Dual-fiber solid-phase microextraction coupled with gas chromatography–mass spectrometry for the analysis of volatile compounds in traditional Chinese dry-cured ham

Journal Pre-proofs Dual-fiber solid-phase microextraction coupled with gas chromatography– mass spectrometry for the analysis of volatile compounds in traditional Chinese dry-cured ham Huan Liu, Junlong Huang, Qingkun Hu, Yan Ping Chen, Keqiang Lai, Jianqiao Xu, Gangfeng Ouyang, Yuan Liu PII: DOI: Reference:

S1570-0232(19)31403-5 https://doi.org/10.1016/j.jchromb.2020.121994 CHROMB 121994

To appear in:

Journal of Chromatography B

Received Date: Revised Date: Accepted Date:

19 September 2019 1 December 2019 14 January 2020

Please cite this article as: H. Liu, J. Huang, Q. Hu, Y. Ping Chen, K. Lai, J. Xu, G. Ouyang, Y. Liu, Dual-fiber solid-phase microextraction coupled with gas chromatography–mass spectrometry for the analysis of volatile compounds in traditional Chinese dry-cured ham, Journal of Chromatography B (2020), doi: https://doi.org/ 10.1016/j.jchromb.2020.121994

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Dual-fiber

solid-phase

microextraction

coupled

with

gas

chromatography–mass spectrometry for the analysis of volatile compounds in traditional Chinese dry-cured ham

Huan Liu1,2, Junlong Huang3, Qingkun Hu3, Yan Ping Chen2, Keqiang Lai1, Jianqiao Xu3,*, Gangfeng Ouyang3, Yuan Liu2,*

1

College of Food Science & Technology, Shanghai Ocean University, Shanghai

2001306, China 2

Department of Food Science & Technology, School of Agriculture and Biology,

Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] (Y.L.) 3

MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of

Chemistry, Sun Yat-sen University, Guangzhou 510275, Guangdong, China; [email protected] (J.X.)

*To whom correspondence should be addressed: E-mail: [email protected] (J.X.); [email protected] (Y.L.) Tel.: +86-021-34208536 (Y.L.)

1 / 34

Abstract For the purpose of obtaining a more comprehensive flavor profile, volatile compounds in traditional Chinese dry-cured hams were studied by dual-fiber solid-phase microextraction (SPME) using two fibers simultaneously. By using the selected pair of fibers and under the optimal extraction time, there were total seventy-two volatile compounds identified, which was higher than the mono-fiber SPME method using a single fiber. Out of the seventy-two compounds, twenty-six compounds were not detected by using mono-fiber SPME and five among them are classified as the major aromatic compounds in the literatures. Due to the higher coverage and less tendency for the occurrence of competition among the volatiles, the total amount of volatiles extracted by dual-fiber SPME (510.02 ng/kg) was higher than mono-fiber SPME. Three grades of dry-cured hams were successfully distinguished based on dual-fiber SPME. The volatile compounds belonged to nine chemical families and differed in different grades of dry-cured hams. These results show that dual-fiber SPME is capable of analyzing flavor profiles more comprehensively and distinguishing traditional Chinese dry-cured hams.

Keywords Dual-fiber solid-phase microextraction; Volatile compounds; Traditional Chinese dry-cured ham; Distinction of grade

1. Introduction 2 / 34

Dry-cured ham is a kind of value-added meat products with high levels of consumer acceptance due to their unique flavor and long-term preservation. In the Mediterranean area, Iberian and Serrano of Spanish, Parma and San Daniele of Italian and French Bayonne hams are the most important dry-cured hams. While among the traditional Chinese dry-cured hams, Jinhua, Rugao and Xuanwei hams are more well-known. They are generally produced by traditional production processes including raw material selection, salting, soaking and washing, sun drying and shaping, fermentation, ripening, post-ripening, grading and storage [1]. Some biochemical reactions in processing, such as lipolysis and proteolysis, are responsible for the development of a wide range of volatile compounds and precursors. These volatiles include aldehydes, alcohols, ketones, sulfur and nitrogen compounds and others. Meanwhile, they are closely related to the distinctive flavor of dry-cured hams [2,3] and could be different under the alterations of muscle types and maturing conditions [4,5]. Aroma and flavor as the key attributes reflect the quality traits of hams and would impact the overall acceptance. Therefore, a study on the volatile components is necessary to help monitor flavor quality of dry-cured hams. Solid-phase microextraction (SPME) is a relatively mature sample pretreatment method and has gained many interests because of the solvent-free, low-cost and time-saving merits. Furthermore, a sensitive analysis would be achieved without using large amounts of samples and be developed for non-lethal analysis in living fish [6]. This technique has been proved to be effective for the analysis of volatile components in meat products, especially combined with gas chromatography–mass spectrometry (GC-MS) apparatus [7,8]. The extraction performance depends on the distribution of analytes among different phases (sample matrix, headspace and fiber coating) [9]. Analytes tend to distribute to 3 / 34

the fiber coatings when the coatings possess high affinities to the analytes. Thus, in order to get more satisfactory results, sometimes the adsorption properties of various fibers would be screened prior to the formal experiment [10]. Nevertheless, these SPME fibers coated with different stationary phases give different gas chromatographic

profiles.

