Volatile Profile of Sea Buckthorn Wines, Raw Juices and Must in Qinghai (China)

Abstract

Headspace solid phase micro extraction was used for extraction of volatile compounds characterizing sea buckthorn wine, raw juice, and new fermented must in Qinghai province (China). Extracted compounds were identified by mass spectrometry after gas-chromatographic analysis. There were 53, 48, 37, and 38 compounds in the volatile fraction of sweet, dry sea buckthorn wine, juice, and must comprising of higher alcohols, ethyl esters, acetates, fatty acids, and carbonyl compounds, which may contribute to the typical flavor of sea buckthorn wine. After cluster analysis, the concentration of volatile compounds showed good ability to describe the volatile characterization of various products of different storage time, juices, and new fermented must.

Keywords:

• Sea buckthorn wine
• HS-SPME
• Volatile compounds
• GC-MS

INTRODUCTION

Sea buckthorn (genus Hippophaë) is a berry-bearing, hardy bush of the family Elaeagnanceae, naturally distributed in Asia and Europe,[1–3] which has a long history of application as food and medicinal ingredients in eastern countries, especially in traditional Tibetan and Mongolian medicine in China.[4] In recent years, sea buckthorn has attracted considerable attention from researchers around the world mainly for its nutritional and medicinal value. The berries contain many bioactive substances, including organic acids, amino acids, flavones, and vitamins.[5] Besides its nutritious and medicine value, it is also a good plant to grow in dry areas to keep vegetation. A large area of sea buckthorn has been planted as a way of vegetation reconversion and environment protection in Qinghai, located in the northeast of Qinghai-Tibet highland in northwest China. The priority has been given to sea buckthorn utilization and product development as its function of economy and dietary constitution for local people in recent years.

Sea buckthorn wine is one of the sea buckthorn products with special taste and flavor. Present researches about sea buckthorn wine were mainly focused on fermentation technologies; there were few reports about its aroma characteristics.[6–8] Hirvi studied volatile components of sea buckthorn fruit by combining gas chromatography-mass spectrometry (GC-MS); sixty components, composed of several aliphatic esters, were identified.[9] Tiitinen had identified 45 volatile compounds from frozen berries of seven sea buckthorn varieties from two growing seasons with solid phase micro extraction (SPME) and GC-MS.[10] Tiitinen also investigated the volatile compounds of sea buckthorn juice before and after malolactic fermentation. The results showed that three acetic esters were increased and four aliphatic esters were decreased during fermentation.[11] Malolactic fermentation was used to improve fruity flavor as well as fermented flavor of sea buckthorn juice and an optimal process was developed.[12, 13] However, the above mentioned papers were all about the research of volatile compounds in sea buckthorn juice and its malolactic fermentation, and there was no similar report about sea buckthorn wine.

Our purpose was to characterize the main volatile compounds and find out the basic information about the aroma profile of Qinghai sea buckthorn wine. The sources of volatile compounds in sea buckthorn wine were also discussed by comparing the difference of volatile characteristics among sea buckthorn wine, raw juice, and new ferment must. The diversity among varieties of sea buckthorn wines as well as the effect of storage time on the volatile compounds were also studied by chemometric method, which could be a feasible way to differentiate varieties of sea buckthorn wines and establish criteria of genuineness to improve the quality, prevent fraud, and guarantee their origin of the sea buckthorn as in wine.[14] The information in this article is helpful to further studies of aroma profile of sea buckthorn wine.

MATERIALS AND METHODS

Samples

A total of 24 bottles (600 ml) of sweet and dry sea buckthorn wines produced in different years (from August 2006 to April 2008) were analyzed. At the same time, one new fermented must (NF) and two kinds of juice sample (J1 and J2) that were produced in different counties in Qinghai Province in 2008, were also analyzed. The samples were kindly donated by the Qinghai Qinghua Biotry Bio-Technology Company (Xining, Qinghai Province, China).

