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Analyses of the volatile compounds in cherry wine during fermentation and aging in bottle using HS-GC-IMS
2021 年 27 巻 4 号 p. 599-607
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2021 年 27 巻 4 号 p. 599-607
Flavor is one of the most important characteristics and qualities of wines and fruit wines. In this study, HS-GC-IMS combined with the analytical method of principal component analysis (PCA) were used to inquire the volatile compounds in cherry wine during fermentation and aging in bottle. The fingerprints of flavor substances were established by the topographic plots, and identified 36 signal peaks that corresponded to 29 compounds. The volatile compounds were mainly esters, alcohols, aldehydes and ketones. Aldehydes were the main volatile compounds of cherry juice. Due to the metabolism of yeast, there was significantly increase in volatile compounds during fermentation. After the fermentation, cherry wine was stored in bottles for wine age. The volatile compounds changed little, and the flavor became more ethereal during aging in bottle. Through the evaluation, a spontaneous malolactic fermentation (MLF) happened during aging in bottle within two years, bringing about quality stability, deacidifying and sensory promotion of the cherry wine.
Cherry is one of the most favorite popular fruits worldwide. It is in relatively high content of nutriment, such as carbohydrates, organic acids, amino acids, vitamins, anthocyanidin and mineral substance (Maria et al., 2015; Sun et al., 2012). Due to their nutritional properties and beneficial healthy effects, cherries are greatly appreciated worldwide (Souza et al., 2014; Blando and Oomah, 2019). In China, Chinese cherry (Prunus pseudocerasus L.), sweet cherry (Prunus avium L.), Prunus tomentosa Thumb. and sour cherry (Prunus cerasus L.) were the most widely grown varieties of cherry.
Flavor is the first feeling when enjoying cherry products for consumers. In some researches, some aldehydes and alcohols were the major volatile compounds in sweet cherry (Maria et al., 2015). The volatile compounds became more complicated after fermentation. The volatile compounds was a basic index in evaluating the quality of wines, as its contribution to the final properties. The special flavor causing from the volatile compounds in wines lies on the processed materials, the culture, the fermentation processing method and the process during aging (Fleet, 2003; Esteban et al., 2019; Benito, 2018).
In previous researches, thousands of volatile compounds had been tested from wines. However only a few could be smelled. A number of volatile compounds consisting of esters, alcohols, terpenoids, organic acids and phenolic acids could be found in cherry wines by using GC-MS, gas chromatography-olfactometry (GC-O), odor activity values (OAV) and other analysis software (Niu et al., 2019; Sun et al., 2012). After malolactic fermentation using lactic acid bacteria (LAB) in cherry wines, esters were the main volatile compounds. Esters, alcohols, aldehydes, organic acids, ketones and phenolic acid were detected by using GC-MS (Li et al., 2019; Sun et al., 2016). Hence many research methods have been successfully established by measuring volatile compounds using various analytical techniques. People can use volatile compounds to differentiate wines in the raw materials, the cultures, the processing method and the substances formed during aging, aiming to enhance and differentiate the characters of cherry wines (Ferreira et al., 2013; Li et al., 2019; Simon et al., 2014; Sun et al., 2014; Sun et al., 2018; Xiao et al., 2017; Yin et al., 2009).
Ion Mobility Spectrometry (IMS) is an analysis apparatus and could separately monitor gasiform compounds in a mixture state that perform very fast. The volume of IMS equipment is small and with a light weight. When it works, it expends less energy. This analytical instrument is based on coupling GC with IMS, and could detect the compounds without any other pretreatment for trace analysis. GC-IMS can provide a quick, sensitive, nondestructive and economical tool to detect trace substances for food process, stability, adulteration, quality, safety control and off-flavor detection (Cavanna et al., 2020; Daniele et al., 2019; Vautz et al., 2006; Witkiewicz et al., 2016). The GC-IMS in combination with headspace sampling (HS) could be operated at atmospheric pressure, no sample pretreatment, fast analysis time, low detection limits and good selectivity (Natalie et al., 2017; Roman et al., 2017; Wang et al., 2020; Yang et al., 2020). For example, HS-GC-IMS has more specific identification of substances and no need of vacuum pump compared with GCMS. Additionally, it is less cost expensive (Roman et al., 2017). HS-GC-IMS could detect plentiful volatile compounds including alcohols, esters, aldehydes, ketones and aromatics from food and agricultural products (Zheng et al., 2018).
