate of 5 °C/min, where it was maintained for 1 min. Finally, the temperature was increased to 270 °C at 10 °C/min, where it was maintained for 5 min. High purity He was used as the carrier gas at a flow rate of 1 mL/min, and the inlet split ratio was 20:1. For the full-scan MS recordings, standard spectrum tuning was applied using an electron ionization (EI) source at 70 eV and 280 °C. The transmission line temperature was also 280 °C and the mass scan range was m/z 45–600.

Data processing and analysis

Microsoft Excel was used for data processing. The variations between the data were analyzed by single factor analysis of variance (ANOVA) and multiple comparisons using the Duncan test were performed. Moreover, to describe the odor of the volatile aroma substances in “Zaoheibao” wine, their relative content was determined and the description of odor from existing reports [15,16] were combined.

Results and discussion

Effect of yeast on the relative content of ester volatile aroma compounds in “Zaoheibao” wine

As the yeast dosage increased, the relative content of ester volatile aroma compounds also increased and reached a maximum of 39.08% when the yeast amount was 200 mg/L (Figure 2(a)). However, adding a large amount of yeast may increase the nutrients used as the yeast propagation medium in the fermentation system, which would in turn not favor the production and accumulation of volatile aroma compounds. Conversely, a very small yeast dosage can delay the start of fermentation and increase the time and cost of the process. Furthermore, it has been reported that the increase in the alcohol content during fermentation inhibits the yeast growth and induces the autolysis of the yeast cells, thus terminating the alcohol fermentation [17]. Consequently, as the yeast dosage increases, the inhibitory effect is enhanced and the continuous fermentation that generates aroma substances is impaired. Higher alcohols are generated in the amino acid biosynthesis pathway of yeast, and the amount of yeast added in the fermentation broth is changed to alter the corresponding amino acid content and further alter the higher alcohol content [18]. One of the synthetic pathways of ester substances such as ethyl acetate and isoamyl acetate is to synthesize alcohol and fatty acids produced by yeast under the action of esterase (reversible enzyme)[19]. Thus, a yeast dosage of 200 mg/L was identified as the optimal amount for the next test.

Figure 2. Effect of different factors on the generation of ester volatile aroma compounds during fermentation of “Zaoheibao” wine. Amount of yeast (a); SO₂ amount (b); fermentation time (c). Note: A, B, C, D indicate significant differences (p < 0.01).

Effect of SO₂ on the relative content of ester volatile aroma compounds in “Zaoheibao” wine

According to the graph in Figure 2(b), as the amount of SO₂ increased, the relative content of ester volatile aroma compounds in wine first increased and then decreased. In particular, when an amount of 30 mg/L SO₂ was added, the relative content of ester aroma substances reached a maximum of 37.76%. Moreover, the addition of an appropriate amount of SO₂ can affect the sterilization and enzyme killing during the wine brewing process [20]. However, a significant excess of SO₂ can greatly reduce the yeast activity and inhibit mixed bacteria, thus impairing the alcohol fermentation process and the formation of ester volatile aroma substances. Therefore, in this study, a SO₂ amount of 30 mg/L was indicated as appropriate for the next test.

Effect of fermentation cycle duration on the relative content of ester volatile aroma compounds in “Zaoheibao” wine

During the wine fermentation process using the previously determined yeast and SO₂ amounts, the content of esters initially increased with fermentation time and then decreased (Figure 2(c)). After fermentation for six days, the wine ester volatile aroma substances reached a maximum of 39.68%. Wine is a co-fermentation product of Saccharomyces cerevisiae in the early stage and non-Saccharomyces cerevisiae in the later stage. The fermentation cycle is short, the non-Saccharomyces cerevisiae has not completed the fermentation work, it produces less terpenoids and ketones, and the fermentation cycle is too long, bacteria cause the wine to rot and deteriorate, and produce acetaldehyde and hydrogen sulfide that affect the aroma of the wine [21]. It should be noted that a short fermentation time does not allow the completion of the fermentation process, leading to an insufficient production of volatile esters. In contrast, a very long fermentation time increases the growth of miscellaneous bacteria, thus preventing the generation of the desired ester volatile compounds.