Analytical Reagents

The retention index probes (alkane mixture consisting of C₁₀ to C₂₅ straight-chain alkanes, concentration 1000 mg L⁻¹ in hexane) are products of Dr. Ehrenstorfer (Augshurg, Germany). Ethyl acetate, isoamyl acetate, 2-phenylethyl acetate, ethyl butyrate, ethyl decanoate, ethyl isovalerate, ethyl octanoate, 3-methyl-1-butanol, isopropanol, 2-butanol, 2-methyl-1-propanol, (s)-2-methyl-1-butanol, hexyl alcohol, 2-phenylethanol, 2,3-butanediol, benzyl alcohol, 3-hydroxy-2-butanone, β-damascone, 2-furaldehyde, 2,5-dimethyltetrahydrofuran, acetic acid, hexanoic acid, isobutyric acid, octanoic acid, and n-decanoic acid are products of Sigma-Aldrich (Buchs, Switzerland). Ethyl L(-)-lactate, ethyl hexanoate, ethyl benzoate, 1-heptanol, furfuryl alcohol, β-ionone, cis-citral, and dibutyl phthalate are products of Acros Organics (Morris Plains, NJ, USA). Diethyl succinate, 1-propanol, and hydroxyacetone are products of Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). 2-Octanol is a product of Chem Service, Inc. (West Chester, PA, USA).

Standard Solutions

Ten microliters of 2-octanol as internal standard and all reference compounds were dissolved in absolute ethanol and made up to a volume of 100 mL as stock solution. These solutions were then diluted in synthetic wine to calculate the emendation factor to 2-octanol (8.22 mg L⁻¹ in synthetic wine). These reference compounds were dissolved in synthetic wine at typical concentrations found in wines. The final alcohol content of the synthetic wine is 12% (v/v). The synthetic wine has 6 g L⁻¹ of tartaric acid and its pH is 3.3 adjusted with 1 mol L⁻¹ NaOH. All of these solutions were stored at 4°C.

Solid Phase Micro Extraction (SPME)

The PDMS device (Supelco Inc., Bellefonte, PA, USA) coated with polydimethylsiloxane (30 μm) was used. Eight milliliters of each sample containing 2 g NaCl was first equilibrated at 40°C for 40 min in a 15 mL vial and then fiber was exposed to the sample for 40 min at the same temperature.[15] Eight microliters of 2-octanol (8.22 mg L⁻¹ in the sample) solution was added into samples as an internal standard before the equilibration. The fiber was desorbed for 5 min in a gas chromatograph injector, equipped with deactivated SPME glass insert and analysis was carried out on a DB-WAX (30 m × 0.25 mm i.d. × 0.25 μm) fused silica capillary column (Agilent Inc., Santa Clara, California). Before using, fiber was conditioned, following instructions from manufacturers, which was cleaned at 250°C for 1 h by inserting it into the GC injector.

Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis was carried out in a TRACE DSQ (Thermo-Finnigan, San Jose, California, USA). The carrier gas was helium at 1 mL/min. The temperature program was as follows: 40°C for 2.5 min, raised to 200°C at 5°C/min, then raised to 240°C at 10°C/min, and finally kept for 5 min. Transfer line temperature was 230°C. Injection temperature was 250°C. Mass spectra were recorded in electron impact (EI) ionization mode and the mass range was m/z 35–400 amu. Ion source temperature was 250°C. An Xcalibur Chemstation equipped with the NIST 2002 MS library (Thermo-Finnigan, San Jose, California) was used for components identification. Identification of the components was done on the basis of retention index and the comparison of EI mass spectra with published data or with reference compounds.

