Analysis of Volatile Flavor Compounds of Jujube Brandy by GC-MS and GCO Combined with SPME

To identify the unique flavor compounds in jujube brandy and changes in flavor compounds in the process of aging, Gas Chromatography-Mass Spectrometry (GC-MS) and GC-Olfactometry (GC-O) combined with solid-phase micro-extraction were used for the analysis of the volatile flavor compounds of fresh and aged jujube brandy. The equilibrium of the flavor compounds required divinylbenzene/carboxen/polydimethylsiloxane fiber for 40 min at 40°C. A total of 72 compounds were positively or tentatively identified by GC-MS, including 34 esters, 12 alcohols, 2 acids, 7 hydrocarbons, 3 aldehydes, 3 ketones and 8 terpenes in jujube brandy. Among them, ethyl laurate, ethyl caproate, ethylbenzoate and ethyl hexanoate were the main components. The flavor components of jujube brandy were identified by GC-O and 47 flavors were detected. Among them, orange-like (ethyl acetate), apple-like (butanoic acid, ethyl ester), fermented (hexanoic acid, ethyl ester), chocolate-like (nonanoic acid, ethyl ester) and red date-like (dodecanoic acid, ethyl ester) were strongly sensed. Changes in the flavor compounds in the process of aging were detected. During the period of aging, the contents of alcohols, aldehydes and ketones generally decreased, whereas those of esters and acids increased.


INTRODUCTION
Brandy, one of the world's six distilled wines, mostly uses grape as a raw material.Jujube brandy, a unique brandy product in China, has a long history.Jujube brandy is produced by fermentation, distillation and aging using jujube as a raw material.Jujube brandy has strong healthcare functions because of the high nutritional value of jujube (Song and Zhao, 2011).The sensory characteristics of jujube brandy are heavily influenced by its volatile flavor components.Therefore, the volatile flavor components of jujube brandy are often subjected to analysis.
Currently, research progress on volatile compounds in jujube brandy is still very scarce.A previous study used the liquid-liquid extraction method to study the golden-silk jujube wine aroma and identified 35 compounds, of which alcohol had the highest content (Lv et al., 2011).Simultaneous distillation extraction was carried out to study the influence of ultra-high pressure treatment on dry red wine aroma components; 53 compounds were identified and the contents of senior alcohols, esters, organic acids, aldehydes and ketones changed after ultra-high pressure treatment (Zhang et al., 2007).Research about HS-SPME combined with Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the aroma composition of jujube brandy is almost nonexistent at home and abroad.
Wine aroma, one of the most important characteristics of wine quality, represents a good balance of several hundred volatile compounds.The quality of wine is closely related to its aroma components, so flavor compounds can be used as one of the wine classification standards.Different groups of volatile compounds, such as alcohols, esters, aldehydes, lactones, terpenes and phenols, have been identified in wines in a wide concentration range.These groups affect wine aroma even at low concentrations.Among the volatiles, alcohols and esters have the highest contents in wines.Esters are important constituents of wine aroma and they possess high fruity nuances (Fan and Qian, 2005).
Qualitative and quantitative characterizations of volatile compounds in wine are usually performed by GC-MS, one of the most sensitive techniques for the analysis of aroma in different samples (Fan and Qian, 2006a;Zhu et al., 2007).By contrast, solid-phase micro-extraction (SPME is a relatively new and simple adsorption technique for the isolation of headspace flavor compounds (Arthur and Pawliszyn, 1990;Arthur et al., 1992;Zhang and Pawliszyn, 1993).Headspace SPME sampling requires neither solvent extraction and purification steps nor a complicated purge-and-trap apparatus.The SPME-GC method is simple to use, inexpensive and does not require solvent extraction.However, SPME analysis is quite sensitive to experimental conditions, such as heating temperature and time, sample volume, concentration and sample matrix and uniformity (Yang and Peppard, 1994;Fan and Qian, 2006b).The application of this technique to flavor analysis of foods and beverages still requires further modification to improve the reproducibility, sensitivity and resolution of the chromatogram.This technique shows high repeatability and possibility of carrying out simultaneous extractions, which is one of its advantages over other solvent-free techniques.
In this study, we evaluated the flavor components in jujube brandy using GC-MS and GC-Olfactometry (GC-O) combined with SPME to identify the compounds that contributed to the unique odor of jujube brandy and changes in aromatic compounds in the process of aging.

