Influence of Low Temperature Enzyme Maceration Techniques on Volatile Compounds of Semi-dry Wine Made with cv . Premier of Rabbiteye Blueberries ( Vaccinium ashei )

A low temperature enzyme maceration treatment was conducted during fermentation process of semi-dry cv. Premier blueberry wine. The aim of this study was to investigate the influence of maceration conditions on the wine aroma. As a pre-treatment, blueberry must was divided into 6 samples which were respectively treated by pectinase with 6 different maceration conditions at 6°C 1day, 6°C 2 days, 6°C 3 days, 16°C 1 day, 16°C 2 days and 16°C 3 days. After that wines were obtained by fermentation with Saccharomyces cerevisiae. Volatile compounds of wines were analyzed by GC-MS. Overall, the typical aroma compounds of semi-dry cv. Premier wines were constituted by three groups of organic compounds including esters, alcohols and fatty acids. Isoamyl acetate, ethyl caprylate, ethyl decanoate, 2-phenethanol and 3-methylbutanoic acid, which occupied 60% of the typical volatile aromatic compounds, all had higher Odor Activity Values (OAVs) in 6°C 3 days than other conditions. Maceration temperature and time had a significant effect on concentration and varieties of wines aroma substances. The results presented will help to better understand the aroma winemaking potential of this variety.


INTRODUCTION
Blueberry belongs to the Vaccinium family.The fruit of the blueberry has a good taste and it has been widely used to make jam, juice and wine in the market.Rabbiteye blueberry (Vaccinium ashei) is a type of blueberry that famous for its high antioxidant activity and radical scavenging capacity and is valued for their thick skin and high phenolics (Wang et al., 2011;Su and Chien, 2007).Cv.Premier is one of the rabbiteye cultivars, which has been widely cultivated in China.
According to recent studies, Flavor of the wine has been found to be an important aspects when consumers consider purchasing wines (Butkhup et al., 2011).Wine aroma and flavour are composed of hundreds of volatile chemical compounds generating from the berry, winemaking and aging process (King et al., 2010).Highclass wines have complicated mutual effect between the volatile aromatic compounds.
Harvesting fruit at specific stages of ripeness decides the style of wine.Once harvested, specific processing techniques and fermentation strategies will further determine the aroma and flavor development of the wine.Some studies have been carried out to determine the volatile compounds contributing to various character of wine (San-Juan et al., 2011;Escudero et al., 2007).Over 800 aroma compounds have been identified in wine.The combination of them forms the character of wine and differentiates one wine from another (Komes et al., 2006).
Wine aroma was affected by various factors.Some influencing factors, such as pre-fermentative maceration, wine yeast strains and application of malolactic fermentation, would make a big difference during vinification (Styger et al., 2011).Enzymetreated wines show significant improvement which terpenes and 2-phenylethanol concentration increased in four white varieties (Valcárcel and Palacios, 2012).Strain with higher nitrogen demand produces higher content of esters during wine fermentation while the concentration of alcohols decreases (Torrea et al., 2002).
The reasonable choices of temperature and skin contact time are important factors to be considered in the low temperature enzyme maceration, for example, color quality is improved with lower temperature (Gómez-Plaza et al., 2000), polysaccharide and proanthocyanidin concentrations increase during longer maceration when color and anthocyanin concentration do the opposite (Gil et al., 2012).Enzyme maceration treated wines can give a promotion of polymeric pigment, but less monomeric anthocyanins than nonenzyme-treated wines (Parley et al., 2001).The concentration of esters and terpenols would increase in wine with low-temperature prefermentative maceration process (Salinas et al., 2003).So the maceration condition is considered to be one of the major factors of the wine quality.
No previous report is seen regarding the aroma components analysis on blueberry wine and the influence on blueberry wine aroma quality caused by different fermentation strategies.The present work is to monitor the volatile aromatic components in semi-dry cv.Premier blueberry wine made in different low temperature enzyme maceration techniques and to choose a better process and optimize the brewing technology of blueberry wine.

Blueberry samples:
All experiments were performed in triplicate.Fresh cv.Premier of rabbiteye blueberries were obtained during a harvest in 2011 from blueberry plantation located in the central-eastern of China.A portion of the sample was analyzed immediately for oenological parameters, while the remainder was frozen and stored at -20°C for subsequent making wine.Reducing sugar concentration of the berry was 99.8 g/L, The pH was 3.6 and the concentration of titratable acid was 7.2 g/L in the berry.

Wine samples:
The berries were crushed into mash and then treated with 0.2 g/kg pectinase (Lafase He Grand Cru from Laffort) and 60 mg/L SO2.The prepared mash was divided into 6 flasks (500 mL) equally.Six samples were separately treated with low temperature maceration condition of 6°C 1 day, 6°C 2days, 6°C 3 days, 16°C 1 day, 16°C 2 days and 16°C 3 days.Once maceration step was completed, the crushed blueberry masses underwent the following process.After maceration, the wine samples were adjusted to 180.0 g/L of fermentable sugar with sugar and added 0.2 g/L yeast, which was activated by blueberry juice for about 15 min at 35°C.Then the main fermentation was carried out at 20°C and ended when the sugar content was below about 10.0 g/L.After centrifugation (4000 rpm) and press, the wine samples were carried out fining separation and sediment removal.Then the wines were sealed and stored at 16°C for one week before being proceeded with preliminary treatment for GC-MS.Winemaking process flow path chart was presented in Fig. 1.