For

instance,

the

commercial

divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber exhibited high affinity for volatile compounds with low retention index (RI), while the extracted amounts of volatiles with high RI were satisfying using the commercial polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber in the analysis of sauced duck neck meats [11]. In relation to dry fermented sausages, the DVB/CAR/PDMS fiber extracted large amounts of high molecular weight volatile compounds. By contrast, several low molecular weight compounds were only extracted by the carboxen/polydimethylsiloxane (CAR/PDMS) fiber [12]. J. Stephen Elmore and co-authors have once put forward a SPME technique to utilize two fibers for collecting flavor compounds from cooked porks. In that study, two fibers coated with different stationary phases were inserted into one vial simultaneously to adsorb volatile compounds and the desorbed compounds were collected in a prototype of cooled injection system (CIS). As a result, the chromatographic result of two fibers was displayed in one gas chromatogram and a more comprehensive flavor profile was obtained [13]. However, the fibers were selected based on pure experientialism. In the present study, to investigate profiles of traditional Chinese dry-cure hams, two out of five SPME fibers were selected and the extraction time was optimized for the implement of dual-fiber SPME. Meanwhile, the thermal desorption unit (TDU) and CIS were applied for trapping and focusing the desorbed volatile compounds, which have been proven to be simple and effective in 4 / 34

food flavor analysis [14,15]. Furthermore, the flavor profiles of three different grades of hams were distinguished based on the dual-fiber SPME technique.

2. Materials and methods 2.1 Chemicals and materials 4-Methyl-2-pentanol (99%) was purchased from J&K Scientific Ltd. (Beijing, China). Methanol, n-hexane, n-heptane, n-octane and n-nonane were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Alkanes solution (C10-C25) was purchased from Smart Solutions. NaCl was obtained from Guangzhou Chemical Factory (Guangzhou, China). Commercial Jinhua hams were purchased from Jinzi Corporation Limited Company of Zhejiang Province which have three grades according to their producing time and technology (JTY, JY and J1). After purchase, they were vacuum-packed and stored at -20 ℃ . Before experiment the visible fat layer and cortex on the surface were removed following by overnight thawing at 4 ℃. Five commercial SPME fibers (Supelco, Bellefonte, PA, USA), including 85 μm polyacrylate (PA), 100 μm polydimethylsiloxane (PDMS), 65 μm PDMS/DVB, 50/30 μm DVB/CAR/PDMS and 85 μm Carboxen/PDMS were preconditioned before extraction at the recommended temperature and time according to manufacturers. 2.2 Optimization of SPME 30 g of biceps femoris muscle of JY was ground with a commercial grinder. Then 4 g of minced muscle and 7 g of distilled water saturated with NaCl [5] were placed into 20-mL vial, tightly capped with a PTFE septum. The vial containing sample was 5 / 34

firstly incubated at 40 ℃ for 30 min. Then one of the five commercial SPME fibers pierced vial to extract 20 min, 40 min and 60 min, respectively, under the same temperature as incubation. After extraction the fiber was immediately introduced into 7890B GC coupled to a 5977A MS (Agilent technologies, CA, USA) for about 5 min with splitless mode. 2.3 Dual-fiber SPME The sample preparations were same as optimization of SPME. In this section, after incubation at 40 ℃

for 30 min, 50/30 μm DVB/CAR/PDMS and 85 μm

Carboxen/PDMS were inserted into 20-mL vial at the same time for 40 min. After extraction, DVB/CAR/PDMS was introduced into TDU (Gerstel) at 250 ℃ for about 2 min, followed by desorbing Carboxen/PDMS for another 2 min with splitless mode. CIS was used to refocus volatile compounds at -40 ℃ which connected with 7890B GC coupled to a 5977B MS (Agilent technologies, CA, USA). The CIS heating rate was 12 ℃/s to 260 ℃, holding for 6 min with low split mode. Finally, the other two grades of hams (JTY and J1) were also analyzed by dual-fiber SPME under the same conditions of JY. 2.4 Dynamic headspace extraction (DHS) 1 g of JY was added in a 20-mL headspace vial. After 30 min of equilibrium, the volatile compounds were trapped onto a Tenax TA tube for 60 min with nitrogen stream at a flow-rate of 100 mL/min. The temperature was 40 ℃ all the time. The adsorbed compounds were then released at 280 ℃ by desorbing Tenax TA tube in TDU system for 5 min. CIS was used to refocus these compounds with heating rate of 6 / 34

