Analysis of Aroma Volatile Compounds in Fuji Apple Using SPME with Different Fiber Coatings

Jie LI Jicheng HAN Haie ZHANG Chaohong ZHANG Minmin LI Yu WANG

Abstract The aroma volatile compounds in Fuji apple were isolated by solid-phase microextraction (SPME), and detected by gas chromatography-mass spectrometry (GC-MS). The results showed that the optimal retention time of the heating up of capillary columns was 5 min. Of the seven fibers used in this work, DVB/CAR/PDMS was found to be the most suitable to extract the aroma volatile compounds in Fuji apple. A total number of 43 volatile compounds were identified according to their retention time on capillary columns and their mass spectra, including eleven esters, ten alcohols, ten aldehydes, seven alkenes, two anhydrides, one ketone, one phenol and one ether. This detection method will provide a new foundation for analysis of volatile compounds in apple were identified.

Key words Fuji apple; Aroma volatile compounds; SPME Fiber; GC/MS

Received: March 9, 2020Accepted: May 8, 2020

Supported by Innovative Projects of Hebei Academy of Agriculture and Forestry Sciences (2019-3-4-5).

Jie LI (1985-), female, P. R. China, assistant researcher, devoted to research about analysis and identification of aroma substance in fruits.

*Corresponding author. Yu WANG(1992-), female, China, research assistant, devoted to research about fruit yield and quality. E-mail: wangyu20200310@163.com.

As one of important traits of fruit quality, aroma has gained increasing attention in recent years. The aroma composition is a complex mixture of large numbers of volatile compounds, whose composition is specific to different kinds of fruits, and tends to be diversified［1-2］. The aroma of fruit can stimulate the epidermal cells of human, and make the feeling of light-hearted, which is related to the nutrition and health of human closely［3］.

It has been reported that there are many factors that influence the measurement of aroma compounds, such as the extraction method of aroma compounds, the detection conditions of aroma compounds, the qualitative and quantitative methods of aroma compounds, etc. ［4］.

The extraction methods include steam distillation extraction (SDE), supercritical fluid extraction (SFE), static headspace extraction (SHE), static headspace extraction (SHE), dynamic headspace extraction (DHE), solid-phase microextraction (SPME), and so on［5］. Among these methods, the technology of SPME has many advantages such as low cost and simple and convenient operation. Therefore, it has become a widely used pretreatment technology for the determination of volatile substances in samples［6］. The key component of the SPME device is the fiber coatings, and their role of extracting volatile substances from fruits are much crucial. There are many fibrous materials that can be chosen to be used in the fiber coatings, which make the polarity of the fiber coatings diversified. Polydimethylsiloxcane (PDMS) is non-polar and can only extract non-polar semi-volatile substances; polyacrylate (PA) is polar and can extract polar and semi-volatile substances; and divinylbenzene/carboxen/polydimethylsiloxcane (DVB/CAR/PDMS) is bipolar and can extract volatile and semi-volatile substances. According to the principle of "similar miscibility", the adsorbability of the same kind of volatile substances by different fiber coatings is different either. Besides, the retention time of the heating up of capillary columns can also affect the detected volatile substances, and it餾 rarely reported in previous studies. Therefore, the objective of this study is to find the suitable fiber for the detection of aroma components and the best retention time of the heating up of capillary columns of Fuji apple.

In the respect of the detective conditions of aroma compounds, it consists of the category of capillary columns, the retention time of the heating up on capillary columns, the time of extraction and analysis, etc. However, there are few reports about the retention time of the heating up on capillary columns. Therefore, in this paper, we selected several different retention time for comparison. There are also many different methods about the qualitative and quantitative detection of aroma compounds. In regard to the qualitative detection of aroma compounds, it mainly depends on the retention indexes (RIs) and the mass spectrometry library. However, about the quantitative detection of aroma compounds, more methods are used, such as the method of areal normalization, the method of external standard, the method of internal standard, the method of standard addition, etc.

Fuji apple is one of the many varieties of apples, which tastes sweet especially. It not only contains a lot of vitamin C, but also contains a lot of carotene, and is very good to the health of people. Not only in China, but all over the world, the production and position of Fuji apple can not be replaced by other varieties. In this study, we aimed to find a way which is much more accurate and faster to detect more aroma volatile compounds, and provide scientists a better way to detect the aroma volatile compounds in Fuji apple.