Analysis of the “Zaoheibao” wine orthogonal test results

According to the range of difference between the parameters of each factor presented in Table 3, the amount of added yeast affected the most the content of ester volatile aroma compounds generated in “Zaoheibao” wine during fermentation, followed by the SO₂ amount and the fermentation time (B > A > C). Moreover, based on the Mean1, Mean2 and Mean3 values (Table 3), the optimal fermentation conditions were A₂B₃C₂. More specifically, as previously determined, the optimal SO₂ amount was 30 mg/L, the yeast amount was 200 mg/L, and the fermentation time was 6 days. Therefore, the fermentation of “Zaoheibao” grapes under the conditions of treatment 6 can produce “Zaoheibao” wine with strong ester and fruit aroma characteristics with a relative ester content of 45.59% out of the total volatile aroma content.

Furthermore, the F value obtained from ANOVA was higher for the yeast amount, followed by the SO₂ amount and the fermentation time (Table 4), while its trend was the same as that of the Mean values in Table 3. However, the F values of all the parameters affecting the ester content were not significant, suggesting that there may be interactions among various factors.

Table 4. ANOVA of the orthogonal test for “Zaoheibao” wine.

Source of variance	Sum of squared deviations (SS)	Degrees of freedom (df)	Mean square (MS)	F	P
SO2 amount	164.13	2	82.065	5.627	0.15
Yeast amount	286.45	2	143.225	9.82	0.09
Fermentation cycle	29.11	2	14.555	0.09	0.50
Error	29.17	3	14.585
Variation	508.86	8
R^2=0.943

Note: Source of variance represents the total variance of all observations, including the inter-treatment variation and error. SS represents the sum of the total square of the total variation of all observations. Degrees of freedom are defined as df = n-1 (n represents the number of levels for each factor). MS represents the degree of variation of observations, MS = SS/df.

Analysis of the volatile aroma components in “Zaoheibao” wine

The aroma compounds detected in this study were synthesized by yeast during alcohol fermentation. It is known that esters are mainly produced by the metabolism of fatty acids, amino acids, alcohols and aldehydes [22], while acetyl coenzyme A (acetyl-CoA) reacts with higher alcohols produced after the degradation of amino acids or sugars and has a typical fruity and ester flavor [23].

As shown in Table 5, almost the same types of volatile aroma substances were detected after the nine treatment processes for the fermentation of “Zaoheibao” wine. In particular, a total of 47 volatile aroma substances were detected, among which 16 were esters, such as isoamyl acetate and ethyl acetate, nine were alcohols, such as linalool, and five of them were acids. In addition, six aldols, five aromatic compounds, four alkanes and two furans were identified. However, their relative contents were significantly different, and the higher values were observed mainly for esters, alcohols and acids, with esters being the most volatile, providing the fruity aroma to “Zaoheibao” wine [24]. Esters play an important role in the mellow and pleasant wine aroma [25], making the odor of the wine more balanced and harmonious. The types of esters detected in this study were not considerably different, but their relative content significantly varied. Specifically, the highest relative content of ethyl acetate was obtained in the grape juice (treatment 0) and after treatments 3, 4 and 7 (Table 5). Moreover, the highest values for isoamyl acetate were detected after treatments 2, 5, 6 and 8, which improved the wine aroma and flavor, giving a strawberry and banana aroma. This sensory characteristic can be found in other non-elaborate grapes and wine [26,27], while these results were also consistent with the findings of other studies on BRS Rúbea and Cora wines [28]. Furthermore, treatment 1 produced a relatively high content of ethyl octanoate. Given that its threshold concentration is 5 µg/g [29], ethyl octanoate provides caramel and fruit flavors that give the “Zaoheibao” wine an aroma of wine brandy.

Table 5. Composition of volatile aroma substances (in %) in “Zaoheibao” wine, as determined from the orthogonal test.

