2020 年 26 巻 1 号 p. 25-37
Spiced beef is a representative traditional Chinese processed beef product. In order to establish quality evaluation of aroma profile, the widely accepted and characteristic volatile flavor of seven typical spiced beef was investigated using headspace solid-phase microextraction combined with gas chromatography-olfactometry-mass spectrometry. A total of 67 volatile flavor compounds were identified in the spiced beef, with 23 compounds in common and 30 aroma-active compounds. The common key aroma-active compounds of the seven types of spiced beef included p-allyl-anisole, 1-methoxy-4-(1-propenyl)-benzene, 3-methyl- 1-butanol, linalool, chavicol, α-phellandrene, myristicin, (Z)-3-hexenol, 1-terpinen-4-ol, furfuryl alcohol, dimethyl pyrazine, and 3-mercaptothiophene. The seven types of spiced beef could be divided into three categories based on their characteristic odors. These three categories had rich odors of sulfur-containing compounds, aldehydes, and spice, respectively. The characteristic odor reflects the geographical differences in consumption habits in China.The results provided a theoretical reference for quality evaluation of packaged spiced beef aroma profile.
Meat products are an important source of high-quality protein. In China, meat consumption has been dominated by pork for a long time (Hu et al., 2015). In recent years, with the upgrading of the consumption structure, the structure of meat consumption has been gradually promoted and beef consumption has increased significantly. It has been reported that beef consumption increased from 6.52 million tons in 2010 to 8.23 million tons in 2017 in China, indicating a significant growth potential. However, beef consumption is still dominated by the use of fresh beef for cooking, with a lack of processed beef products. Monotonous forms of consumption are not conducive to the growth of beef consumption and cannot meet the consumer's demand. Spiced beef is a representative and popular traditional Chinese processed beef product. It is characterized by an emphasis on usage of soybean paste and spices to achieve a rich taste and volatile flavor using water as a heating medium. Although Chinese dietary habits display obvious geographical characteristics, spiced beef is widely accepted by people across China (Yao et al., 2013). The typical traditional Chinese spiced beef primarily includes seven types of products, including Beijing spiced beef, Beijing halal spiced beef, Hong Kong spiced beef, Tianjin spiced beef, Tianjin spiced beef of southern flavor, Shanghai spiced beef, and Inner Mongolia spiced beef (Shi et al., 1999). However, the lack of quality evaluation affects the standardized production and consumer acceptability of packaged spiced beef. The establishment of quality evaluation was helpful to promote its consumption in China. The aroma profile is an important quality evaluation index of spiced beef, which has still been insufficiently researched at present due to the complex reaction in beef boiling and interaction among beef, soybean paste, and spices. Beef volatile flavors are primarily derived from the Maillard reaction between reducing sugars and amino acids, the Strecker degradation that belongs to a sub-reaction within the Maillard reaction, and lipid oxidation (Shahidi, 1998; Werkhoff et al., 1990). Previous research has identified a total of 880 volatileflavor compounds in cooked beef, of which 25 compounds have a meaty odor, including aldehydes, sulfur compounds, and furan compounds (Shahidi, 1998). Regarding spiced beef, volatile flavor compounds and their changes during processing have been investigated for some specific brands. In addition to the beef volatile flavor, several hydrocarbons, ethers, and alcohols with low odor thresholds produced from added seasonings and spices have been used to enhance the overall volatile flavor of spiced beef (Liu et al., 2015; Gong et al., 2017; Sun et al., 2014). The contents of aldehydes, alcohols, and ketones in spiced beef were found to increase significantly with specific pretreatment such as ultrasonic treatment of raw beef (Zou et al., 2018). However, the aroma profileespeciallywidely accepted and characteristic flavors of spiced beef still remain rarely investigated with a lack of systemicity, affecting the quality evaluation and standardized processing of packaged spiced beef.
Currently, techniques such as simultaneous distillation extraction (SDE), solvent-assisted flavor evaporation (SAFE), dynamic headspace sampling, stir bar sorptive extraction (SBSE), and solid-phase microextraction (SPME) are regularly used for the extraction of volatile flavor substances (Kataoka et al., 2000; Watkins et al., 2012; Tian et al., 2018). SDE and SAFE are typically time- and labor-intensive, involving the use of solvents (Polášková et al., 2008). Compared with SDE and SAFE, SPME and SBSE are rapid and efficient at addressing both at-laboratory and on-site requirements without the use of solvents (Vas and Vékey, 2004; Vas et al., 1998; Pawliszyn, 2012). The headspace solid-phase microextraction (HS-SPME) method involves the placement of an SPME fiber in the upper space of the sample to extract the analyte and does not require an organic solvent and set sampling, extraction, and concentration in one step. The method used for analyzing high volatile compounds has the advantages of simplicity, convenience, and sensitivity. Furthermore, gaschromatography-mass spectrometry (GC-MS) is the most important method for identifying volatile flavors in meat products. However, GC-MS cannot determine the contribution of various volatile compounds to the overall flavor. Gas chromatography-olfactometry (GC-O) combined with GC separation ability and human olfaction can effectively evaluate the aroma-active compounds from complex matrix. Combined with GC-MS and GC-O, gas chromatography-olfactometry-mass spectrometry (GC-O-MS) can identify volatile flavor compounds and also analyze the contribution of volatile flavors (Plutowska and Wardencki, 2008; Zhao et al., 2018; Hu et al., 2018).