Material and Methods

Materials

The Fuji apple trees were planted on the base of Changli Fruit Research Institute, Hebei Academy of Agriculture and Forestry Sciences, Northeast of Hebei Province, China. Fuji apple fruits were collected in late October. These samples were smooth and uniform in size. After peeling, 50 g of pulp sample was frozen with liquid nitrogen, added with 1 g of PVPP (to remove polyphenols and prevent samples from oxidating) and 0.5 g of D-gluconic acid lactone (to inhibit the activity of glucoside enzymes), and then rapidly crushed into powder. These powdery samples were stored in the refrigerator at -80 ℃ for the determination of aromatic substances.

Seven kinds of SPME fibers with different coats were purchased from Supelco Inc. (Bellefonte, PA, USA). They are polydimethylsiloxane/divinylbenzene (PDMS/DVB, 65 μm thick, blue color/pink color), divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm thick, gray color), polydimethylsiloxane (PDMS, 7 μm, green color/30 μm, yellow color/100 μm thick, red color), polyacrylate (PA, 85 μm thick, white color). The fibers used were preconditioned prior to the analysis in the injection port of the gas chromatograph according to the instructions suggested by the manufacturer.

C7-C30 normal alkanes for calculating the retention indices (RI) were purchased from Aldrich Chemical Co. Authentic reference aroma compounds were obtained from Beijing Peking University Zoteq Co., LTD.

Methods

SPME sampling

First, the samples that stored at -80 ℃ were taken out and thawed quickly. Second, they were centrifuged at 8 000 rpm and 4 ℃ for 10 min. Third, 4 ml of each supernatant was transferred to a 15 ml vial (special for SPME). Fourth, 10 μl of 4-methyl-2-amyl alcohol (4M2P) aqueous solution (1.038 8 g/L) was added as internal standard. Fifth, before the SPME fiber was inserted into the vial, the vial was sealed with one Teflon cover and equilibrated for 30 min at a 40 °C magnetic stirrer. Finally, the fiber was exposed in the upper space of the sealed vial to extract compounds for 30 min.

Analysis by GC-MS

A GCMS-QP2010 equipment was used. The GC was equipped with an HP-INNOWax capillary column (60 m×0.25 mm×0.25 μm , Agilent Technologies). Helium was the carrier gas with a constant flow of 1 ml/min to the column. The injection port temperature was at 250 ℃. It was parsed for 5 min at the injection port. The initial oven temperature was at 50 ℃, which was held for 5 min and then increased at 3 ℃/min to 120 ℃, which was held for 5 min and finally increased at 6 ℃/min to 250 ℃, which was held for 5 min. The injection port was in splitless mode. Electron impact ionisation was used at 70 eV (EI). The acquisition of mass spectra was performed in a mass range of 30-500 m/z. The ion source temperature was at 230 ℃. MS was detected with 2 min solvent delay. The analysis of the sample at each condition was repeated 3 times. C7-C30 n-alkanes were run under the same chromatographic conditions used in the separation of the compounds in our samples in order to calculate the retention indices (RI) of detected compounds. The system of AMDIS（Automatic Mass Spectral Deconvolution and Identification System）was used to analyze the mass spectrogram. Compounds were identified by comparing their mass spectra with those included in the NIST11 database, and confirmed by comparison of the retention time of the separated constituents with those of the authentic samples and by comparison of retention indexes (RIs) of the separated constituents with the RIs reported in literatures.

In order to find better retention time of the heating up of capillary columns for the eseven SPME fibers, the aroma volatile compounds in Fuji apple were detected for four different retention time (1, 5, 8 and 10 min, respectively) in each heating process.

Results and Analysis

The optimization of retention time of the heating up on capillary columns

The retention time was optimized, and the results were listed in Table 1.The results indicated that 5 min was more suitable for the seven SPME fibers among the four chosen retention time of the heating up of capillary columns. In this retention time, the time of the heating up of capillary columns had little impact on the compounds extracted from different SPME fibers. Thus, under the same experimental conditions, in order to save time and improve the efficiency of the experiment much better, we chose the retention time of 5 min in the next experiments.

Identification and analysis of aromatic substances extracted from different fiber coatings

The total ion chromatograms of aroma volatile compounds in Fuji apple are shown in Fig. 1. The analysis results of aroma volatile components extracted from Fuji apple by different fiber coatings are shown in Table 2.