Aroma substance	Juice and treatments										Odor description
	0	1	2	3	4	5	6	7	8	9
Ethyl palmitate	0.01	–	0.01	–	0.02	0.01	0.01	0.01	0.01	0.01	Wax, fruit and cream
Ethyl hexanoate	1.06	4.7	4.55	4.14	3.88	5.5	12.89	4.66	4.36	5.73	Green apple, floral, violet
Ethyl laurate	–	–	–	–	–	0.02	–	–	–	–	Sweet, floral, fruity, cheese
Ethyl isovalerate	–	–	0.02	0.01	–	–	–	–	–	–	Banana, sweet fruit
Isoamyl acetate	0.95	1.75	7.93	3.5	6.44	10.15	22.81	2.95	6.66	8.34	Banana, sweet fruit
Isobutyl acetate	0.07	0.06	0.17	0.16	0.17	0.22	0.49	0.1	0.17	0.14	Strawberry, fruity, floral
Ethyl acetate	27.28	6.03	7.06	19.66	7.07	9.19	5.43	6.23	6.13	4.8	Fruity, sweet
Hexyl acetate	–	–	0.05	–	0.02	0.09	0.32	–	–	0.06	Fruity, pear
Ethyl octanoate	0.36	6.15	4.38	2.62	3.48	4.09	2.67	4.14	3.06	4.83	Pineapple, pear, floral
Crystal violet lactone	–	–	–	–	–	0.02	0.01	–	–	–	–
Ethyl nonanoate	–	0.02	–	0.01	–	–	0.02	0.02	0.04	0.01	Fruit, banana
Ethyl caprate	0.18	3.3	1.62	0.58	2.24	1.58	0.46	1.65	1.61	2.84	Fruity, fat
Trans-4-decenoic acid, ethyl ester	–	0.04	–	–	–	0.01	–	–	–	–	Wax, pear, leathery
Ethyl butyrate	0.09	0.35	0.43	0.39	0.36	0.68	0.48	0.43	0.08	0.49	Cranberry, strawberry, fruity
Ethyl propionate	–	–	–	3.1	–	–	–	–	–	–	Pineapple
Isoamyl n-octanoate	0.02	0.02	0.01	0.01	0.01	0.01	–	0.01	0.01	0.01	Brandy aroma, sweet
Isoamyl alcohol	31.95	27.89	0.02	0.06	28.33	28.85	0.01	29.11	30.59	27.58	Mellow, pungent, bitter
Ethanol	0.09	0.57	0.05	–	0.71	0.18	0.02	0.49	0.22	1.19	Alcohol
Linalool	0.09	0.33	0.15	0.29	0.24	0.16	0.32	0.29	0.29	0.17	Musk, floral, fruity
α-Terpineol	–	–	–	0.1	–	–	–	–	–	–	Haitong flowers, sweet lilac, lily of the valley
2,3-Butanediol	–	–	–	0.19	–	0.1	–	0.01	–	–	Butter, cheese
(2R,3R)-(−)-2,3-butanediol	–	–	–	0.04	–	–	0.01	–	–	–	Butter, cheese
1-Pentanol	–	0.02	–	–	–	–	–	–	–	–	Bitter almond, fat
Phenethyl alcohol	–	–	–	–	–	0.05	–	–	–	–	Sweet rose
2-Cetyl alcohol	–	–	–	–	–	0.01	–	–	–	–	–
Oxalic acid	29.79	–	24.34	18.1	30.02	20.7	17.92	28.32	28.15	27.94	–
N-Benzoyl-L-isoleucine	–	–	33.79	30.64	–	–	20.33	–	–	–	–
N^6-Anisoyl adenylate	–	0.42	0.37	0.5	0.5	0.02	0.38	0.55	0.52	0.36	–
2-(Aminooxy)acetic acid	–	26.19	0.11	0.64	0.07	–	0.14	0.22	0.09	–	–
L-homocysteine	–	–	–	–	–	0.01	–	–	–	–	–
2,4-Di-tert-butylphenol	–	–	–	0.02	–	–	–	–	–	–	–
3,5-Di-tert-butylphenol	–	–	0.01	–	–	–	–	–	–	–	–
Phenol	1.02	–	–	–	0.18	–	–	–	0.15	–	Special smell
2-Methyltetrahydrothiophene-3-one	0.02	–	0.06	0.08	0.05	–	–	–	0.05	0.04	–
4-Hydroxy-2-butanone	–	0.02	–	–	–	0.01	0.04	0.03	0.01	–	Cream, fat
3-Hydroxybutyraldehyde	–	–	0.01	–	0.01	–	–	–	0.01	–	–
Ethylbenzene	0.02	0.13	0.16	0.16	0.11	0.03	0.16	0.17	0.16	0.16	Fragrant smell
m-Xylene	–	–	–	–	0.49	–	0.65	–	–	–	–
o-Xylene	0.01	–	–	–	–	0.23	–	–	0.57	–	–
p-Xylene	0.05	0.62	0.76	0.85	0.11	0.94	0.1	0.86	0.24	0.77	Fragrant smell
1,3-Di-tert-butylbenzene	0.08	0.08	0.13	0.26	0.1	0.16	0.09	0.12	0.09	0.09	–
2,4-Dimethyl-1-heptene	–	–	–	1.01	–	0.19	–	–	–	–	–
1,1-Diethoxyethane	0.15	3.15	0.36	0.9	0.32	0.05	0.42	2.2	0.66	0.33	Fruity
2,5-Dimethylhexane-2,5-hydrogen peroxide	–	–	–	0.06	–	–	–	–	–	–	–
2,4,5-Trimethyl-1,3-dioxolane	0.13	1.25	0.22	0.82	0.08	–	–	0.7	0.09	–	–
4-Methyl-2-(2-methylpropenyl) tetrahydro-2H-pyran	–	0.02	–	0.06	–	–	0.01	0.02	–	0.01	–
2,5-Diethoxytetrahydrofuran	–	0.01	–	–	–	–	–	–	–	–	–