In this study, the volatile flavor compounds and key aroma-active compounds of seven types of traditional Chinese spiced beef were identified using HS-SPME combined with the GC-O-MS.The objectives of this study were to investigate the wildly accepted and characteristic flavor by identifying the volatile flavor of the typical spiced beef and provide a theoretical reference for quality evaluation of packaged spiced beef aroma profile.
Materials and chemicals Raw beef (Angus beef chuck, 2 d postmortem) of seven carcasses and processing seasonings and spices, including salt, white sugar, soy sauce, Baijiu, and others, were purchased from the local supermarket. A homologous series of straight-chain alkanes (C8–C20) were purchased from Sigma-Aldrich (St. Louis, USA).
Sample preparation Each raw beef carcass was cut into a piece (8.00 × 8.00 × 8.00 cm3) weighing 500.0 g after removing the connective tissue and the visible fat. The basic formulas of the seasonings and spices of the seven types of spiced beef are shown in Table 1. Beijing spiced beef, Beijing halal spiced beef, Hong Kong spiced beef, Tianjin spiced beef, Tianjin spiced beef of southern flavor, Shanghai spiced beef, and Inner Mongolia spiced beef were labeled B1, B2, B3, B4, B5, B6, and B7, respectively. Unseasoned beef was labeled B0 as control. The seasonings were first added into water until boiling, and then the beef and mixed spices were put into the water and boiled for 30 min. Next, the water temperature was adjusted to 95 °C and the beef was boiled for 3 h. After the thermal treatment, the beef was cooled to room temperature. The beef samples were vacuum-packaged and stored at −4 °C.
| No. | The basic formulas |
|---|---|
| B1 | 200 g kg−1 soybean paste, 30 g kg−1 salt, 1 g kg−1 cardamom, 3 g kg−1 star anise, 1.5 g kg−1 cinnamon, 1.5 g kg−1 Sichuan pepper, 1 g kg−1 angelica dahurica, 1 g kg−1 clove. |
| B2 | 100 g kg−1 soybean paste, 37 g kg−1 salt, 1.5 g kg−1 cardamom, 3 g kg−1 star anise, 2 g kg−1 cinnamon, 2 g kg−1 Sichuan pepper, 1.5 g kg−1 angelica dahurica, 3 g kg−1 clove, 1.5 g kg−1 fructus amomi. |
| B3 | 20 g kg−1 salt, 80 g kg−1 soy sauce, 10 g kg−1 star anise, 40 g kg−1 crystal sugar, 10 g kg−1 scallion, 20 g kg−1 Baijiu, 5 g kg−1 cinnamon, 4 g kg−1 ginger. |
| B4 | 25 g kg−1 salt, 30 g kg−1 soy sauce, 4 g kg−1 star anise, 0.002 g kg−1 fragranc, 2 g kg−1 cinnamon, 0.002 g kg−1 Sichuan pepper, 0.004 g kg−1 cumin, 2 g kg−1 ginger, 1 g kg−1 trinaphthalene, 2 g kg−1 garlic, 1 g kg−1 angelica dahurica, 1 g kg−1 scallion, 0.004 g kg−1 tsao-ko, 0.003 g kg−1 clove. |
| B5 | 100 g kg−1 soy sauce, 2 g kg−1 star anise, 70 g kg−1 sugar, 1.5 g kg−1 clove, 30 g kg−1 rice wine, 2 g kg−1 cinnamon, 0.7 g kg−1 monosodium glutamate. |
| B6 | 80 g kg−1 soybean paste, 20 g kg−1 salt, 30 g kg−1 soy sauce, 50 g kg−1 sugar, 3 g kg−1 star anise, 2 g kg−1 cinnamon, 0.005 g kg−1 Sichuan pepper, 3 g kg−1 clove, 20 g kg−1 Baijiu. |
| B7 | 30 g kg−1 salt, 100 g kg−1 soy sauce, 0.005 g kg−1 cardamom, 3 g kg−1 star anise, 2 g kg−1 cinnamon, 1 g kg−1 Sichuan pepper, 5 g kg−1 monosodium glutamate. |
No. B1, B2, B3, B4, B5, B6, and B7 represent Beijing spiced beef, Beijing halal spiced beef, Hong Kong spiced beef, Tianjin spiced beef, Tianjin spiced beef of southern flavor, Shanghai spiced beef, and Inner Mongolia spiced beef, respectively (the same below).
Headspace solid-phase microextraction The volatile flavor compounds were analyzed using an HS-SPME apparatus (Supelco, Bellefonte, USA). Approximately 3 g of ground sample was placed into one SPME extraction bottle. The bottle was placed in a water bath pot at 60 °C for 30 min after tightening the lid (Machiels and Istasse, 2003; Ruiz et al., 1998). Then, the SPME fiber assembly (50/30 µm DVB/CAR/PDMS) after thermal cleaning was inserted in the bottle and the fiber was penetrated into the headspace to adsorb the volatile flavor compounds. After adsorption for 30 min, the SPME fiber assembly was removed from the bottle and inserted into the injection port of GC for thermal desorption for 10 min. Then, the GC effluent was split in a ratio of 1:1 between the mass spectrometry detector and the olfactory detector port.