As shown from Table 2, it could be seen that total of 43 volatile compounds were detected in Fuji apple, including eleven esters, ten alcohols, ten aldehydes, seven alkenes, two anhydrides, one ketones, one phenol and one ether. The predominant volatile compound in Fuji Apple was hexyl acetate, followed by butyl acetate, 1-butanol, 1-hexanol and (E)-2-hexanal.

Discussion

It has been reported that there are more than 300 volatile molecules in fresh apples［7］. The identity, concentration and total number of volatile compounds emitted from mature apple are cultivar specific［8］. The contribution of each compound to the specific aroma of each cultivar depends on the activity of the relevant enzymes, the specificity and availability of the substrate and the existence of other compounds［9］. It has been proposed for cultivar classification with α-farnesene and esters, and the esters are the most abundant volatile compounds emitted by apple［10］.

The number of identified compounds with different fiber coatings

From the point of view of the number of identified compounds, it is clear that the amount of identified compounds was the largest in using DVB/CAR/PDMS which contained thirty-two in Fuji apple. Wang et al. ［11］ reported that using the capillary column of Rtx-1MS (30 m×0.25 mm×0. 25 μm), the SPME fiber of DVB/CAR/PDMS showed the best results. Xiao et al. chose the DVB/CAR/PDMS fiber in Cherry wines. The effects of PDMS/DVB (blue) and PDMS/DVB (pink) are moderate which contain twenty-one and fourteen compounds, separately［12］. They can extract esters, alcohols, aldehydes and alkenes compounds. However, the amount of esters and alkenes was less than that of DVB/CAR/PDMS, which might be due to two reasons. One reason is that the contents of esters and alkenes are lower, and the other is that PDMS/DVB may be not suitable for extracting esters and alkenes compounds. The effect of PA which detected six compounds was poorer than that of PDMS/DVB. With this fiber coating, the alkenes, ketones, phenols and ethers were not identified. And meanwhile, the number of ester, alcohol and aldehyde compounds that identified was also much less. The fibers of PDMS (100 μm) and PDMS (30 μm) which only contained four and two compounds separately, were poorer than PA, and phenols and acetic acid are only identified by using PDMS (100 μm). The effect of PDMS (7 μm) was the worst, and it did not detect any compounds. When the three kinds of PDMS were used for extracting volatile compounds, the identified compounds were the least. Ester, ketone, ether and alkene compounds were not identified. PDMS may be not suitable for extracting these four kinds of aroma compounds. Therefore, of the seven fibers used in this work, DVB/CAR/PDMS was the most suitable fiber for extracting volatile aroma compounds in the fruit.

The Species of Identified Compounds with Different Fiber Coatings

The number of ester compounds was the largest among the identified compounds. According to the type of ester, "ester flavor" apple species were divided into four categories: acetate, propionate, butyrate and ethanol type, respectively ［13］. They are formed by the esterification reaction of alcohols with organic acids under catalysis of enzymes. In Fuji apple samples, these esters that formed with the four kinds of organic acids and most of alcohols were also identified. These ester compounds can impart Fuji apple with fruity notes and make the odor of Fuji apple enhanced and diffusive. Hexyl acetate exists as the highest ester concentration at μg/kg FW, and provides a sweet, fruity fragrance that is rich in apricots, peaches, and apples ［14- 22］. Hexyl acetate has fruity, green, sweet odor; ethyl butanoate has sweet, fruity odor; and ethyl propanoate has fruity, fermented and pineapple aroma［23］. DVB/CAR/PDMS is the most suitable for the extraction of ester compounds. When it was used, eleven esters were all identified.

n-butyl butyrate1249RI, MSNDNDNDNDNDND5.01

Aldehyde compounds and ketones were also isolated from Fuji apple, and their formations may be due to the beta-oxidation of fatty acids ［24-26］. Among them, (E)-2-hexenal has a fragrant smell of apple, which is popular to people［27-29］. In this paper, (E)-2-hexenal was also identified. Among the seven fibers used in this paper, DVB/CAR/PDMS and PDMS/DVB (blue) were more suitable for the extraction of these compounds.

Alcohols might be formed during the fermentation of carbohydrates from fruits during the ripening step. 1-Hexanol has a pleasant floral fragrance and is found extensively in plum, cherry, blueberry ［30-34］. However, studies have showed that substances such as butanol have a negative effect on the aroma of apples［8, 35］. In this paper, although the amount of characteristic aroma of (E)-2-hexenal and ethyl butyrate was higher, their flavor was poor, and it might be due to the higher content of 1-butanol that reduces the overall aroma. DVB/CAR/PDMS and PDMS/DVB (blue) were also suitable for the extraction of alcohol compounds.