12 ℃/s from -40 ℃ to 260 ℃, holding for 5 min. Split ratio was 40﹕1. 2.5 GC-MS analysis GC oven program was 40 ℃ for 2 min, ramped at 4 ℃/min to 70 ℃, and then ramped at 1 ℃/min to 80 ℃ and ramped at 3 ℃/min to 100 ℃, ramped at 7 ℃ /min to 240 ℃ , holding for 3 min. Chromatographic separation was performed with a HP-5ms (30 m × 0.25 mm I.D. × 0.25 μm thickness) fused silica column from Agilent and ultra-high purity helium was used as the carrier gas. Electron-impact mass spectra was generated at 70 eV with m/z scan range from 35 to 550 amu. The ion source temperature was 230 ℃. For the sake of quantifying extraction amounts of analytes, 1 μL of 4-methyl-2-pentanol solution (C = 0.3 mg/kg) was directly injected into GC-MS with a split ratio of 30﹕1. The volatile compounds were identified by NIST 17 mass spectra library installed in the GC-MS equipment. Triplicate analyses were performed on each sample and all compounds were identified by target ion. Furthermore, a C6–C25 n-alkane series was analyzed under the same chromatographic conditions for calculating RIs of target analytes, as shown the following expression: 𝑅𝑡(𝑥) ― 𝑅𝑡(𝑛)

𝑅𝐼 = 100 (𝑅𝑡(𝑛 + 1) ― 𝑅𝑡(𝑛) +𝑛) where 𝑅𝑡(𝑥), 𝑅𝑡(𝑛) and 𝑅𝑡(𝑛 + 1) are the retention times of target analyte, Cn and Cn+1, respectively and 𝑅𝑡(𝑛) < 𝑅𝑡(𝑥) < 𝑅𝑡(𝑛 + 1). 2.6 Statistical analyses The extraction amounts of volatile compounds were studied by one-way analysis of variance (ANOVA) using the IBM SPSS Statistics 19.0 program (IBM Corporation, Somers, NY, USA). Student-Newman-Keuls (SNK) test was used for mean 7 / 34

comparison. Statistical significance was set at p < 0.05.

3. Results and discussions 3.1 Optimization of SPME Five commercial fibers (85 μm PA, 100 μm PDMS, 65 μm PDMS/DVB, 50/30 μm DVB/CAR/PDMS and 85 μm Carboxen/PDMS) and extraction time (ranging from 20 min to 60 min) were optimized for high efficient extraction. Throughout the whole experiment, extraction temperature was 40 ℃ to avoid the formation of artifacts caused by thermal degradation. As Ruiz et al. reported that an increase in area of thermally degraded compounds would be found at 60 ℃ , but not at 40 ℃ under long extraction time [16]. As shown in Figure 1, two- and three-phase fibers extracted more volatile compounds than PA or PDMS. Given combination of multiphase fibers, compounds unique to each fiber were filtered and the largest number was obtained in the extraction by Carboxen/PDMS followed by DVB/CAR/PDMS. Moreover, the total number of compounds extracted by these two fibers was largest as well (data not shown). Regarding the major aromatic compounds (Figure 1) which contribute significantly to ham flavor, Carboxen/PDMS and DVB/CAR/PDMS extracted more compounds than the other three fibers. In addition, the largest number was obtained at 40 min for all five fibers. Thus, DVB/CAR/PDMS and Carboxen/PDMS would be applied for dual-fiber SPME with 40 minutes of extraction. Fig. 1 8 / 34

3.2 Comparison between dual-fiber SPME and mono-fiber SPME In order to compare the analysis results of dual-fiber SPME and mono-fiber SPME, extractions using DVB/CAR/PDMS and Carboxen/PDMS separately were conducted and the adsorbed compounds were directly desorbed in 7890B GC coupled to a 5977B MS detector. There were eighty volatile compounds detected in JY which belonged to nine chemical families: aldehydes, alcohols, ketones, terpenes, esters, acids, aliphatic hydrocarbons, aromatic hydrocarbons and others. The number of volatile compounds extracted by DVB/CAR/PDMS-Carboxen/PDMS (DCP-CP) (seventy-two compounds) is higher than that extracted by DVB/CAR/PDMS (sixty-three compounds) and Carboxen/PDMS (fifty-three compounds) individually (Figure 2a). Beside that, there is a marked increase in the number of aromatic hydrocarbons extracted by DCP-CP, whereas, the extraction of acids is not very effective. In Figure 2b, DCP-CP performed the maximum extraction capacity of volatile compounds reaching to 510.02 ng/kg which may be caused by the decrease of competitive adsorption effect among volatiles. More total amount of volatile compounds was obtained by DVB/CAR/PDMS than by Carboxen/PDMS, which is in disagreement with previous studies of pork loin [17,18]. To estimate reproducibility of DCP-CP , the whole experiment was repeated in triplicate and relative standard deviation (RSD) for most quantified peak was less than 25%. Fig. 2 Table 1 shows the detailed information of volatile compounds analyzed by monoand dual-fiber SPME. Among the total eighty volatile compounds, twenty-six 9 / 34