Liquid-liquid extraction of wine volatile compounds:
Wine samples (100 mL) were extracted three times by shaking with 1 mL of internal standard (a solution of 41.75 ug/mL 2-octanol in dichloromethane) and 50, 30, 30 portions of dichloromethane.The extracts were mixed and dehydrated with anhydrous sodium sulfate.Then the dichloromethane extracts were stored at -5°C for 24 h in order to separate the frozen water from the organic phase.After 24 h, the extracts were slowly concentrated to 2 mL and then to 1 mL with a rotary evaporator at 0~5°C.The concentrate (1 mL) was stored in a glass screw-top vial at 5°C.

GC-MS analysis condition:
The extracts were analysed by GC-MS on SHIMADZU GC-MS-QP 2010 gas chromatography-mass spectrometry, equipped with a 30 m×0.25 mm (i.d.), 0.25 µm coating thickness DB-5 fused silica capillary column.The carrier gas was helium at 1 mL/min and split ratio was 30:1.The temperature program was kept at 40°C for 2 min and raised to 180°C at 4°C/min and then raised to 250°C at 10°C/min for 6 min.The transfer line temperature was 230°C and the injection port temperature was 250°C.
Compound identification: The mass spectrometric data of GC-MS analysis was got from the standard database of NIST147, NIST27 and WILEY7.The identification of volatile compounds was confirmed by injection of pure chemicals as reference for retention times and comparison of their Retention Indices (RI), determined by using an alkane standard mixture (C 8 -C 20 ).The pure chemicals and n-alkanes were injected at the same condition as the samples.
Classic enological parameters: Reducing sugars, pH and total acidity were measured according to International Organization of Vine and Wine (O.I.V.) methods.
Most of the compounds identified in this study were also found in relevant studied wines (Aznzr et al., 2003;Angioni et al., 2012).Compared with the wine samples after the low-temperature treatment at 6°C 1 day, 6°C 2 days and 6°C 3 days, the blueberry wine samples after the treatment at 16°C 1 day, 16°C 2 days and 6°C 3 days can be detected with volatile aroma components including pantolactone, ethyl laurate, 4hydroxybutanoate methyl, 2-phenylethyl acetic acid ester and benzyl alcohol, which have not been identified in the low-temperature treatment process at 6°C.Five compounds were only identified in the samples at 6°C, which included 2-hydroxy-propanoic acid ethyl ester, 2-6-dimethyl-7-octene-2-6-diol and quinic acid.As a result, pre-treatments at 6°C and 16°C both had their own characters and advantages.In addition to esters, alcohols and fatty acids, the two other chemical substances including 2 h-pyran-2, 6 (3 h)-dione and 4-ethenyl-2-methoxy phenol have been detected in all wine samples.But the difference of these two low-content chemical compounds was not obvious among 6 wine samples.In order to choose a better maceration condition, the analysis was based on the concentration of the aroma substances and relevant OAVs.
OAVs are calculated to evaluate the influence of low temperature maceration on wine aroma.The OAV is obtained as the ratio of compound concentration to its Odor Perception Threshold (OPT) value, which is to assess the influence of the single aromatic compounds produced during maceration (Chaves-Lopez et al., 2009).The OAVs of aroma components and sensory characteristics of six samples played an important role in reflecting the aroma typicity of cv.Premier blueberry wine and gave the wine distinctive style and its feature (Table 2).On the basis of odor description and threshold, the most powerful odorants were established and only those displaying OAVs greater than 1 were deemed to contribute to wine aroma (Mingorance-Cazorla et al., 2003).In this study only the main aroma compounds at OAVs values ≥1 were considered.OAVs (OAVs≥1)of main esters, alcohols and fatty acids in 6 samples are shown in Fig. 2.
Esters: Ester compounds were the most abundant chemical substances in all samples, including isoamyl acetate, ethyl caprylate, ethyl hexanoate, butyrolactone, ethyl decanoate and ethyl laurate (Table 2).Isoamyl acetate, which contributes with fruity notes to the wine, has a kind of banana flavor.The content of isoamyl acetate was up to 0.7 mg/L (Table 1).The similar experimental figure of such high content has been found in several literatures about wine aroma (Viana et al., 2009;Pla et al., 2003).Among the six wine samples, the OAV of Isoamyl acetate showed the highest activity in the 6°C 3 days, reached to 4.6 (Fig. 2).Ethyl caprylate with the aroma characteristics of ripe fruits, pear and sweety, also gives blueberry wine fruity flavor (Rocha et al., 2003), its OAVs were between 1.55 and 2.54 and also was the most outstanding in 6°C 3 days.Ethyl hexanoate (strawberry and apple aroma characteristics, the OAVs of which were between 26 and 36 in the six samples, showed the highest odor activity among all the volatile compounds.Especially in the sample after 16°C 2 days treatment, the OAV (36.3) was the highest of all the compounds (Fig. 2) and it reached to 35.4 in the 6°C 3 days sample.The aroma of γ-Butyrolactone is of burnt smell type and it is associated with a toasted and rubber descriptor (Lerma et al., 2012;Krist et al., 2004).
Ethyl decanoate is the fruital ester series (Santos et al., 2004).The OAVs was at a low level.Ethyl laurate has only been detected in the wine sample macerated at 16°C 1 day, 16°C 2 days and 16°C 3 days.The OAV of Ethyl laurate was almost at the same level among three 16°C wine samples while this compound can not be detected in the 6°C wine samples.Thus pretreatment temperature may be one of the main influencing factors on ethyl laurate in the blueberry wine.Ethyl laurate pertains to oily, fatty, floral aroma series (Butkhup et al., 2011), which bring delightful flowery and fatty odor to blueberry wine.
In conclusion, the fruity aroma of cv.Premier semi-dry blueberry wine showed the most intense and outstanding after 6°C 3 days maceration treatment by analyzing the content, aroma type and OAV of esters.Due to the effect of ethyl laurate, blueberry wine would have advantages of the flowery and fatty odor when 16°C maceration treatment was taken.