MATERIALS AND METHODS
Optimization of SPME analysis for headspace flavor compounds of jujube brandy: Fresh jujube brandies (Hebei, Fuping) were analyzed by GC with SPME using Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), DVB/PDMS and PDMS.These three types of SPME fibers were compared for their adsorption capabilities on the volatile compounds of jujube brandy.Jujube brandy was diluted with distilled water (10% alcohol content).Sodium chloride (1 g) was added to 7.5 mL of sample solution in a 20 mL sealed glass vial.
To determine the effects of heating temperature and time on the equilibrium of flavor compounds between the SPME coating and headspace of the sample bottle, the sample bottles were maintained at 30, 40, 50 and 60°C for 30, 40, 50 and 60 min, respectively.

GC-MS analysis of volatile flavor compounds:
Flavor compounds of jujube brandy were detected by GC-MS with semi-quantitative method.The contents of flavor compounds were quantified using an internal standard (3-octanol, 99%, Sigma-Aldrich).Wine volatile compounds were analyzed using an Agilent 5975 Mass Spectrometer coupled to an Agilent 7890A Gas Chromatograph (Agilent, Santa Clara, USA).A DB-WAX column (60 m×0.25 mm ID and 0.25 µm film thickness) was used for separation.The working parameters were as follows: injector temperature of 250°C, EI source of 230°C, MS Quad of 150°C and transfer line of 250°C.The initial temperature was 50°C for 3 min, which was increased to 80°C at a rate of 3°C/min.The temperature was further raised to 230°C at 5°C/min and maintained at 230°C for 6 min.The carrier gas had a flow rate of 1.0 mL/min.Samples were injected using the splitless mode.A mass range of 50-550 m/z was recorded at 1 scan/sec.

GC-O analysis of volatile flavor compounds:
Characteristic flavor compounds of jujube brandy were specified by GC-O with aroma intensity method by 3 persons 3 times each.GC analysis of volatile compounds was carried out on a GC-7890A equipped with a Flame Ionization Detector (FID) and sniffing port.The column and temperature program was identical to GC-MS analysis.The effluent from the capillary column was split 1:1 between the FID and sniffing port using a "Y" splitter.Sniffing was carried out using OSS-9000 sniffer.

GC-MS analysis of volatile flavor compounds in different aging ages:
Jujube brandies in different ages (1, 2, 4, 7, 8, 10 and 20 years, respectively) were detected by GC-MS to determine the changes in volatile flavor compounds during aging ages.

RESULTS AND DISCUSSION
Optimization of SPME analysis for headspace flavor compounds of jujube brandy: Three types of SPME fibers were compared for their adsorption capabilities (Fig. 1).DVB/CAR/PDMS, PDMS and PDMS/DVB extracted 118, 119 and 88 aroma compounds in the sample wine, respectively.Of the three types of SPME fibers, DVB/CAR/PDMS extracted the most flavor compounds (Table 1).The abilities of the three fibers in extracting aroma compounds differed.After comparison on compounds amounts and concentration, DVB/CAR/PDMS was more sensitive to absorbing alcohols, terpene, aldehyde and ketone; PDMS/DVB was more sensitive to esters and least sensitive to organic acids; and PDMS was more sensitive to organic acids and least sensitive to most aroma compounds.DVB/CAR/PDMS was sensitive to most aroma compounds, but the other two fibers both least sensitive to specific compounds.In consideration of the aforementioned factors, DVB/CAR/PDMS was the ideal fiber to extract more aroma compounds in wine for GC analysis among the three fibers.
The balance time of analytes into the stationary phase is related to the extraction temperature.An appropriate extraction temperature should be selected to obtain satisfactory sensitivity in GC analysis.Table 2 shows the results of jujube brandy aroma components at different extraction temperatures.Esters and alcohols contained the largest amounts at 40 and 30°C, with the highest contents at 60 and 40°C, respectively.Acids could only be detected at 40°C.Although hydrocarbons, aldehydes and ketones were best adsorbed at 50 and 60°C, the extraction contents demonstrated no differences at 40°C.Thus, 40°C was considered the most appropriate extraction temperature for jujube brandy.SPME was used to measure analytes under a state of equilibrium.When volatile components obtained adsorption equilibrium between two phases, redundant extraction times were not beneficial on the extraction effect.By contrast, the excrescent time could increase the chances of components reacting chemically and reduce the lifetime of SPME fiber.As Table 3 shows, most of the compounds reached maximum extraction quantity in 40 min.Small molecular substances decreased when the time was extended.Therefore, 40 min was the optimal extraction time.