Quantification

The quantification was carried out following the internal standard quantification method. Quantitative data of the identified compounds were obtained by the formula:

The concentration of volatile compounds for which there was no pure reference available was obtained by using the same emendation factor as one of the compounds with the most similar chemical structure.[14, 15]

Statistics

All samples were analyzed in triplicate. The concentrations of volatile compounds in sea buckthorn wines (dry and sweet) were reported by average value of products from different producing times. The average value from two kinds of juice represents the concentrations of volatile compounds of sea buckthorn juice. The concentration of volatile compounds of new must is a mean of triplicate analysis. The chemical data of volatile compounds obtained from the assays were standardized and used as variables for object description for cluster analysis. The objects were all samples including juices (J1 and J2), new ferment must (NF), and two kinds of product having different ageing from 2006 to 2008. The data matrix was used for computerized multivariate analysis by the software package SAS (2004) from StatSoft, Inc. (Tulsa, OK, USA).

RESULTS AND DISCUSSION

Volatiles Characterization by SPME/GC-MS

There were 53, 48, 37, and 38 compounds determined in the volatile fraction of sweet, dry product, juice, and must (Table 1), respectively. As shown in Table 1, the volatile compounds in sea buckthorn wine were mainly composed of acetates, ethyl esters, higher alcohols, and fatty acids, as well as other minor components, such as ketenes and carbonyl compounds. Acetate and ethyl esters were richer in sea buckthorn wine than in raw juice. This could be attributed to acetyl-coenzyme activity produced by yeast during fermention. Whereas, the norisoprenoids derived from the breakdown of carotenoids and having remarkable impact on the bouquet of wine are less in sea buckthorn wine. It was not in conformity with the previous reports that the contents of carotenoids are rich in sea buckthorn berry and juice.[2] The reasons why norisoprendoids were low in sea buckthorn wine still needs to be studied.

Table 1 Concentrations of volatile compounds in sea buckthorn wine from 2006 to 2008, juice, and must in 2008 (mg L⁻¹)