Thus far, few researchers monitored volatile compounds in cherry wines during fermentation and aging in bottle. In this study, HS-GC-IMS combined with the analytical method of PCA were used to detect the volatile compounds in cherry wine during fermentation and aging in bottle. The fingerprints of flavor substances were established the topographic plots, furthermore the changes of volatile compounds could be obtained. Compared with the other classical techniques, the results will provide a fingerprints of flavor substances, which is more visualized and accurate.
Preparation of cherry wine Healthy and ripe cherry of “Tieton” from a local farms in Rizhao city (Shandong) were selected as main materials in this study. Cherry were washed, manually removed the pit, crushed and destemmed. After the enzymolysis of pectinase, sugars was added to make the percentage of soluble solids (Brix) to 25, thus the alcohol content of final product could up to 12 % (v/v). 150 mg/L of sulfur dioxide (SO2) was added to inhibit spoilage organisms and prevent oxidation (Mandrilea et al., 2020). Commercial Saccharomyces cerevisiae strains of Active Dry Wine Yeast (300 mg/L, Angel Yeast Co., Ltd, China) was added as the promoter of fermentation. Alcohol fermentation was conducted under the static condition at 23 °C over 7–10 days. During fermenting, the fermented cherry pulp shake twice per day. After fermentation, the fermented cherry pulp was centrifuged (2808 G; 10 min) to get rid of cherry pomace and yeast. Afterwards, it was bottled and placed in the shade until further studies.
Collection of samples Samples were collected on day 0 (CJ, cherry juice, 0 % v/v), day 2 (SPF, start of primary fermentation, 2.37 % v/v), day 5 (MPF, middle of primary fermentation, 8.63 % v/v), day 8 (EPF/AY0, end of primary fermentation, 12.38 % v/v), age 1 (AY1, aging in bottle for one year, 12.25 % v/v), age 2 (AY2, aging in bottle for two years, 12.20% v/v), age 3 (AY3, aging in bottle for three years, 12.15 % v/v), age 4 (AY4, aging in bottle for four years, 12.16 % v/v). All the raw cherry was collected from the same farm for those years. Samples of cherry juice and primary fermentation were stored recently in bottling at 4 °C until analysis. Samples of aging in bottle were produced in corresponding year within four years, and stored in a cool dark place at room temperature.
Analytical Methods The volatile compound analysis of cherry wine samples were performed on a GC-IMS instrument (FlavourSpec, Gesellschaft für Analytische Sensorsysteme mbH, Dortmund, Germany) combined with a headspace sampling unit (CTC Analytics AG, Zwingen, Switzerland) that can be directly sampled without other pretreatments. The analysis conditions of gas phase-ion mobility spectrometry unit and automatic headspace sampling unit were given on Table 1. All samples were tested in triplicate.
| Gas phase-ion mobility spectrometry unit | |
| Analysis time | 20 min |
| Column type | FS-SE-54-CB-1 15 m ID:0.53 mm |
| Column temperature | 60 °C |
| Carrier gas/drift gas | N2 |
| IMS temperature | 45 °C |
| Automatic headspace sampling unit | |
| Injection volume | 100 µL |
| Incubation time | 10 min |
| Incubation temperature | 35 °C |
| Syringe temperature | 40 °C |
| Incubation speed | 500 rpm |
Volatile compounds were detected by matching retention index (RI) and the drift time of standard compounds with the built-in NIST database and IMS database in the GC-IMS library (Gesellschaft für Analytische Sensor-systeme mbH, Dortmund, Germany).
The instrumental analysis software includes VOCal and three plugins (G.A.S., Dortmund, Germany) and GC-IMS Library Search. VOCal can analyse the spectrogram, in which one peak expresses a kind of volatile compound. The spectral differences can be directly compared in samples by the Reporter plugin using the topographic plots. The Gallery plot plugin can intuitively and quantitatively show the fingerprint comparison in cherry wines during the process of fermentation and aging in bottle. The dynamic PCA plugin can be used to analyze and determine the unknown quickly by cluster and similarity analysis.
HS-GC-IMS topographic plots of cherry wines during the process of fermentation and aging in bottle Flavor is one of the most important characteristics for an alcoholic beverage. In order to compare with the 2D-topographic plot of cherry wine visually, HS-GC-IMS exploring results of cherry wines were showed in a pseudocolor map (Fig. 1). The color of the figure stands for signal value, in which the red perpendicular line (left side) is a reactive ion peak. The red or blue color manifests strong or weak of the signal, which means the content of the volatile compound is higher or lower. Thirty-six signal peaks were identified in cherry wine including twenty-nine detected qualitative compounds and seven unknowns. It was composed of eleven esters, seven aldehydes, five alcohols, four ketones, one pyrazine and one acid.
Changes in the two-dimensional (2D)-topographic of volatile compounds in cherry wine. (A) the process of fermentation, (B) the process of aging in bottle.