Volatile flavor compounds The identification of the volatile flavor compounds was performed by a GC-MS system. A TSQ8000 gas chromatograph combined with HS-SPME and mass selective detector (MSD) (Shimadzu, Kyoto, Japan) fitted with a DB-5MS fused silica capillary column (30 m × 0.25 mm, 0.25 µm) was used for analyzing the volatile flavor compounds (Wang et al., 2012; Wang et al., 2018). The carrier gas was high-purity helium (≥99.99%) supplied at a constant flow rate of 1.5 mL/min. The injection port was set in a splitless mode for 2 min at 250 °C. The initial temperature of the column was held at 40 °C for 3 min, ramped at a rate of 5 °C/min to 150 °C for 1 min, and again ramped at rate of 15 °C/min to 270 °C, which was maintained for 1 min. The detection temperature was 270 °C.
The MSD was used for chemical identification. The transfer line temperature was 230 °C. The electron ionization energy was 70 eV, and the electron impact ion source temperature was set at 230 °C. The quality scan range was set to m/z 40–600 with full scan mode. The volatile compounds were identified by comparing the retention indices (RIs) and searching the mass spectra from the National Institute of Standards and Technology (https://www.nist.gov/). The compounds with a positive and negative matching degree >800 were reported. The RIs of unknown compounds were determined via sample injection with a homologous series of straight-chain alkanes (C8–C20). The RI values were reported in the literature, and the data were listed in authentic online databases (http://www.flavornet.org/). The relative contents of the volatile flavor compounds were obtained using the normalization method of peak area.
Aroma profile An ODP-2 olfactory detector port (Thermo Fisher Scientific, Waltham, USA) was used for obtaining the aroma profile. The interface temperature of the ODP was set at 200 °C. Moist nitrogen was continuously pumped into the sniffing port to prevent drying of the nasal cavity of the assessors. Each ODP user was trained to recognize odors using the treated sample and the standard aroma compounds before identifying the aroma. The aroma-active compounds were recorded as the time for onset and end, odor characteristics, and intensities of the aroma extracts. The description and the time when each aroma-active compound was identified were confirmed consistently by at least two assessors.
Statistical analysis Aroma profiles of the seven types of spiced beef were analyzed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) using the MSpectrumPattern software (PERSEE, Beijing, China).
Identification of volatile flavor compounds A total of 87 volatile flavor compounds present in the seven types of spiced beef and unseasoned beef were identified using HS-SPME-GC-O-MS, and the results are shown in Table 2. In unseasoned beef, there were 28 identified volatile flavor compounds, including 10 aldehydes, 2 ketones, 7 hydrocarbons, 1 esters, 3 alcohols, and 5 nitrogen- or oxygen- or sulfur-containing and heterocyclic compounds. The amount of volatile flavor compounds was significantly increased due to the use of seasonings. A total of 67 volatile flavor compounds were identified in the seven typical spiced beef, including 15 aldehydes, 5 ketones, 10 hydrocarbons, 4 esters, 1 acid, 4 ethers, 10 alcohols, and 18 nitrogen- or oxygen- or sulfur-containing and heterocyclic compounds, of which acid and ethers were not detected in unseasoned beef. The numbers of the volatile flavor compounds identified in the seven types of spiced beef were sorted out as follows: B1 (51)> B6 (50)>B2 (46)>B3 (45)>B4 (44)>B7 (42)>B5 (34). The types of flavor volatile compounds varied, but the numbers and the relative contents of the volatile flavor compounds were primarily concentrated in aldehydes, hydrocarbons, ethers, alcohols, and nitrogen- or oxygen- or sulfur-containing and heterocyclic compounds. Among the 67 volatile flavor compounds, 30 aroma-active compounds were perceived by the assessors (these 30 aroma-active compounds, shown in Table 2, were sequentially labeled from F1 to F30), and 27, 27, 27, 28, 25, 27, and 26 aroma-active compounds were respectively identified in the seven types of spiced beef. The primary aroma characteristics included malt, almond, grass, fat, green, citrus, turpentine, camphor, pepper, pine, anise, licorice, nut, etc.