		Sweet		Dry		Juice		Must
Compounds	KI ^a	Mean ^b	sd	Mean ^b	sd	Mean ^c	sd	Mean ^d	sd
Acetates
Ethyl acetate	nc	2.289	0.57	2.194	0.40	0.684	0.07	3.961	0.84
2-Methybutyl acetate	1027	0.029	0.03	0.008	0.01	nd		0.090	0.03
Isoamyl acetate	1137	0.608	0.33	0.209	0.11	0.024	0.01	2.868	0.58
Hexyl acetate	1281	0.034	0.02	0.026	0.02	nd		0.052	0.03
1-Methylbutyl acetoacetate	1650	0.041	0.04	0.014	0.02	0.156	0.11	0.209	0.06
2-Phenethyl acetate	1815	0.097	0.05	nd		nd		0.350	0.15
Glycerol 1-acetate	2274	0.070	0.07	0.031	0.02	nd		nd
Sub total		3.168		2.482		0.864		7.530
Ethyl esters
Ethyl isobutyrate	nc	0.062	0.06	0.098	0.01	0.030	0.03	nd
Ethyl butyrate	1050	0.203	0.07	0.214	0.05	0.095	0.03	0.281	0.06
Ethyl 2-methylbutyrate	1068	0.011	0.02	0.051	0.01	0.138	0.02	nd
Ethyl isovalerate	1088	0.257	0.16	0.181	0.07	0.445	0.14	0.560	0.26
Ethyl hexanoate	1243	0.195	0.06	0.162	0.11	0.179	0.12	0.388	0.17
Ethyl heptylate	1328	0.044	0.04	0.030	0.02	0.139	0.04	0.141	0.05
Ethyl lactate	1363	0.042	0.04	0.247	0.20	nd		0.208	0.11
Ethyl octanoate	1441	0.151	0.14	0.754	0.27	0.168	0.01	0.671	0.11
Ethyl decanoate	1643	0.198	0.17	0.163	0.12	0.081	0.08	1.988	0.32
Ethyl benzoate	1678	0.011	0.02	0.153	0.22	0.346	0.11	0.212	0.09
Diethyl succinate	1688	0.768	0.18	1.243	0.38	0.249	0.05	nd
Ethyl phenacetate	1750	0.009	0.02	nd		0.066	0.01	0.075	0.03
Ethyl laurate	1825	0.026	0.05	0.169	0.17	0.097	0.10	1.110	0.12
Diethyl malate	2063	0.076	0.06	0.031	0.02	0.128	0.06	0.206	0.08
Monoethyl succinate	2418	0.154	0.12	0.125	0.07	nd		nd
Sub total		2.207		3.621		2.161		5.840
Higher alcohols
2-Butanol	1041	4.667	3.92	1.376	1.95	5.322	5.32	10.538	2.20
2-Methyl-1-propanol	1123	25.755	10.97	18.420	3.98	7.397	4.08	30.366	4.43
3-Methyl-1-butanol	1240	237.532	49.65	251.880	17.69	80.293	2.29	292.692	15.31
1-Hexanol	1368	1.754	1.45	3.285	0.64	nd		nd
1-Heptanol	1451	0.076	0.10	0.123	0.03	0.356	0.09	0.647	0.33
dl-6-Methyl-5-hepten-2-ol	1489	0.062	0.08	0.043	0.05	0.169	0.12	0.291	0.19
2,3-Butanediol,[R-(R*,R*)]-	1553	0.837	0.88	0.347	0.27	0.179	0.05	nd
1-Nonanol	1666	0.054	0.08	0.048	0.07	0.245	0.04	0.185	0.04
1-Dodecanol	1979	0.220	0.18	0.095	0.13	nd		nd
Benzyl alcohol	1847	0.402	0.80	1.351	1.91	4.227	0.48	4.756	0.91
Phenylethyl alcohol	1929	13.380	3.66	11.188	1.23	4.043	0.42	14.962	1.71
Glycerin	2307	3.294	2.92	3.553	1.78	0.504	0.14	0.387	0.07
Hydroquinone	2403	0.016	0.03	0.045	0.03	0.091	0.00	0.185	0.05
1,2-Benzenediol	2462	0.133	0.17	nd		0.120	0.02	0.076	0.02
Sub total		288.182		291.754		102.946		355.085
Fatty acids
Acetic acid	1472	16.607	13.78	3.390	1.12	1.162	1.16	1.873	0.64
3-Hydroxy-3-methyl butyric acid	1505	0.018	0.02	nd		0.074	0.07	0.059	0.06
Hexanoic acid	1806	1.841	0.23	2.181	0.65	1.370	0.31	nd
Octanoic acid	2081	8.086	1.83	7.960	4.45	3.421	0.69	5.237	0.79
n-Decanoic acid	2257	4.445	3.89	4.525	2.62	nd		5.262	0.37
n-Hexadecanoic acid	nc	0.380	0.51	0.885	0.94	nd		1.273	0.02
Sub total		31.377		18.941		6.027		13.704
Carbonyl compounds
1-Hydroxy-2-propanone	1321	2.785	2.56	0.208	0.29	nd		nd
Furfural	1481	0.561	0.47	0.035	0.05	0.199	0.11	nd
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one	1544	4.216	3.64	nd		nd		nd
5-Methyl-2-furancarboxaldehyde	1586	6.783	6.23	0.531	0.75	5.207	3.32	nd
2,5-Dimethyl-4-hydroxy-3(2H)-furanone	2054	0.142	0.17	0.014	0.02	nd		nd
5-(hydroxymethyl)-2-furancarboxaldehyde	2341	1.780	1.09	0.113	0.10	nd		0.191	0.04
Sub total		16.267		0.901		5.406		0.191
Norisoprenoid
Ionone	1531	0.002	0.01	0.003	0.001	nd		nd
2,7-dimethyl-naphthalene	2015	0.007	0.01	0.036	0.03	nd		0.080	0.01
Sub tatol		0.009		0.040		0.000		0.080
Terpenes
Linalool oxide	1453	0.006	0.01	0.019	0.02	0.074	0.04	0.078	0.02
Linalol	1556	0.009	0.02	0.120	0.12	0.120	0.07	0.063	0.01
Terpineol	1704	0.021	0.01	0.016	0.02	0.044	0.01	0.051	0.01
Sub total		0.036		0.155		0.238		0.192
^aRentention indices on a DB-Wax column.
^bAverage content of compounds in product (dry or sweet) from 2006 to 2008.
^cAverage content of compounds in two kinds of juices from different counties at 2008.
^dAverage contents of compounds in one new must from triplicate analysis at 2008.
nc: Rentention indices cannot be calculated in this analysis; nd: Compounds is not be detected.