compounds are considered to be major aromatic compounds according to previous researches on Jinhua ham. There were fourteen compounds only extracted by DCP-CP and except for butanal, benzene and pyridine with high volatility (RI<750), the others were detected at long retention time (RI>1060). Hashimoto et al. have studied the volatile components in orange Juice by SPME cryofocusing and results reveal that the introduce of cold trap is effective to analyze the trace volatile compounds [19]. In addition, twelve compounds missing in the extraction of DVB/CAR/PDMS or Carboxen/PDMS were also extracted by DCP-CP. Table 1 The most abundant compounds in JY are aldehydes (DVB/CAR/PDMS, 60.07 ng/kg; Carboxen/PDMS, 56.86 ng/kg; DCP-CP, 204.4 ng/kg), which is in accordance with the analysis of Dalmatian smoked dry-cured ham (35.6%) [20]. Six aldehydes were extracted by DCP-CP while not by either DVB/CAR/PDMS or Carboxen/PDMS or both. Three of them are butanal, 2-methylbutanal and (E)-2-octenal defined as the major aromatic compounds. Butanal has ever been detected in Jinhua ham by applying a cold trap at -30 ℃ [21]. Also, cooking and experimental conditions would affect its extraction from salami samples [22,23]. To our knowledge, this may be the first time to report the presences of 2-chloro-hexanal, 2-phenylpropenal and 2-butyl-2-octenal in Jinhua ham as the flavor components. Yet, 2-chloro-hexanal was only extracted by Carboxen/PDMS with 0.49 ng/kg. Alcohols generally have a high threshold value except for mushroom-flavored 1-octen-3-ol. In this study, 1-octen-3-ol represents 76.12%, 78.57% and 59.01% of 10 / 34

alcohols for DVB/CAR/PDMS, Carboxen/PDMS and DCP-CP, respectively. It is generally the most abundant alcohol in meat products such as Istrian dry-cured ham [24] and dry-cured ‘lacón’ at the end of drying–ripening stage [25]. On the contrary, 1-penten-3-ol accounts for the minimum proportions (0.04%; 0; 0.04%). (Z)-2-Octen-1-ol was only extracted by DCP-CP with amount of 1.06 ng/kg. Nevertheless, existence of split flow in TDU-CIS and relatively low concentration in ham may be responsible for the absence of phenylethyl alcohol in the extraction results of DCP-CP. As reported in other researches, the relative amount of phenylethyl alcohol is low with only 0.04% in Jinhua ham or lower than 0.03% in Toscano dry-cured ham within a ripening time of three months [26,27]. Regarding ketones, 2-heptanone is the predominant ketone at the end of process [25] exerting great influence on meat samples. In this study, it shows a significant difference (p<0.05) in extraction amount between two fibers and single fiber. In addition, three compounds (3,5-octadien-2-one, acetophenone and 9H-fluoren-9-one) were extracted by DCP-CP which missed in the extraction of Carboxen/PDMS or DVB/CAR/PDMS. Whereas, 2-methyl-cyclopentanone was only extracted by DVB/CAR/PDMS at a very low amount (0.03 ng/kg). It could be explained in the bases of injectors, because split ratio with TDU-CIS may lead to the loss of ketones. These differences the volatile extraction methods, Among the three terpenes, d-limonene is determined as a dominant compound to ham flavor imparting sweet /orange note in Jinhua ham [24,28] or giving a bit of lemon aroma in półgęsek [29]. What’s more, a significant statistical difference is showed in the extraction amount of 11 / 34

DVB/CAR/PDMS, Carboxen/PDMS and DCP-CP. There was hexanoic acid methyl ester extracted in this study and it has been found to be the most abundant ester at the end of the process of Celta dry-cured ‘lacón’ [25]. Carboxen/PDMS had a higher affinity for esters (3.3 ng/kg) than DCP-CP (1.64 ng/kg) and DVB/CAR/PDMS (1.16 ng/kg). As reported by Marco et al, CAR/PDMS coating had a higher affinity for esters while DVB/CAR/PDMS showed higher affinity for aldehydes [28]. On the other hand, low content of esters in the analysis of DCP-CP may be caused by the competitive adsorption with large amount absorbed compounds. Although the extraction of acids by DCP-CP is not satisfactory since it is failed to extract three acids, the successful extraction of 2-methylpentanoic acid anhydride was achieved by DCP-CP. Furthermore, DCP-CP showed good extraction capability of aliphatic hydrocarbons, especially tetradecane is absent in the extraction of Carboxen/PDMS. In aromatic hydrocarbons, six low-volatility compounds with RI varying from 1080.29 to 2009.47 were only extracted by DCP-CP. This may be due to the effective extraction of compounds by applying a cold trap [19]. In addition, high-volatility benzene and pyridine were also captured by DCP-CP because of cold trap and more absorbent materials. To our knowledge, the presence of pyridine in Iberian dry-cured ham has been previously reported using a 2 cm DVB/CAR/PDMS fiber and it was found in dry-cured loins, albeit tiny amounts [7,30]. In turn, high temperature during sampling probably favors the thermal reactions and generation of pyrazines from Maillard reaction in cooked meats [31,32]. Detected pyrazines herein are 12 / 34