All samples were tested in triplicate.
CJ: cherry juice; SPF: start of primary fermentation; MPF: middle of primary fermentation; EPF/AY0: end of primary fermentation; AY1: aging in bottle for one year; AY2: aging in bottle for two years; AY3: aging in bottle for three years; AY4: aging in bottle for four years.
The aldehydes was most prevalent in cherry juice (CJ). Respectively benzaldehyde, (E)-2-hexenal, butanal, hexanal, ethyl acetate, butanol, 3-methyl-1-butanol and 2-methyl-1-propanol were the main volatile compound. Those compounds including benzaldehyde, hexanal, butanol, 3-methyl-1-butanol, butanal and (E)-2-hexenal were reported as the key volatile compounds of the cherry in some studies (Maria et al., 2015; Zlatic et al., 2017). The volatile compounds became more complicated after fermentation. The esters were the most prevalent, respectively ethyl acetate, ethyl butanoate, ethyl propionate, ethyl octanoate, isoamyl acetate, ethyl hexanoate and isobutyl acetate were the key volatile compounds of cherry wine. Ethyl propionate and isobutyl acetate were not reported previously in cherry wines (Sun et al., 2012; Li et al., 2019; Xiao et al., 2017; Sun et al., 2018; Xiao et al., 2014). Most of esters contribute toward the pleasant and fruity fragrance of cherry wine.
Despite of ethanol, propanol, 3-methyl-1-butanol and 2-methyl-1-propanol were the key volatile compounds of alcohols, which can impart fermented aroma produced during alcoholic fermentation in wines (Xiao et al., 2017). Furthermore, 3-hydroxy-2-butanone, acetone and pentanal were also playing a significant role in cherry wines (Fan et al., 2020). Propanol, butanol, hexanol, 3-methyl-1-butanol and 2-methyl-1-propanol were detected as higher alcohols, which were produced through glycolysis of sugar or the Ehrlich pathway by saccharomyces cerevisiae (Hazelwood et al., 2008). Formation of higher alcohols primarily depends on fermentation temperature, amino acids, fermenter size and process of aging (Mendes et al., 2019; Liu et al., 2018).
Fingerprint analysis of volatile compounds during fermentation The peak intensity of some volatile compounds varied greatly in cherry wine during fermentation, which were chosen to build the Gallery Plot (Fig. 2). The light of each square was based on the peak intensity, and it was visualized and accurate to compare the changes of fermentation, which provided a fingerprints of flavor substances. One row represents a type of sample, and one column represents a type of volatile compound in the fingerprint.
The Gallery Plot of volatile compounds in cherry wine during fermentation.
All samples were tested in triplicate.
CJ: cherry juice; SPF: start of primary fermentation; MPF: middle of primary fermentation; EPF: end of primary fermentation;
As described above, the aldehydes can be directly detected that benzaldehyde, hexanal and (E)-2-hexenal formed the main volatile compounds as showed in Fig. 2. Those aldehydes might be derived from lipids through some ways, including oxidization and β-oxidization through lipoxygenottom enzymes (Maria et al., 2015). Butanol, 2-methyl-1-propanol, 3-methyl-1-butanol, butanal and ethyl acetate also existed in the fruit of cherry. After fermentation process, the content of most volatile compounds increase, except for some aldehydes. Three aldehydes (benzaldehyde, hexanal and (E)-2-hexenal) decreased due to the volatilization and degradation during fermentation.
On account of the metabolism of saccharomyces cerevisiae, the majority of esters and alcohols increased, including ethyl acetate, ethyl octanoate, ethyl hexanoate, ethyl propionate, ethyl butanoate, isoamyl acetate, isobutyl acetate, propanol, 3-methyl-1-butanol and 2-methyl-1-propanol. Furthermore, 4-methyl-2-pentanone, acetone and pentanal also increased obviously. Some esters produced during sluggish substrates esterification. Thereinto, ethyl esters probably generated through yeast enzymatic metabolism and ethanol decomposition of acetyl-CoA generated through the synthesis and degradation of aliphatic acid (Niu et al., 2011).
At the start of primary fermentation (SPF), 3-hydroxy-2-butanone, butanal, propyl acetate increased obviously. Along with fermentation process, those three compounds decreased, which might be as the mesostate to transfer into other compounds of metabolism in fermentation. The critical factor might be the effect of yeasts type (Cullere et al., 2004). All in all, the content of esters and alcohols got a large promotion during the fermentation process, and played an important role as the key volatile compounds in cherry wine.