Note: T0, grape juice; T1–T9, wine (see Table 2); - represents not detected;- in the odor description indicates lack of information about odor in the known literature; the values in boldface indicate the highest values of isoamyl acetate, ethyl acetate and ethyl octanoate among the treatments.

The fermentation under the optimized conditions of treatment 6 afforded the highest relative content of volatile aroma esters. In particular, a total of 26 volatile aroma substances were detected, which corresponded to 86.19% of the total detected compounds in treatment 6. In addition, 45.59% out of 86.19% were esters, indicating the efficiency of this method to prepare a wine with improved ester and fruity aroma features. In general, the four major esters that are mainly identified in the main body of fermented wine are isoamyl acetate, ethyl hexanoate, ethyl octanoate and ethyl acetate [30,31]. The same ester volatile compounds were observed in this study as well (treatment No. 6). Among them, the highest relative content was detected for isoamyl acetate (22.81%), which provided banana and sweet fruit flavors and together with ethyl octanoate and ethyl acetate determined the aroma characteristics of “Zaoheibao” wine.

Earlier studies [7] have reported that β-ionone, damascone and various terpenes are the main aroma components of “Cabernet Sauvignon” wine. However, β-ionone and damascone were not detected in this study. Instead, 2-methyltetrahydrothiophene-3-one, 4-hydroxy-2-butanone and 2,4-dimethyl-1-heptene were identified along with two types of terpenoids, which also significantly contributed to the characteristic aroma of “Zaoheibao” wine.

Alcohols are also an important component of aroma in wine. Herein, the relative content of alcohols obtained for the nine different treatments significantly varied due to the fermentation conditions of each experiment. However, additional effects should be considered as well, because alcohol derivatives are also produced by the decarboxylation and deamination of amino acids of sugar metabolism [32]. Among the detected alcohols of this study, the highest content was found for isoamyl alcohol (Table 5), which has been shown to increase the taste of herbs in wine [33].

A few types of acids were also detected after all the fermentation treatments, but, in some cases, their relative content was relatively high. In general, acid substances give a pungent odor and bad taste, which does not favor the production of wine with high flavor quality. Other compounds that were identified during the experiment were alkanes, aromatics and aldehydes, which also significantly contribute to the aroma and characteristic flavor of wine.