Flavor analysis of jujube brandy by GC-MS:
Flavor compounds of jujube brandy were detected by GC-MS.A total of 72 compounds were positively or tentatively identified by GC-MS, including 34 esters, 12 alcohols, 2 acids, 7 hydrocarbons, 6 aldehydes and ketones and 8 terpenes in jujube brandy.With the most contents, ethyl laurate, ethyl decanoate, ethyl octanoate and ethyl hexanoate were the main components.

Flavor analysis of jujube brandy by GC-O:
Characteristic flavor compounds were identified by GC-O.The gas chromatogram of volatile compounds in fresh jujube brandy is shown in Fig. 2. The identified volatile compounds in fresh jujube brandy are listed in Table 3 to 5. A total of 47 compounds were definitely or tentatively identified by GC-MS and 26 flavors were sensed in GC-O analysis.Among them, 27 esters were sensed, of which the contents of decanoic acid ethyl ester, dodecanoic acid ethyl ester, octanoic acid ethyl ester and hexanoic acid ethyl ester were the highest.Two alcohols, namely, ethanol (alcohol-like) and 1dodecanol, 3, 7, 11-trimethyl-(green), were sensed.
Among the 26 flavors sensed in GC-O analysis, esters with the fragrance of fruits and flowers were sensed most strongly, followed by alcohols and terpenes with the fragrance of green.Dodecanoic acid ethyl ester, benzenepropanoic acid ethyl ester andtetradecanoic acid ethyl ester gave this wine the scent of red dates, which constituted the unique feature of jujube brandy.
Flavor analysis of jujube brandy in different ages by GC-MS: Several differences between the fragrance of fresh and aged jujube brandies were observed, especially in terms of the contents of alcohols, acids and terpenes.Most aroma compounds in fresh jujube brandy could be detected in aged wine, whereas ncapric acid isobutyl ester, (+)-4-carene, 1-undecanol, 2octanol, 1-nontanol, 1-dodecanol and 2-methylpropionic acid could only be detected in fresh wine.2tridecanone was not detected in fresh wine, but it was detected in all aged wines.Nine, 12-Octadecadienoic acid started to appear in wine aged after 7 years.Three-Furaldehyde could only be detected in wine aged for 8 years and 1-heptanol began to appear in wine aged after 10 years.Benzoic acid propylester, 3-furaldehyde and 1-heptanol form late in the aging process.Therefore, jujube brandies at different ages showed varying aromatic characteristics because of dynamicchanges, such as production, replacement and disappearance of aromatic compounds, in aging years.
Among esters, except n-caprylic acid isobutyl ester, decanoic acid, dodecanoic acid and ethyl ester decreased in the process of aging, whereas the contents of other esters demonstrated an upward trend.1-Butanol, 2-methyl-and 1-octen-3-ol were the main alcohols and the contents of alcohols demonstrated an overall reducing trend.Among the acids, the levels of decanoic acid and dodecanoic acid were the highest, whereas the contents of acids and aldehydes only slightly increased.The decrease in the alcohols and an increase in the esters would be expected due to slow acid catalyzed esterification reactions.Terpenes initially decreased and then increased with the aging year, but could not be detected in 20-year-old jujube brandy.

CONCLUSION
DVB/CAR/PDMS fiber was the optimal choice to extract aroma compounds of jujube brandy.The vial containing the sample was incubated at 40°C for 10 min.The flavor compounds of jujube brandy were detected by GC-MS.A total of 72 compounds were positively or tentatively identified by GC-MS, including 34 esters, 12 alcohols, 2 acids, 7 hydrocarbons, 3 aldehyde, 3 ketones and 8 terpenes, in jujube brandy.Among them, ethyl laurate, ethyl caproate, ethyl benzoate and ethyl hexanoate were the main components.
During the period of aging, 81 aroma components were detected.The contents of 11 types of common components (e.g., hexanoic acid, ethyl ester, octanoic acid, decanoic acid, dodecanoic acid and benzaldehyde) were the highest.At each stage of aging, esters had the most content, followed by alcohols, terpene, aldehydes and ketones.Acids had the least content by this fiber.However, the main aroma composition types and their contents differed.The contents of alcohols, aldehydes and ketones generally decreased, whereas those of esters and acids increased during the process of aging.

Table 1 :
Comparison of SPME fibers on aroma component amount and content DVB

Table 2 :
Changes in volatile compounds amount and content at different equilibrium temperature Fig. 2: Gas chromatogram of volatile compounds in fresh jujube brandy

Table 3 :
Changes in volatile compounds peak area at different equilibrium time

Table 5 :
Changes in flavor content during aging process