Acetates and ethyl esters produced by the yeast during fermentation are important in young wine aroma and contribute to the fruity flavor of wines.[16, 17] High levels of acetates were observed in all samples, especially in new fermented must (Tables 1 and 2). Acetate esters are the result of the reaction of acetyl-CoA with higher alcohols formed from degradation of amino acids or carbohydrates.[18] As shown in Table 1, the content of ethyl acetate and isoamyl acetate was higher in wines and fermented must than that in juice. Seven acetate esters, such as ethyl acetate, 2-methybutyl acetate, and isoamyl acetate, etc., were detected. The relative content of seven esters accounted for 0.93, 0.78, 0.73, and 1.97% of the total volatile compounds in sweet, dry product, juice, and new fermented must (Table 2), respectively. Ethyl acetate, isoamyl acetate, and 1-methylbutyl acetoacetate were present in all samples, but other acetates can just be detected in some samples. Must had all acetates except for glycerol 1-acetate, and juices just had ethyl acetate, isoamyl acetate, and 1-methylbutyl acetoacetate. This might be proof that most acetate was produced from yeast fermentation. Some acetates cannot be detected in old wines due to long-time storage.[17]

Table 2 Percentage of seven series compounds relative to total contents of volatile compounds in sea buckthorn wines from 2006 to 2008, juice, and must in 2008 (%)

Compounds	Sweet	Dry	Juice	Must
Acetates	0.93	0.78	0.73	1.97
Ethyl ester	0.65	1.14	1.84	1.51
Higher alcohols	84.41	91.82	87.51	92.82
Fatty acid	9.19	5.96	5.12	3.58
Carbonyl	4.77	0.28	4.60	0.05
Norsoprenoid	0.01	0.00	0.00	0.02
Terpenes	0.05	0.01	0.20	0.05
Total	100	100	100	100

A total of 15 ethyl esters were identified, which accounted for 0.65, 1.14, 1.84, and 1.51% of the total volatile compounds in sweet, dry product, juice, and new must as shown in Table 2. The influence of their concentration was mainly dependent on several factors: yeast strain, fermentation temperature, aeration degree, and sugar content.[14] In this study, the contents of ethyl esters were higher in sea buckthorn wines and must than that in juice as shown in Table 1. The fermentation process could increase the contens of ethyl ester in sea buckthorn wine. Sea buckthorn juices had a higher relative concentration of ethyl than products and fermented must, which indicated that ethyl esters were main volatile compounds in sea buckthorn juice, the result was in accordance with previous reports.[9, 10, 11] Ethyl esters of fatty acids have positive contribution to the general quality of wine. Most of these compounds have the nuance flavor of mature fruit. Ethyl butyrate (sour fruit, strawberry, fruity), ethyl hexanoate (green apple, fruity, strawberry, anise), ethyl octanoate (pineapple, pear, floral), and ethyl decanoate (fruity, fatty, pleasant) are responsible for the “fruity” and “floral” sensory properties of wine.[19, 20] From this view, the ethyl esters are important volatile compounds that have great influence on the aroma characteristics of sea buckthorn.