| No. | Basics of identity | Compounds | RId | Odors | Derived from | Relative contents (%) | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| B0 | B1 | B2 | B3 | B4 | B5 | B6 | B7 | ||||||
| 1 | MSa, Ob | Aldehydes 3-Methyl-butanal | malt | leucine | ND | 0.66 | 7.78 | 2.88 | 0.9 | 3.7 | 0.98 | 2.22 | |
| (F1) | |||||||||||||
| 2 | MS, O | Pentanal | almond | 13-linoleic acid hydroperoxide | ND | NDe | 0.49 | ND | 0.67 | 1.29 | 0.17 | 0.76 | |
| (F2) | |||||||||||||
| 3 | MS, O | Hexanal | grass | linoleic acid | 1.94 | 0.54 | 2.06 | 1.95 | 3.39 | 7.93 | 1.58 | 5.81 | |
| (F3) | |||||||||||||
| 4 | MS, RIc | Furfural | 829 | ND | 0.22 | 0.6 | 0.12 | 0.41 | 0.67 | 0.19 | 0.28 | ||
| 5 | MS, RI, O | Heptanal | 903 | fat | oleic acid | 1.37 | 0.28 | 0.34 | 0.65 | 1.31 | 2.41 | 0.38 | 2.32 |
| (F4) | |||||||||||||
| 6 | MS, RI | Benzaldehyde | 993 | 2.24 | 0.05 | 0.16 | 0.15 | 0.41 | ND | 0.44 | 1.1 | ||
| 7 | MS, RI, O | Octanal | 1 004 | green | oleic acid | 2.18 | 0.16 | 0.37 | 0.66 | 1.14 | 0.81 | 0.41 | 2.6 |
| (F5) | |||||||||||||
| 8 | MS, RI, O | Nonanal | 1 102 | citrus | oleic acid | 6.81 | 0.38 | 0.97 | 1.09 | 2.15 | 3.47 | ND | 3.86 |
| (F6) | |||||||||||||
| 9 | MS, RI | Decanal | 1 200 | 1.71 | ND | ND | ND | ND | ND | ND | 0.17 | ||
| 10 | MS, RI | Geranial | 1 272 | ND | ND | ND | ND | ND | ND | 0.08 | ND | ||
| 11 | MS, RI | 2,4-Decadienal | 1 281 | ND | ND | ND | 0.03 | 0.07 | ND | 0.07 | 0.09 | ||
| 12 | MS, RI | Undecanal | 1 291 | 0.33 | ND | ND | ND | ND | ND | ND | ND | ||
| 13 | MS, RI | Dodecanal | 1 409 | 0.55 | ND | ND | ND | ND | ND | ND | ND | ||
| 14 | MS, RI | (E)-2-dodecenal | 1 464 | ND | ND | ND | 0.02 | ND | ND | ND | 0.05 | ||
| 15 | MS, RI | Tridecanal | 1 561 | ND | 0.04 | 0.11 | 0.05 | 0.06 | 0.09 | 0.06 | ND | ||
| 16 | MS, RI | 12-Methyltridecanal | 1 585 | ND | ND | ND | ND | ND | ND | ND | 1.29 | ||
| 17 | MS, RI | Palmitaldehyde | 1 808 | ND | 0.05 | ND | ND | 0.28 | ND | ND | ND | ||
| 18 | MS, RI | Hexadecanal | 1 813 | 3.19 | ND | ND | ND | ND | ND | ND | ND | ||
| 19 | MS | 2-Methylundecanal Ketones | 0.15 | ND | ND | ND | ND | ND | ND | ND | |||
| 20 | MS | 2-Butanone | 0.09 | ND | ND | ND | ND | ND | ND | ND | |||
| 21 | MS | Acetoin | ND | 0.29 | 1.58 | ND | 1.4 | ND | ND | ND | |||
| 22 | MS, RI | 2-Heptanone | 900 | ND | ND | ND | ND | ND | ND | ND | 0.16 | ||
| 23 | MS, RI | Acetophenone | 1 041 | 0.29 | ND | ND | ND | ND | ND | ND | ND | ||
| 24 | MS, RI | 2-Nonanone | 1 098 | ND | ND | ND | ND | ND | ND | 0.02 | ND | ||
| 25 | MS, RI | Geranyl acetone | 1 454 | ND | ND | 0.04 | 0.03 | ND | ND | ND | 0.02 | ||
| 26 | MS, RI | Hexadecanone Hydrocarbons | 1 781 | ND | 0.13 | 0.31 | 0.16 | 0.12 | ND | 0.08 | 0.54 | ||
| 27 | MS, RI | Toluene | 1.59 | ND | ND | ND | ND | ND | 0.11 | 0.96 | |||
| 28 | MS, RI | M-xylene | 0.16 | ND | ND | ND | ND | ND | ND | ND | |||
| 29 | MS, RI | O-xylene | 880 | ND | 0.1 | 0.11 | ND | 0.74 | ND | 0.09 | 0.17 | ||
| 30 | MS | (R)-1-methyl-5-(1-methylvinyl) cyclohexene | 0.18 | ND | ND | ND | ND | ND | ND | ND | |||
| 31 | MS, RI | Styrene | 893 | 0.09 | ND | ND | ND | ND | ND | ND | ND | ||
| 32 | MS, RI, O | α-Pinene | 937 | turpentine | star anise, cinnamon, fructusamomi | ND | 4.36 | 0.94 | 0.36 | 0.63 | 0.31 | 0.8 | 0.34 |
| (F7) | |||||||||||||
| 33 | MS, RI, O | Camphene | 950 | camphor | star anise, cinnamon, fructusamomi | ND | 0.03 | 1.61 | 0.77 | 1.57 | 1.23 | 0.46 | 0.44 |
| (F8) | |||||||||||||
| 34 | MS, RI, O | Sabinene | 969 | pepper | ND | 20.09 | 4.33 | 3.39 | 4.51 | 4.69 | 2.24 | 1.54 | |
| (F9) | |||||||||||||