The detected alkanes might be formed due to the degradation of amino acid. DVB/CAR/PDMS was the most suitable for the extraction of these compounds.

Acetic acid and acetic anhydride were isolated from Fuji apple. They are considered from the action of deaminase of amino acids. DVB/CAR/PDMS and PDMS (100 μm) were more suitable.

Anisole was the only ether compound identified in Fuji apple. This substance was also identified in Huaguan and Golden apples［35］. Only the fiber of PDMS/DVB (blue) was detected with it.

1, 2-Benzenediol, O-(2-furoyl)-O-(pentafluoropropionyl)- is the only phenol that detected with the fiber of PDMS (100 μm). As a flavor compound, it has phenolic, plastic and rubber odor［23］.

References

［1］ SANZ C, OLIAS JM, PEREZ AG. Aroma biochemistry of fruits and vegetables. In phytochemistry of fruit and vegetables［J］. Oxford University Press Inc.: New York, NY, USA. 1997: 125-155.

［2］ SCHWAB W, DAVIDOVICH-RIKANATI R, LEWINSOHN E. Biosynthesis of plant-derived flavor compounds［J］. Plant J., 2008(54): 712-732.

［3］ ZHANG SL, CHEN KS. Molecular physiology of fruit quality development and regulation［J］. Beijing: China Agriculture Press, 2007.

［4］ LI GP. Studies on volatiles in fruit of Chinese pear cultivars and expression of ester biosynthesis related genes［J］. Hangzhou: Zhejiang University, 2012.

［5］ EBELER SE, THOMGATE JH. Wine chemistry and flavor: looking into the crystal glass［J］. Journal of Agricultural and Food Chemistry, 2009(57): 8098-8108.

［6］ PAWLISZYN J. Solid phase microextraction: theory and practice［M］. New York: John Wileyand Sons, 1997.

［7］ NIJSSEN LM, VAN INGEN-VISSCHER CA, DONDERS JJH. VCF volatile compounds in food: database (Version 13.1.)［J］. Zeist (The Netherlands): TNO Triskelion Recuperato da, . 2011

［8］ DIXON J, HEWETT EW. Factors affecting apple aroma/flavour volatile concentration: A review［J］. N. Z. J. Crop Hortic. Sci, 2000(28): 155-173.

［9］ RIZZOLO A, GRASSI M, ZERBINI PE. Influence of harvest date on ripening and volatile compounds in the scab-resistant apple cultivar "Golden Orange"［J］. Hortic. Sci. Biotech, 2006(81): 681-690.

［10］ HOLLAND D, LARKOV O, BAR-YABKOV I, et al. Developmental and varietal differences in volatile ester formation and acetyl-CoA: Alcohol acetyl transferase activities in apple ( Malus domestica Borkh.) fruit［J］. Agric. Food Chem, 2005(53): 7198-7203.

［11］ WANG CZ, ZHANG YM, XU YT, et al. Optimization of extracting conditions for apple aroma by SPME-GC-MS［J］. Shandong Agricultural Sciences, 2012, 44(7): 116-120

［12］ XIAO Z, ZHOU X, NIU Y, et al. Optimization and application of headspace-solid-phase micro-extraction coupled with gas chromatography-mass spectrometry for the determination of volatile compounds in Cherry wines［J］. Journal of Chromatography B, 2014: 122-130

［13］ PAILLARD NMM. The flavour of apples, pears and quinces［J］. Amsterdam: Elsevier Science Publishing CompanyInc, 1990: 1-41.

［14］ GOKBULUT I, KARABULUT I. SPME-GC-MS detection of volatile compounds in apricot varieties［J］. Food Chemistry, 2012(132): 1098-1102.

［15］ NIKFARDJAM MARTIN POUR MAIER, DANIEL. Development of a headspace trap HRGC/MS method for the assessment of the relevance of certain aroma compounds on the sensorial characteristics of commercial apple juice［J］. Food Chemistry, 2011, 126(4): 1926-1933.

［16］ WANG Y, YANG C, LI S, et al. Volatile characteristics of 50 peaches and nectarines evaluated by HP-SPME with GC-MS［J］. Food Chemistry, 2009, 116(1): 356-364.

［17］ RIZZOLO A, CAMBIAGHI P, GRASSI M, et al. Influence of 1-methylcyclopropene and storage atmosphere on changes in volatile compounds and fruit quality of conference pears［J］. Agric. Food Chem, 2005(53): 9781-9789.