Fingerprint analysis of volatile compounds during aging in bottle The fingerprints analysis of volatile compounds in cherry wine during aging in bottle was given in Fig. 3. After the primary fermentation, cherry wines were stored in bottles for several years. Some compounds increased, including ethyl isobutanoate, ethyl isovalerate, ethyl 2-methylbutanoate, hexanol and acetic acid. The content of all ketones and three esters decreased obviously, including of ethyl octanoate, isoamyl acetate, isobutyl acetate, 3-hydroxy-2-butanone, 4-methyl-2-pentanone and acetone. Furthermore, the content of some volatile compounds including of 2-methyl-1-propanol, 3-methyl-1-butanol, ethyl butanoate, ethyl propionate, ethyl acetate and propanol were high and constant in cherry wines. Excessively high content of fusel oil may bring about unpleasant flavor, so generally discarded. However, fusel oil may as the raw material to synthesize or esterify other compounds (Lee et al., 2011). Whereas, the content of 2, 6-dimethylpyrazine, butanol, pentanal and 2-pentanone increased during AY0 to AY2, then decreased after AY2. This agreed with the phenomena of malolactic fermentation (MLF) (Sun et al., 2018), which mean that a spontaneous malolactic fermentation might happen during AY0 to AY2. As the secondary fermentation process, MLF is usually undertaken after the completion of primary fermentation. Consequently, it increases quality stability, deacidifying and sensory promotion of the wines (Li et al., 2019; Sumby et al., 2019).
The Gallery Plot of volatile compounds in cherry wine during aging in bottle.
All samples were tested in triplicate.
AY0: end of primary fermentation; AY1: aging in bottle for one year; AY2: aging in bottle for two years; AY3: aging in bottle for three years; AY4: aging in bottle for four years.
Cluster analysis of cherry wines Principal component analysis (PCA) is an important technique in data analysis that uses multivariate linear transformation and discrimination analysis to choose some interrelated variables. When there was a high degree of correlation between the original variables, new variables required the less (Sebzalli and Wang, 2001). The PCA model could be used as the separation model, by the time of the cumulative contribution rate exceeded 60% (Zhang et al., 2020).
As shown in Fig. 4A, the cumulative contribution rate was 98%. The volatile compounds of cherry juice (CJ) were obviously different from processed primary fermentation (SPF, MPF, EPF), and the compounds of SPF, MPF and EPF were very similar. The main contributing volatile compounds of CJ to the differentiation were hexanal, acetone and (E)-2-hexenal. Propyl acetate, 3-hydroxy-2-butanone, butanal, propionaldehyde and other compounds contributed to SPF, MPF and EPF.
The PAC analysis in cherry wine during fermentation (A) and aging in bottle (B).
All samples were tested in triplicate.
CJ: cherry juice; SPF: start of primary fermentation; MPF: middle of primary fermentation; EPF/AY0: end of primary fermentation; AY1: aging in bottle for one year; AY2: aging in bottle for two years; AY3: aging in bottle for three years; AY4: aging in bottle for four years.
Differing from cherry juice, it indicated that the volatile compounds changed not much after the start of primary fermentation. As presented in Fig. 4B, the cumulative contribution rate was 79%. The volatile compounds of cherry wine were different from aging in bottle (AY0-4) in the distribution map, and the compounds of AY1, AY3 and AY4 were relatively similar. The major contributor of AY0 were isobutyl acetate and isoamyl acetate, simultaneously 2, 6-dimethylpyrazine, pentanal and propanal to AY2. It demonstrated that the content of volatile compounds mainly changed in the process of AY2, indicating that a spontaneous MLF happened. The dimensionless data had a fine representation of the original data.
In this study, changes of volatile compounds were researched by HS-GC-IMS and PCA in cherry wines during fermentation and aging in bottle. The fingerprints of flavor substances were established by the topographic plots, and 29 compounds were identified. In the CJ, the aldehydes were the most prevalent volatile compounds. Fermentation results showed that the volatile compounds were complex detected in cherry wines, particularly the esters and alcohols. Among them, ethyl propionate and isobutyl acetate had not been reported previously. Furthermore, some compounds (including butanal, 3-hydroxy-2-butanone, propyl acetate) increased obviously at the SPF, but decreased along with fermentation process. During aging in bottle, the content of some compounds were high and constant. All ketones and three esters decreased obviously, while some others increased. In addition, clustering analysis ulteriorly demonstrated the differences and similarities during the production of cherry wines. It indicated that the compounds of SPF, MPF and EPF were similar, meanwhile AY1, AY3 and AY4 were relatively similar. Through the evaluation of wine during bottle storage, a spontaneous MLF happened during aging in bottle within two years. It meant that the wine should be stored in bottles at least two years for winemaker.