| 35 | MS, RI, O | β-Pinene | 975 | pine | star anise, cinnamon, fructusamomi | ND | 11.81 | 3.31 | 1.8 | 2 | 1.17 | 1.87 | 1.24 |
| (F10) | |||||||||||||
| 36 | MS, RI | Myrcene | 998 | ND | 0.11 | ND | ND | ND | ND | 0.04 | ND | ||
| 37 | MS, RI, O | α-Phellandrene | 1 001 | turpentine | Sichuan pepper | ND | 2.98 | 5.66 | 0.32 | 1.1 | 0.48 | 1.19 | 1.72 |
| (F11) | |||||||||||||
| 38 | MS, RI, O | D-limonene | 1 028 | citrus | star anise, cinnamon, fructusamomi, Sichuan pepper | ND | 1 | ND | 0.13 | 0.22 | ND | 0.32 | 0.17 |
| (F12) | |||||||||||||
| 39 | MS, RI | α-Cubebene | 1 355 | ND | 1.13 | 0.23 | 0.06 | 0.36 | ND | 0.24 | 0.35 | ||
| 40 | MS, RI | Pentadecane | 1 500 | 0.27 | ND | ND | ND | ND | ND | ND | ND | ||
| 41 | MS, RI | Hexadecane | 1 600 | 0.64 | ND | ND | ND | ND | ND | ND | ND | ||
| 42 | MS, RI | Heptadecane esters | 1 700 | 0.21 | ND | ND | ND | ND | ND | ND | ND | ||
| 43 | MS, RI, O | Ethyl methylbutyrate | 837 | fruit | esterification of alcohols and acids | ND | ND | ND | 0.65 | 1.42 | ND | 0.23 | 0.28 |
| (F13) | |||||||||||||
| 44 | MS, RI | Methyl tetradecanoate | 1 661 | ND | 0.02 | ND | ND | ND | 0.28 | ND | ND | ||
| 45 | MS, RI | Citronellylvalerate | 1 633 | ND | 0.05 | ND | ND | ND | ND | ND | ND | ||
| 46 | MS, RI | Ethyl benzoate | 1 185 | ND | 0.1 | 0.5 | 0.1 | ND | 1.05 | 0.27 | ND | ||
| 47 | MS | Morpholin-4-yl-acetic acid methyl ester acids | 0.43 | ND | ND | ND | ND | ND | ND | ND | |||
| 48 | MS, RI | Tetradecanoic acid ethers | 1 992 | ND | 0.1 | ND | ND | 0.1 | 0.32 | 0.04 | 0.19 | ||
| 49 | MS, RI, O | p-Allyl-anisole | 1 192 | anise, licorice | star anise | ND | 0.61 | 1.24 | 28.7 | 2.34 | 2.5 | 3.69 | 2.75 |
| (F14) | |||||||||||||
| 50 | MS, O | 1-Methoxy-4-(1-propenyl)-benzene | 1 291 | anise, licorice | star anise | ND | 20.31 | 10.59 | 38.61 | 29.42 | 33.09 | 23.77 | 25.42 |
| (F15) | |||||||||||||
| 51 | MS, RI | Methyl eugenol | 1 426 | ND | ND | 0.29 | ND | ND | ND | ND | ND | ||
| 52 | MS, RI, O | Myristicin | 1 550 | spice | cardamom | ND | 0.28 | 0.45 | ND | ND | ND | ND | ND |
| (F16) | |||||||||||||
| 53 | MS, O | Alcohols 3-Methyl-1-butanol | burnt | ND | ND | ND | 1.76 | ND | 1.07 | 0.29 | 1.79 | ||
| (F17) | |||||||||||||
| 54 | MS, RI, O | (Z)-3-hexenol | 858 | grass | ND | 3.82 | 3.32 | 0.49 | 1.67 | 1.75 | 0.61 | 0.54 | |
| (F18) | |||||||||||||
| 55 | MS, RI | 1-Octen-3-ol | 982 | mushroom | 0.33 | ND | ND | ND | ND | ND | ND | ND | |
| 56 | MS, RI, O | Octanol | 987 | nut | ND | 0.41 | 3.5 | 1.45 | 0.88 | 1.29 | 0.39 | 0.91 | |
| (F19) | |||||||||||||
| 57 | MS, RI | Isooctanol | 1 032 | 2.91 | ND | ND | ND | ND | ND | ND | ND | ||
| 58 | MS, RI | 2-Ethyl-1-hexanol | 1 052 | ND | 0.03 | 0.09 | 0.06 | 0.06 | 0.12 | 0.06 | 0.16 | ||
| 59 | MS, RI | trans-Sabinene hydrate | 1 076 | ND | 0.02 | 0.08 | 0.02 | 0.04 | ND | 0.06 | ND | ||
| 60 | MS, RI | 1-Octanol | 1 095 | ND | 0.02 | ND | 0.06 | 0.09 | 0.17 | 0.06 | ND | ||
| 61 | MS, RI, O | Linalool | 1 109 | flower | ND | 0.88 | 0.86 | 4.51 | 1.16 | 2.04 | 3.36 | ND | |
| (F20) | |||||||||||||
| 62 | MS, RI | Dehydrolinalool | 1 127 | ND | 0.04 | 0.1 | 0.07 | 0.06 | ND | 0.19 | ND | ||
| 63 | MS, RI, O | 1-Terpinen-4-ol | 1 173 | must | ND | 1.93 | 4.2 | 0.76 | 1.04 | 0.92 | 2.99 | 1.37 | |
| (F21) | |||||||||||||
| 64 | MS, RI | α-Terpineol | 1 205 | ND | 0.19 | 0.36 | 0.38 | 0.24 | 0.25 | 0.75 | 0.46 | ||
| 65 | MS | 2-Methyl-1-hexadecanol nitrogen- or oxygenor sulfur-containing and heterocyclic compounds | 0.59 | ND | ND | ND | ND | ND | ND | ND | |||
| 66 | MS, O | Dimethyl disulfide | onion | Strecker degradation | ND | 0.98 | 4.57 | 0.67 | 1.79 | 4.89 | 0.12 | 1.26 | |