Higher alcohols and esters are produced by yeast metabolism during alcoholic fermentation and play an important role in making the flavor of the wines.[21, 22] In this work, it was proven that higher alcohols were the largest group of volatile compounds and 20 higher alcohols were identified in sea buckthorn wines. They were made up of 84.41, 91.82, 87.51, and 92.82% of the total volatile compounds in sweet, dry product, juice, and new must. Both content and relative concentration of these volatile compounds were higher in products and new must than that in juices as shown in Tables 1 and 2. Fusel alcohol can be synthesized by yeast through two mechanisms: anabolic pathway from glucose, or catabolic pathway from their corresponding amino acids (valine, leucine, iso-leucine, and phenylalanine), which influence the production of alcohols.[20, 23] It was well known that amino acid compositions depend heavily on grape variety and for this reason all these volatile compounds are related to grape variety. From this view, these composition contents of sea buckthorn wine should have the relationship with the raw sea buckthorn juice.

Fatty acid production is governed by the initial composition of the must and fermentation conditions.[24] From the results of the work, it was obviously indicated that fatty acid content could be increased during product storage. Table 1 shows that fatty acid content of products was higher than that in new fermented must. Fatty acids make up 9.19, 5.96, 5.12, and 3.58% of total volatile compounds in sweet, dry product, juice, and must, respectively.

In sea buckthorn wine, some other minor volatile compounds, such as ketone and carbonyl compounds, were also found and had different contents in samples. Furfural has been determined in sea buckthorn wine produced from Maillard reactions.[25] These compounds were higher in sweet than in dry product, this may be attributed to high sugar content in sweet product.

Chemometric Difference

The multivariate techniques of data analysis can be a way to explain the wine differentiation in volatile profile;[17, 26, 27] therefore, the analytical data obtained from both samples was used as variable vectors for chemometric analysis in order to obtain more detailed information about product varieties and storage time that could influence the contents of volatile compounds of sea buckthorn wines, as well as the different profile among the products, raw juices, and new fermented must. For this purpose, eight products of sea buckthorn wine (dry and sweet from 2006 to 2008), two juices, and one new fermented must were chosen as object descriptions for cluster analysis. The contents of volatile compounds were used as variables to perform cluster analysis of all the samples to obtain the dendogram of quantitative contents of volatile compounds in sea buckthorn wines, juices, and new ferment must (Fig. 1). This diagram shows that the volatile compounds estalished some groups among products of different storage time. A fair homogeneous group formed by the products produced in 2006 and 2007 can be distinguished from other products, must, and juices. Another heterogeneous group including products produced in 2008 was found. Two old sweet wines formed one group that was different from new product, which reflected the influence of the storage time on the content of volatile compounds. Two kinds of juice also formed one group, which was different from products in volatile characteristic.

Figure 1 Ward's method hierarchical clustering (dendrogram) in function of the contents of volatile compounds in sea buckthorn wines, juices, and new fermented must. *Name of products including information of the variety of products and producing time, for example D0708 means dry wine produced in August 2007.

The difference between sweet and dry products was mainly the content of alcohol and sugar, as the fermentation technology was almost the same.[28] Whereas, the difference of volatile compounds between dry and sweet products still existed. As Fig. 1 shows, old dry products (D0612 and D0708) can be differed from old sweet products (S0608 and S0710). However, the volatile compounds of new dry and sweet products were almost the same, which again reflects the influence of storage time on the profile of volatile compounds of products. The products of the same producing time had similar characteristic of volatile compounds regardless of the product varieties. New fermented must also had a similar profile of volatile compounds with new products. The volatile compounds of sea buckthorn wines changed as storage time increased.

CONCLUSIONS

The higher alcohols, ethyl esters, acetates, fatty acids, and carbonyl compounds were the main volatile compounds in the sea buckthorn wine in Qinghai. SPME–GC has enabled the characterization of the volatile composition in varieties of sea buckthorn wine made from different years. The chemometric analysis of these volatile compounds revealed that the volatile compounds generated during the storage gave a significant difference among products produced in different years. Although old dry and sweet products had different volatile profiles, the new products had no differences between them. The new fermented must had more similar volatile profile with new products than old ones. Storage time had a great influence on the composition of volatile profile of sea buckthorn wine.

ACKNOWLEDGMENTS

The authors are grateful to Qinghai Qinghua Biotry Bio-Technology Company (Qinghai province) for the supply of the samples used in this study.