Alcohols:
The main alcohol aroma compounds included 2-phenylethanol, 2, 3-butanediol, 4-hydroxybenzen-eethanol and 3-(methylthio)-1-propanol. The chemical substances had low OAVs which were all under 1 except 2-phenylethanol (Fig. 2).2phenylethanol played a role in the typical aroma of cv.Premier blueberry wine.2-phenylethanol belongs to aromatic alcohol.It has a rose-like odor above its perception threshold value (Selli et al., 2003).According to determination by GC-MS, the concentrations of 2-phenylethanol found in extracts were on the same order of magnitude as the values found for other wines (Fretz et al., 2005;Jurado et al., 2008;Pinillos et al., 2004;Gamero et al., 2011), had a concentration range from 11~28 mg/L and OAV from 1.1~2.8(Table 1 and 2).The OAVs of 2-phenylethanol from 6°C wine samples all showed higher OAVs than 16°C wine samples.Because of its ability to produce strong flavor, 2-phenylethyl alcohol may be related to a higher capacity to utilise the assimilable nitrogen in must (Torrea et al., 2002).Maceration temperature has a great effect on the leaching of 2-phenylethanol precussor substances.Lower temperature pretreatments bring about higher OAVs of 2phenylethanol.
Fatty acids: Most of the organic acids in blueberry wine are produced by fermentation.The main volatile fatty acids (OVAs≥1)in blueberry wine were decanoic acid, 9-decenoic acid and 3-methylbutanoic acid.decanoic acid plays a role in herbaceous and fatty odor series.It showed higher OVAs (0.94 and 1.0, respectively) in 6°C 3 days and 16°C 2 days sample than the others.The concentration of 9-decenoic acid was detected to be the highest of all fatty acids aroma compounds.The presence of 9-decenoic acid in rabbiteye blueberry wine has been described with waxy, fatty and soapy, which belong to fatty odor (Butkhup et al., 2011).Figure 2 indicated that 9-decenoic acid OAVs in 6°C 1 day, 6°C 2 days and 6°C 3 days (10.09, 21.48 and 12.28, respectively) all had higher activity than 16°C 1 day, 16°C 2 days and 16°C 3 days sample (4.75, 11.48 and 6.45, respectively).9-decenoic acid aroma value reached its highest in 6°C 2 days of all samples.The same situation happened to 3methylbutanoic acid (fatty) OVAs had a range from 4~6 in 6°C samples while from 1~4 in 16°C samples.6°C 3 days had the highest aroma value of 3methylbutanoic acid in all the samples.3methylbutanoic acid was found to be very intense at the sniffing port and its odor descriptors were fatty, rancid and cheesy (Gil et al., 2012).From what have been discussed above, fatty acids samples had higher OAVs when macerated in a temperature of 6°C than 16°C.

CONCLUSION
It can be concluded that, isoamyl acetate, ethyl caprylate, ethyl hexanoate, ethyl decanoate, ethyl lactate, phenylethyl alcohol, decanoic acid, 9-decenoic acid and 3-methylbutanoic acid played an important role to bring the blueberry wine with strong typicity.The results suggested that maceration temperature and time had a significant effect on concentration and variety of aroma substance.Only wine under 16°C did have ethyl laurate which was described by floral and fatty odor, while maceration condition of 6°C was adverse to the production of it.However, 60% of the typical volatile aroma compounds, including isoamyl acetate, ethyl caprylate, ethyl decanoate, 2-phenethanol and 3-methylbutanoic acid, had the highest OAVs when maceration condition at 6°C 3 days.The semi-dry cv.Premier blueberry wine macerated at 6°C 3 days was identified to be more abundant in volatile aroma compounds.

Table 1 :
Average concentration (mean±standard deviation) and Odor Perception Thresholds (OPT) of volatile compounds determined in semidry rabbiteye blueberry wines

Table 2 :
Mean Odor Activity Values (OAVs) and odor descriptor of typical aroma compounds