| (F22) | |||||||||||||
| 67 | MS | Pyridine | ND | ND | 0.63 | ND | ND | ND | 0.1 | ND | |||
| 68 | MS, RI, O | Furfuryl alcohol | 854 | burnt | Maillard reaction | ND | 1.39 | 1.39 | ND | 0.29 | ND | ND | ND |
| (F23) | |||||||||||||
| 69 | MS, RI | Methylfuranthiol | 868 | ND | 0.07 | 0.13 | ND | ND | 0.19 | 0.07 | ND | ||
| 70 | MS, RI, O | Dimethyl pyrazine | 912 | roasted nut | Maillard reaction | ND | 0.75 | 1.58 | 0.07 | 0.4 | 0.25 | 0.3 | 0.19 |
| F24) | |||||||||||||
| 71 | MS, RI, O | 2-Pentylfuran | 944 | green bean | linoleic acid | 0.67 | 5.99 | 0.06 | 0.06 | 0.1 | 0.25 | 0.06 | 0.63 |
| (F25) | |||||||||||||
| 72 | MS, RI, O | Dimethyl trisulfide | 954 | sulfur | Strecker degradation | ND | 0.79 | 3.82 | 1.07 | 0.94 | 2.68 | 0.54 | 0.77 |
| (F26) | |||||||||||||
| 73 | MS, RI, O | 3-Mercaptothiophene | 959 | cooked meat | Maillard reaction | ND | 2.8 | 4.68 | 0.23 | 0.63 | ND | 0.79 | 0.65 |
| (F27) | |||||||||||||
| 74 | MS, RI | (Z)-linalool oxide | 1 068 | ND | 0.03 | ND | 0.05 | ND | ND | 0.04 | ND | ||
| 75 | MS, RI | Maltol | 1 086 | ND | ND | ND | 0.01 | ND | ND | ND | ND | ||
| 76 | MS, RI | Acetylthiophene | 1 092 | ND | 0.02 | ND | 0.06 | ND | ND | 0.17 | ND | ||
| 77 | MS, RI | Dimethyl tetrasulfide | 1 214 | ND | ND | 0.1 | 0.02 | ND | ND | ND | ND | ||
| 78 | MS, RI, O | Chavicol | 1 249 | phenol | star anise | ND | 0.23 | 0.29 | 0.81 | 0.59 | 0.49 | 0.42 | ND |
| (F28) | |||||||||||||
| 79 | MS, RI, O | Benzothiazole | 1 256 | gasoline | ND | 0.37 | 1 | 0.75 | 0.66 | 1.48 | 0.41 | 0.44 | |
| (F29) | |||||||||||||
| 80 | MS, RI | Safrole | 1 284 | ND | 0.16 | 0.12 | ND | ND | ND | ND | ND | ||
| 81 | MS, RI, O | p-Vinylguaiacol | 1 326 | clove | clove | ND | 0.19 | 0.18 | 0.48 | 0.54 | 0.36 | 0.58 | 0.91 |
| (F30) | |||||||||||||
| 82 | MS, RI | Elemicin | 1 510 | ND | 0.03 | ND | ND | 0.22 | ND | 0.41 | 0.31 | ||
| 83 | MS, RI | (E)-Isoelemicin | 1 590 | ND | 0.09 | 0.31 | ND | ND | ND | ND | ND | ||
| 84 | MS | 2-Myristynoyl pantetheine | 0.14 | ND | ND | ND | ND | ND | ND | ND | |||
| 85 | MS | 4-Sec-butyl-2,6-di-tertbutylphenol | 5.3 | ND | ND | ND | ND | ND | ND | ND | |||
| 86 | MS | 6-Undecylamine | 0.95 | ND | ND | ND | ND | ND | ND | ND | |||
| 87 | MS | L-Homoserine | 0.56 | ND | ND | ND | ND | ND | ND | ND | |||
There were 23 common volatile flavor compounds present in the seven types of spiced beef, including 3-methyl-butanal (malt), hexanal (grass), furfural, heptanal (fat), octanal (green), α-pinene (turpentine), camphene (camphor), sabinene (pepper), β-pinene (pine), α-phellandrene (turpentine), p-allyl-anisole (anise and licorice), 1-methoxy-4-(1-propenyl)-benzene (anise and licorice), (Z)-3-hexenol (grass), octanol (nut), 2-ethyl-1- hexanol, 1-terpinen-4-ol (must), α-terpineol, dimethyl disulfide (onion), dimethyl pyrazine (roasted nut), 2-pentylfuran (green bean), dimethyl trisulfide (sulfur), benzothiazole (gasoline), and p-vinylguaiacol (clove). Specific characteristic volatile flavor compounds were also identified in the seven types of spiced beef. Citronellylvalerate was identified only in B1. Methyl eugenol was identified only in B2. Maltol was identified only in B3. No specific compounds were identified in B4 and B5. Geranial and 2-nonanone were identified only in B6. 12-Methyltridecanal and 2-heptanone were identified only in B7. However, these specific volatile flavor compounds were not perceived by the assessors because of the low relative contents, and they also did not contribute to the overall volatile flavor of the seven types of spiced beef.