2,6-dimethylpyrazine, trimethylpyrazine and tetramethylpyrazine among which 2,6-dimethylpyrazine was just not extracted by Carboxen/PDMS. Another major flavoring compound in ham is 2-pentylfuran the proportion of which varies between 2.6% and 0.41% in charqui samples [33]. In this study, 2-pentylfuran represents 7.81% for DCP-CP, while represents 41.54% and 27.27% for DVB/CAR/PDMS and Carboxen/PDMS, respectively. Though pentyl oxirane and methoxy-phenyl oxime have the similar RI (about 900), only the former was detected by DCP-CP. In turn, between the two identified sulfur compounds, dimethyl trisulfide is absent in the extraction of DVB/CAR/PDMS which has been proved to be the cooked cabbage flavor [34]. Yet methylene chloride is absent in the extraction of Carboxen/PDMS. Finally, the volatile compounds studied by DHS are consistent with that by dual-fiber SPME, exceptions are three major compounds (acetic acid，butyrolactone and 2,6-dimethylpyrazine) unique for the former and five major compounds (butanal， 3-methylbutanal, 2-methylbutanal, dimethyl disulfide and dimethyl trisulfide) unique for the latter. Nevertheless, large split ratio (40﹕1) may be one reason for the losses of some compounds with high volatility or low concentration before injecting into column. In terms of above results, it could be concluded that dual-fiber SPME is successful to comprehensively analyze the flavor profile of dry-cure hams. 3.3 Distinction of dry-cured ham Displayed in Figure 3 are the proportions of volatile compounds in JTY, JY and J1 which are mainly derived from smoking, lipid oxidative reactions and seasoning [35]. 13 / 34

Results indicate that aldehydes are the most abundant compounds in JY and J1, approximately ten and eight times as many as JTY. Similarly, Ge et al. have reported that contents of aldehydes in three Jinhua hams from different factories reach to 49.8%, 36.6% and 33.2%, respectively [36]. However, the methional only presents in JTY which is the product of L-methionine reaction with a cooked potato-like flavor [3,34]. Acids account for the largest proportion in JTY reaching to 29.78% with 2.2-fold of J1. In contrast with linear chain acids, branched chain acids generally play a crucial role in aroma development of JY because of their characteristic cheesy odor [3,37]. Fig. 3 Moreover, different from JY and J1, esters represent the second largest proportion (22.23%) for JTY, in which three methyl esters and butyrolactone defined as fruity aroma [12,37] were detected. Furthermore, the content of lactones could be related to intramuscular fat contents [38]. In turn, the tetramethylpyrazine characterised as rosted nuts or chocolate [12,37] is absent in JTY and the extraction amounts of alcohols and ketones differ among three grades of hams. These results show that dual-fiber SPME described herein allows for the efficient distinction of volatile compounds in different grades of dry-cured hams.

4. Conclusions In this study, an efficient sample pretreatment technique termed dual-fiber SPME was employed to analyze the volatile compounds of traditional Chinese dry-cured 14 / 34

hams. Out of the eighty volatile compounds in JY, twenty-six compounds were successfully extracted by DCP-CP which were absent in the extraction of DVB/CAR/PDMS or Carboxen/PDMS and among them, butanal, 2-methylbutanal, (E)-2-octenal, 2,6-dimethylpyrazine and dimethyl trisulfide are described as the major aromatic compounds. In addition, dual-fiber SPME performed a larger extraction capacity than mono-fiber SPME. Further study was also conducted to distinguish three grades of dry-cured hams based on dual-fiber SPME. Results reveal that the aldehydes are the most abundant compounds in JY and J1, while acids account for the largest proportion in JTY reaching to 29.78%. Moreover, the second largest proportion in JTY, fruity esters only represent about 0.4% for JY and J1. Also, the proportions of the other chemical groups differ among three grades of dry-cured ham. In conclusion, dual-fiber SPME is a feasible technique allowing for comprehensive study of volatile compounds in dry-cured hams and effective distinction of flavor profiles among different grades of dry-cured hams. In the future, for the analysis of the volatile profiles of food as thoroughly as possible, the present study indicates that mixed-mode SPME fiber coatings might need to be developed.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (J.X.); [email protected] (Y.L.)