PCA of “Zaoheibao” wine orthogonal test

Although the composition of different volatile aroma substances in various samples can be identified by GC–MS analysis, the main aroma substances in each sample cannot be determined [34]. PCA is a unified tool designed to reduce the number of variables with little information loss [35]. Here, the main component analysis was performed on the volatile aroma substances of “Zaoheibao” grape juice and wine. The selection criteria were 26 types of volatile aroma substances with a relative content of ≥0.1%. Two principal components explained 83.8% of the total variability, i.e. 61.41% and 22.39%, respectively. As shown in Figure 3, all the ten treatment samples clearly covered the entire PCA diagram. Treatment 6, whose characteristic aroma substance is isoamyl alcohol, occupied the upper right corner of the diagram.Grape juice (treatment 0) was detected at the lower left corner of the PCA diagram. The related aroma substance is A4 (ethyl acetate). Treatments 1, 3 and 7, and 2, 4, 5, 8 and 9 were grouped into two different categories, and the representative aroma substances individually are A1 (ethyl caproate), A7 (ethyl decanoate) and A2 (isoamyl acetate), A5 (hexyl acetate) and A26 (2,4,5-trimethyl- 1,3-dioxolane).

Figure 3. “Zaoheibao” wine and grape juice main components. Note: Left panel, treatments (wine T1–T9; grape juice T0; see Table 2). Right panel, aroma substances.

Volatile aroma and odor of “Zaoheibao” wine

“Zaoheibao” wine consisted of several types of aroma substances and its odor profile was hierarchical with a complex aroma structure (Figure 4). The aroma profile of all the treatments in the orthogonal test was mainly fruity and floral, while some samples had a strong mellow odor. Esters, aldehydes, ketones, aromatic compounds and alkenes provided mainly floral, fruity and creamy aromas, which greatly enriched the flavor of “Zaoheibao” wine. It should also be noted that no unpleasant odors, such as sour odor or spoilage, were detected [36]. Although the middle-scent profiles of the “Zaoheibao” grape juice and the fermented wine did not differ significantly, the results of the main component analysis indicated that the fermentation process considerably affects the flavor of fermented wine.

Figure 4. Aroma substance and odor contour map of “Zaoheibao” wine based on the orthogonal test. Note: Treat 1–9 represent different treatments (various amounts of SO₂ and yeast starter and fermentation time; see Tables 1 and 2).

Conclusions

In the wine-making process, single factors and their interaction can significantly and differently affect the formation of wine aroma substances. In this study, the influence of the addition of SO₂, the fermentation cycle and the addition of yeasts on the production of aroma substances in the “Zaoheibao” wine showed that the aroma substances of the “Zaoheibao” grape juice are mainly ethyl acetate, and the compound that has the main contribution to the aroma is isoamyl alcohol in treatment 6 (30 mg/L SO₂; 200 mg/L yeast, 6 d fermentation time). The aroma substances in treatments 1 (20 mg/L SO₂; 100 mg/L yeast; 2 d fermentation time), 3 (20 mg/L SO₂; 200 mg/L yeast; 10 d fermentation time) and 7 (40 mg/L SO₂; 100 mg/L yeast; 2 d fermentation time) are mainly ethyl caproate and ethyl caprate. The other group of related characteristic aroma substances are isoamyl acetate, hexyl acetate and 2,4,5-trimethyl-1,3-dioxolane. This study indicated that for the production of high aroma and flavor quality “Zaoheibao” wine with the appropriate amount of aroma components, it is important to control the optimal amount of SO₂ and yeast that are added for fermentation, as well as the fermentation time. Thus, apart from enhancing the quality of “Zaoheibao” wine, the results of the present study could significantly contribute to the improvement of the content of volatile aroma substances in other wine varieties.

Acknowledgements

The authors thank Guofeng Duan (College of Horticulture, Shanxi Agricultural University) and Rufu Wang (College of Food Science and Engineering, Shanxi Agricultural University) for guidance with this experiment.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by Research and Development Plan (Guide) Project Planning Task of Shanxi Privice (201703D221028-1), Shanxi Province Science and Technology Key (Guide) Project (2015-TN-10-10).