Comparison analysis of the aroma-active compounds Aldehydes are primarily derived from lipid oxidation and protein degradation of meat. Due to their low odor thresholds, they are the key component of meat odor (Mottram, 1998). Small molecular aldehydes, especially branched aldehydes, with a high relative content contribute significantly to beef flavor. The numbers and the relative contents of aldehydes identified in the seven types of spiced beef and unseasoned beef are shown in Table 3. The results showed that aldehydes identified in B5 (19.61%), B7 (17.57%), and B2 (12.01%) had higher total relative contents than those of other samples, indicating that they exhibited better meaty odor. Aldehydes are involved in the oxidation of oleic acid and linoleic acid and in the Strecker degradation of leucine. 3-Methyl-butanal is degraded in the Strecker degradation of leucine, which is chocolate-flavored and gives a pleasant fruity odor after dilution (Garcia et al., 1991; Ahn et al., 2016; Ahn et al., 2016). Pentanal is produced by the pyrolysis of 13-linoleic acid hydroperoxide and has a pleasant fruity aroma (Greenberg, 1981). Hexanal with a grassy odor is recognized as an oxidation product of linoleic acid and an important aromatic component of cooked beef. Hexanal is also commonly used as an indicator of lipid oxidation (Jayathilakan et al., 2007). Therefore, B5, B7, and B4 exhibited a higher degree of lipid oxidation than B1, B2, B3, and B6. Heptanal, octanal, and nonanal are oxidation products of oleic acid, which have green and citrus flavors. These aroma-active aldehydes impart a clear flavor to the seven types of spiced beef (Xie et al., 2008). Relatively large molecular aldehydes, such as undecanal, dodecanal, hexadecanal, and 2-methylundecanal, were detected in unseasoned beef. However, these aldehydes contributed less to the overall flavor. The same detected aroma-active aldehydes of hexanal, heptanal, octanal, and nonanal indicated that the addition of the spices did not mask the meaty odor derived from lipid oxidation.
| Group | B1 | B2 | B3 | B4 | B5 | B6 | B7 | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Numbers | Relative contents% | Numbers | Relative contents% | Numbers | Relative contents% | Numbers | Relative contents% | Numbers | Relative contents% | Numbers | Relative contents% | Numbers | Relative contents% | |
| Aldehydes | 5 | 2.02 | 6 | 12.01 | 5 | 7.23 | 6 | 9.56 | 6 | 19.61 | 5 | 3.52 | 6 | 17.57 |
| Hydrocarbons | 6 | 40.27 | 5 | 15.85 | 6 | 6.77 | 6 | 10.03 | 5 | 7.88 | 6 | 6.88 | 6 | 5.45 |
| Esters | 0 | 0 | 0 | 0 | 1 | 0.65 | 1 | 1.42 | 0 | 0 | 1 | 0.23 | 1 | 0.28 |
| Ethers | 3 | 21.2 | 3 | 12.28 | 2 | 67.31 | 2 | 31.76 | 2 | 35.59 | 2 | 27.46 | 2 | 28.17 |
| Alcohols | 4 | 7.04 | 4 | 11.88 | 5 | 8.97 | 4 | 4.75 | 5 | 7.07 | 5 | 7.64 | 4 | 4.61 |
| Nitrogen- or oxygen- or sulfur-containing and heterocyclic compounds | 9 | 13.49 | 9 | 17.57 | 8 | 4.14 | 9 | 5.94 | 7 | 10.40 | 8 | 3.22 | 7 | 4.85 |
Hydrocarbons detected in unseasoned beef generated by the homolytic scission from fatty acid alkoxy radicals do not have any potential odorants, whereas the detected hydrocarbons in the spiced beef are related to the use of spices (Carrapiso et al., 2002). The aroma-active hydrocarbons included α-pinene, camphene, sabinene, β-pinene, α-phellandrene, and d-limonene. These low odor threshold compounds were volatilized from star anise, cardamom, ginger, cinnamon, Sichuan pepper, fructusamomi, etc. and exhibited strong turpentine, camphor, pepper, pine, and citrus odors, which contribute significantly to the overall volatile flavor of the spiced beef (Forss, 1973). For instance, the volatile flavor compounds of star anise, cinnamon, and fructusamomi contain α-pinene, β-pinene, camphene, d-limonene, etc.; the volatile flavor compounds of Sichuan pepper contain α-phellandrene, d-limonene, and others (Deng et al., 2005; Choi, 2003; Jirovetz et al., 2002; Opdyke, 1973; Hognadottir and Rouseff, 2003; Toldrá, 2017; Pérez-palacios et al., 2010). The relative contents of hydrocarbons in B1 (40.27%), B2 (15.85%), and B4 (10.03%) were higher than those in other samples, indicating that the rich volatile components of spices were effectively distilled during processing, thus providing the products a better spicy aroma. Not all of the aroma-active hydrocarbons in each seasoning were detected, possibly due to some of them evaporated quickly or some were involved in the interaction between the beef and the ingredients.