15 / 34

Notes The authors declare no competing financial interest.

Funding This work was financially supported by National Natural Science Foundation of China (Grant No. 31622042, 21806188, 21527813 and 21737006) and Natural Science Foundation of Guangdong Province (2018A030313324).

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analysis of volatile components in orange juice, Food Sci. Technol. Res. 12 (2006) 295-298. [20] N. M. Radovčić, S. Vidaček, T. Janči, H. Medić, Characterization of volatile compounds, physico-chemical and sensory characteristics of smoked dry-cured ham, J. Food Sci. Technol. 53 (2016) 4093-4105. [21]H. Tian, Z. Wang, The use of thermal desorption-GC-MS in volatile flavor profiling of jinhua ham, Food and Mach. 23 (2007) 10. [22]R. Wagner, M. R. B. Franco, Effect of the variables time and temperature on volatile compounds extraction of salami by solid phase microextraction, Food Anal. Method. 5 (2012) 1186-1195. [23]J. S. Pulgar, M. Roldan, J. Ruiz-Carrascal, Volatile compounds profile of sous-vide cooked pork cheeks as affected by cooking conditions (vacuum packaging, temperature and time), Molecules 18 (2013) 12538-12547. [24]N. Marušić, S. Vidaček, T. Janči, T. Petrak, H. Medić, Determination of volatile compounds and quality parameters of traditional Istrian dry-cured ham, Meat Sci. 96 (2014) 1409–1416. [25]J. M. Lorenzo, S. Fonseca, Volatile compounds of Celta dry-cured 'lacón' as affected by cross-breeding with Duroc and Landrace genotypes, J. Sci. Food Agric. 94 (2014) 2978–2985. [26]H. Tian, Z. Wang, S. Xu, Characterization of odor-active compounds in jinhua ham by GC-olfactometry, Food Ferment. Ind. 30 (2004) 117-123. [27]C. Pugliese, F. Sirtori, L. Calamaib, O. Francia, The evolution of volatile 19 / 34

compounds profile of "Toscano" dry-cured ham during ripening as revealed by SPME-GC-MS approach, J. Mass Spectrom. 45 (2010) 1056–1064. [28]A. Marco, J. L. Navarro, M. Flores, Volatile compounds of dry-fermented sausages as affected by solid-phase microextraction (SPME), Food Chem. 84 (2004) 633–641. [29]K. Nowicka, D. Jaworska, W. Przybylski, E. Górska, K. Tambor, A. Półtorak, Determinants of the sensory quality of półgȩsek in relation to volatile compounds and chemical composition, Pol. J. Food Nutr. Sci. 67 (2017) 283-292. [30]S. Ventanas, J. Ventanas, M. Estévez, J. Ruiz, Analysis of volatile molecules in Iberian dry-cured loins as affected by genetic, feeding systems and ingredients, Eur. Food Res. Technol. 231 (2010) 225–235. [31]V. Vasta, V. Ventura, G. Luciano, V. Andronico, R. I. Pagano, M. Scerra, Biondi, M. Avondo, A. Priolo, The volatile compounds in lamb fat are affected by the time of grazing, Meat Sci. 90 (2012) 451–456. [32]J. -C. Xie, B. -G. Sun, S. -B. Wang, Aromatic constituents from Chinese traditional smoke-cured bacon of mini-pig, Food Sci. Technol. Int. 14 (2008) 329–340. [33]M, P. Gianelli, V, Salazar, L, Mojica, M, Friz, Volatile compounds present in traditional meat products (charqui and longaniza sausage) in Chile, Braz. Arch. Biol. Techn. 96 (2014) 179-186. [34]M. Kosowska, M. A. Majcher, H. H. Jeleń, T. Fortuna, Key aroma compounds in smoked cooked loin, J. Agric. Food Chem. 66 (2018) 3683−3690. 20 / 34

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Figure Captions Graphical abstract. Illustration of the experimental design about extraction and desorption. 21 / 34

Figure 1. The number of all and major volatile compounds extracted by five fibers.

Figure 2. (a) The number and (b) extraction amount of volatile compounds in JY analyzed by mono- and dual-fiber SPME.

Figure 3. (a) The relative proportions of nine chemical families in JTY, (b) JY and (c) J1.

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Graphical abstract.

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Figure 1.

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Figure 2.

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Figure 3.