［18］ PORTNOY V, BENYAMINI Y, BAR E. The molecular and biochemical basis for varietal variation in sesquiterpene content in melon ( Cucumis melo L.) rinds［J］. Plant Mol. Biol., 2008(66): 647-661.

［19］ JETTI RR, YANG E, KURNIANTA A, et al. Quantification of selected aroma-active compounds in strawberries by headspace solid-phase microextraction gas chromatography and correlation with sensory descriptive analysis［J］. Food Sci., 2007(72): S487-S496.

［20］ WENDAKOON SK, UEDA Y, IMAHORI Y, et al. Effect of short-term anaerobic conditions on the production of volatiles, activity of alcohol acetyltransferase and other quality traits of ripened bananas［J］. Sci. Food Agric., 2006(86): 1475-1480.

［21］ BERGER RG. Flavours and fragrances-chemistry, bioprocessing and sustainability［J］ Springer-Verlag: Berlin, Germany, 2007.

［22］ VILLATORO C, ALTISENT R, ECHEVERRIA G, et al. Changes in biosynthesis of aroma volatile compounds during on-tree maturation of "Pink Lady" apples postharvest biol［J］. Technol, 2008(47): 286-295.

［23］ AUBERT C, BOURGER N. Investigation of volatiles in Charentais cantaloupe melons ( Cucumis melo var. cantalupensis ). Characterization of aroma constituents in some cultivars［J］. Agric. Food Chem., 2004(52): 4522-4528.

［24］ YU AN, SUN BG, TIAN DT, et al. Analysis of volatile compounds in traditional smoke-cured bacon (CSCB) with different fiber coatings using SPME［J］. Food Chem., 2008(110): 233-238.

［25］ GUADAGNI DG, BOMBEN JL, HUDSON JS. Factors influencing the development of aroma in apple peel［J］. Sci. Food Agric, 1971(22): 110-115.

［26］ DEFILIPPI BG, DANDEKAR AM, KADER AA. Relationship of ethylene biosynthesis to volatile production, related enzymes and precursor availability in apple peel and flesh tissues［J］. Agric. Food Chem., 2005(53): 3133-3141.

［27］ WANG XD, SHI DC, SONG Y, et al. GC-MS analysis of fruit aroma components of organic Fuji apple［J］. Acta Horticulturae Sinica, 2005, 32(6): 27-31.

［28］ GONZáLEZ-AGaERO M, TRONCOSO S, GUDENSCHWAGER O, et al.  Differential expression levels of aroma-related genes during ripening of apricot ( Prunus armeniaca L.)［J］. Plant Physiol. Biochem, 2009(47): 435-440.

［29］ KALUA CM, BOSS PK. Comparison of major volatile compounds from Riesling and Cabernet Sauvignon grapes ( Vitis vinifera L.) from fruitset to harvest. Austra［J］. Grape Wine Res, 2010(16): 337-348.

［30］ CHAI Q, WU B, LIU W, et al. Volatiles of plums evaluated by HS-SPME with GC-MS at the germplasm level［J］. Food Chemistry, 2012(130): 432-440.

［31］ DU XF, KURNIANTA A, MCDANIEL M, et al. Flavour profiling of ‘Marion and thornless blackberries by instrumental and sensory analysis［J］. Food Chemistry, 2010, 121(4): 1080-1088.

［32］ SU SY, JIANG WG, ZHAOYP. Characterization of the aroma-active compounds in five sweet cherry cultivars grown in Yantai［J］. Flavour and Fragrance Journal, 2010, 25(4): 206-213.

［33］ AUBERT C, CHANFORAN C. Postharvest changes in physicochemical properties and volatile constituents of apricot ( Prunus armeniaca L.). characterization of 28 cultivars［J］. Agric. Food Chem., 2007(55): 3074-3082.

［34］ RUDELL DR, MATTINSON DS, MATTHEIS JP, et al. Investigations of aroma volatile biosynthesis under anoxic conditions and in different tissues of "Redchief Dilicious" apple fruit (Malus domestica Borkh.) ［J］. Agric. Food Chem., 2002(50): 2627-2632.

［35］ YAN ZL, ZHANG SN, ZHANG QJ, et al. MS/GC analysis of aromatic components in Huaguan apple fruit［J］. Journal of Fruit Science, 2005, 22(3): 198-202.