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Table 1. The extraction amount of volatile compounds in JY analyzed by mono- and dual-fiber SPME (concentration ± means). RId

Compoundse

606.21 645.55 655.92 695.88 802.35 848.79 900.22 904.54 949.34 951.92 1000.27 1033.84 1048.90 1100.11 1153.07 1156.19 1200.16 1208.65 1264.92 1293.14

Aldehydes Butanal 3-methylbutanal 2-methylbutanal Pentanal Hexanal 2-Hexenal Heptanal 2-Chloro-hexanal (Z)-2-Heptenal Benzaldehyde Octanal Benzeneacetaldehyde (E)-2-Octenal Nonanal 2-Phenylpropenal (E)-2-Nonenal Decanal (E,E)-2,4-Nonadienal (E)-2-Decenal (E,Z)-2,4-Decadienal

Extraction amount, ng/kg 50/30 μm 85 μm DVB/CAR/PDMS DVB/CAR/PDMS Carboxen/PDMS - Carboxen/PDMS not quantified 0.41±0.05b 0.52±0.06 2.06±0.12b 34.15±1.91b 0.1±0b 13.16±3.31a not quantified 0.63±0.02b 1.19±0.17b 0.89±0.14b 1.84±0.31a not quantified 2.35±0.25b not quantified 0.2±0.04b 0.68±0.11a 0.45±0.07b 1.2±0.25a 0.1±0.02

not quantified 0.17±0.02c not quantified 2.37±0.17b 35.93±1.86b 0.16±0.04b 12.16±1.46a 0.49±0.04 0.33±0.04c 0.72±0.16c 0.8±0.12b 0.46±0.12b 0.57±0.07 1.74±0.25c not quantified 0.09±0.02c 0.55±0.08a 0.12±0.03c 0.19±0.26b not quantified

2.42±0.97 0.75±0.26a 3.03±0.81 16.53±1.59a 148.66±8.33a 9.49±4.52a 0.47±0.02b not quantified 1.93±0.08a 4.83±0.32a 1.9±0.17a 3.33±1.72a 2.19±0.18 4.59±0.32a 0.57±0.12 0.38±0.04a 0.13±0.01b 0.87±0.23a 1.75±0.55a 0.18±0.05

pf

*** *** *** *** *** *** *** *** ** *** *** *** ** **

Identificationg

MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS MS, RI MS, RI MS, RI MS, RI MS, RI 27 / 34

1316.03 1375.45

(E,E)-2,4-Decadienal 2-Butyl-2-octenal

0.11±0.03 0.03±0.01b

not quantified 0.01±0b

0.28±0.12 0.12±0.01a

676.40 728.92 762.35 866.03 964.59 975.03 1064.92 1063.39 1106.47

Alcohols 1-Penten-3-ol 3-Methyl-1-butanol 1-Pentanol 1-Hexanol 1-Heptanol 1-Octen-3-ol (Z)-2-Octen-1-ol 1-Octanol Phenylethyl alcohol

0.01±0 0.14±0.01 3.34±0.02b 1.33±0.16b 0.46±0.1b 18.65±3.07b not quantified 0.32±0.08b 0.25±0.03

not quantified 0.05±0 2.74±0.11b 0.91±0.01b 0.38±0.06b 15.62±2.54b not quantified 0.18±0.04c not quantified

0.03±0.03 0.45±0.19 18.61±0.62a 3.75±0.33a 1.17±0.09a 45.27±2.64a 1.06±0.22 6.37±1.15a not quantified

836.18 888.55 946.76 1031.13 1043.76 1060.19 1062.47 1148.43 1259.85 1363.70 1692.85

Ketones 2-Methyl-cyclopentanone 2-Heptanone Dihydro-5-methyl-2(3H)-furanone 3-Octen-2-one 5-Ethyldihydro-2(3H)-furanone Acetophenone 3,5-Octadien-2-one Dihydro-5-propyl-2(3H)-furanone 5-Butyldihydro-2(3H)-furanone Dihydro-5-pentyl-2(3H)-furanone 9H-Fluoren-9-one

0.03±0 1.82±0.24b 0.11±0.02b 0.79±0.18b 2.69±0.31 not quantified 0.32±0.05 0.29±0.05b 0.84±0.13b 1.2±0.16b not quantified

not quantified 2.01±0.29b 0.04±0c 0.39±0.12a 2.1±0.35 not quantified not quantified 0.19±0.03c 0.42±0.1c 0.41±0.12c not quantified

not quantified 5.5±0.66a 0.24±0.22a 2.28±0.08c 2.67±0.21 0.46±0.09 12.07±2.46 0.56±0.05a 1.69±0.17a 2.47±0.25a 0.21±0.09

***

*** *** *** *** ***

*** *** *** 0.082

*** *** ***

MS, RI MS

MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI

MS MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS MS, RI MS 28 / 34

1016.84 1020.07 1170.63

Terpenes o-Cymene D-limonene Terpinen-4-ol

3.1±0.78 1.39±0.45a 0.45±0.05a

1.6±0.42 0.45±0.07b 0.16±0.04b

3.07±0.94 0.08±0.01c 0.47±0.04a

0.08 *** ***

MS, RI MS, RI MS, RI

921.78

Esters Hexanoic acid methyl ester

1.16±0.13b

3.31±0.87a

1.64±0.5b

**

MS, RI

856.33 868.78 898.32 1012.38 1051.04 1178.47

Acids 3-Methylbutanoic acid 2-Methylbutanoic acid Pentanoic acid Hexanoic acid 2-Methylpentanoic acid anhydride Octanoic acid

3.97±0.19 1.91±0.09 1.86±0.51 24.89±4.91b 1.04±1 2.91±2.34a

not quantified 0.97±0.21 1.06±0.42 9.02±4.26c not quantified 0.08±0.04b

not quantified not quantified not quantified 97.01±20.95a 8.27±1.66 14.67±10.2a

1022.59 1095.21 1195.39 1399.09 1498.83

Aliphatic hydrocarbons 3-Ethyl-2-methyl-1,3-hexadiene Undecane Dodecane Tetradecane Pentadecane

0.22±0.04b 0.12±0.01a 1.19±0.15a 0.06±0.01 0.04±0b

0.22±0.05b 0.09±0.01b 0.61±0.15b not quantified 0.02±0b

0.45±0.01a 0.14±0.01a 0.3±0.02c 0.17±0.02 0.18±0.04a

680.42

Aromatic hydrocarbons Benzene

not quantified

not quantified

32±13.83

*** **

*** * *** ***

MS, RI MS, RI MS, RI MS, RI MS MS, RI

MS, RI MS, RI MS, RI MS, RI MS, RI

MS, RI 29 / 34

745.14 862.50 887.00 903.27 906.15 908.42 960.71 987.03 998.03 1080.29 1078.25 1087.71 1109.99 1549.73 1732.81 1741.97 2009.47

Pyridine o-Xylene Styrene Pentyl oxirane Methoxy-phenyl oxime 2,6-Dimethylpyrazine Mesitylene 2-Pentylfuran Trimethylpyrazine 1-Ethenyl-3-ethyl-benzene Tetramethylpyrazine 1-Ethenyl-4-ethyl benzene 1,3-Diethenyl benzene Fluorene Phenanthrene Anthracene Pyrene

not quantified 0.24±0.03b 0.89±0.24b 0.26±0.03 0.39±0.3 0.35±0.03 0.44±0.07 2.38±0.32b 0.38±0.02b not quantified 0.4±0.04b not quantified not quantified not quantified not quantified not quantified not quantified

not quantified 0.12±0.01c 0.24±0.09c not quantified 4.16±0.16 not quantified 0.28±0.06 2.1±0.22b 0.42±0.06b not quantified 0.38±0.04b not quantified not quantified not quantified not quantified not quantified not quantified

0.49±0.3 1.05±0.25a 1.7±0.28a 0.35±0.07 not quantified 6.69±1.3 not quantified 4.52±1.28a 1.27±0.09a 0.4±0.07 5.84±2.85a 0.31±0.06 0.44±0.07 0.14±0.04 1.25±0.43 0.64±0.26 0.81±0.38

*** **

** *** *

MS, RI MS, RI MS, RI MS MS, RI MS, RI MS MS, RI MS, RI MS MS, RI MS MS MS MS MS MS

Others <600 Methylene chloride 0.09±0.01 not quantified 4.18±1.61 MS b c a 737.95 Dimethyl disulfide 4.97±0.93 1.13±0.42 11.31±0.44 *** MS, RI b b a 876.30 Hexanenitrile 0.13±0.01 0.11±0.01 0.32±0.02 *** MS a b b 932.67 N,N-Diethyl formamide 0.46±0.03 0.21±0.05 0.13±0.11 ** MS 960.36 Dimethyl trisulfide not quantified 1.55±2.07 0.47±0.09 MS, RI a-c Means in the same row not followed by a common letter are significantly different (p<0.05; SNK test). d RI calculated for HP-5ms (30 m×0.25 mm I.D.×0.25 μm thickness) fused silica column installed on a gas chromatograph equipped with a 30 / 34

mass selective detector. e Compounds stated in bold are significant compounds within class [ 21, 26, 37, 28, 39-41]. f Exact p-values are shown when the means in the same row are not significantly different. Significant differences between means in the same row are indicated with asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. g Identification method of volatile compounds; MS, mass spectrum identified using NIST 17.L; RI, RI in agreement with literature. .

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Highlights 

The volatile compounds in dry-cured hams were studied by dual-fiber SPME.



More volatiles were identified by using dual-fiber SPME in comparison with using mono-fiber SPME.



The flavor profiles of three grades of dry-cured hams were sucessfully distinguished.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Huan Liu: Methodology, Investigation, Writing - Original Draft Junlong Huang: Data Curation Qingkun Hu: Writing- Reviewing and Editing Yan Ping Chen: Writing- Reviewing and Editing Keqiang Lai: Methodology Jianqiao Xu: Validation, Formal analysis Gangfeng Ouyang: Resources, Project administration Yuan Liu: Conceptualization, Supervision, Funding acquisition

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