Esters are produced from the esterification of alcohols and carboxylic acids. Only one aroma-active ester, ethyl methylbutyrate, was detected in B3, B4, B6, and B7. Ethyl methylbutyrate is an important flavor component in wine and imparts a fruity odor to the spiced beef. These results indicated that in addition to being produced from Baijiu, the interaction between the alcohol and acid products may promote the formation of ethyl methylbutyrate.
Ethers of the detected p-allyl-anisole, 1-methoxy-4-(1- propenyl)-benzene, and myristicin were probably associated with the odorant formation of spices since no ethers were detected in unseasoned beef. p-Allyl-anisole and 1-methoxy-4-(1-propenyl)-benzene are derived from star anise, and has an anise and licorice odor and increases the mellowness to the overall volatile flavor. Therefore, it has been considered as an important characteristic aroma of spiced beef (Gong et al., 2017). Myristicin was identified in B1 and B2, which was related to the addition of cardamom, whereas it was not detected in B7 due to the low addition of cardamom.
Linear alcohols are the primary products of lipid oxidation, whereas branched alcohols are reduced from the corresponding branched aldehydes produced by the Strecker degradation (Gasser and Grosch, 1988; Pérez-palacios et al., 2010; Gaspardo et al., 2008). Alcohols have high odor thresholds that are considered to contribute less to the overall volatile flavor of meat samples (Sabio et al., 1998). The alcohols identified in this study were related to the use of spices. 3-Methyl-1-butanol has a low odor threshold and a stronger burnt odor. It is seldom detected in cooked meat and most likely derived from spices. (Z)-3-hexenol, octanol, linalool, and 1-terpinen-4-ol are naturally occurring alcohols found in several spice plants and have grass, nut, flower, and must odors, respectively (Hognadottir and Rouseff, 2003; Toldrá, 2017; Pérez-palacios et al., 2010; Gaspardo et al., 2008; Sabio et al., 1998; Guth and Grosch, 1991; Silvis et al., 2018). 1-Octene-3-ol with mushroom odor, the important oxidation products of 12-hydroperoxide from arachidonic acid, was detected in unseasoned beef, which may have been masked by the combination of beef and seasonings in the spiced beef.
Nitrogen- or oxygen- or sulfur-containing and heterocyclic compounds are primarily derived from the Maillard reaction between reducing sugars and aminoacids, thermal degradation of amino acids, and degradation of thiamine. Due to the low odor threshold, these compounds are generally regarded as the most important flavor of meat. The detected dimethyl disulfide, furfuryl alcohol, dimethyl pyrazine, 2-pentylfuran, dimethyl trisulfide, 3-mercaptothiophene, chavicol, benzothiazole, and p-vinylguaiacol were the important meaty odor compounds. Dimethyl disulfide and dimethyl trisulfide are produced by the Strecker degradation of amino acids and have sulfur or onion-like odor and, sometimes, a meaty odor under certain conditions (Macleod et al., 1988). Furfuryl alcohol, dimethyl pyrazine, and 3-mercaptothiophene are the key products of the Maillard reaction, having burnt, roasted nut, and cooked meat odors, respectively (Shahidi, 1998). 2-Pentylfuran derived from the autoxidation of linoleic acid has a green bean odor and is an important aroma component of cooked beef (Hornstein and Crowe, 1960). Benzothiazole, which has a gasoline odor, was described by Gasser and Grosch (1988) as an important flavor compound in cooked beef odor using the method of flavor dilution factor (FD-factor) (Gasser and Grosch, 1988). Chavicol with a phenol odor may be derived from star anise, and p-vinylguaiacol with a clove odor was detected in linden honey in a previous study and may be derived from clove (Opdyke, 1973; Blank et al., 1989). The higher relative contents of the meat-derived compounds in B1 (13.07%), B2 (17.10%), and B5 (9.55%) indicate that the samples well showed a meaty odor. Gong et al. (2017) found that inorganic sulfide, nitrogen oxides, aromatic components, and organic sulfides were the key flavor compounds of spiced beef, which was consistent with the results of this study (Liu et al., 2015). Sulfur-containing compounds as the key aroma of beef were not detected in unseasoned beef resulting in the mild Maillard reaction during the boiling process of beef (Kerth and Miller, 2015). The detected sulfur-containing compounds in the spiced beef indicated that the addition of seasonings significantly accelerated the Maillard reaction. The ingredients of reducing sugars were important precursors for the Maillard reaction. Furthermore, NaCl could reduce the binding ability of sarcoplasmic protein on key volatile flavor to promote the release of it (Pérez-Juan et al., 2007).
PCA and HCA of the aroma-active compounds PCA was performed on 30 aroma-active compounds of the seven types of spiced beef (Fig. 1). The results demonstrated that the cumulative contribution rate of PC1 and PC2 was 80%, which could represent the volatile flavor principal components. The score plot depicting the distribution of the samples (Fig. 1a) and the loading plot depicting the distribution of the 30 aroma-active compounds (Fig. 1b) are shown in Fig. 1. The PCA method showed good distinction among the samples and the aroma-active compounds. B3 scored higher than others on PC1, indicating that it consisted of more types and higher relative contents of the aroma-active compounds, rendering the samples to be rich in flavor. The distances between B4, B5, B6, and B7 were less, indicating that the key aroma-active compounds of the samples were relatively similar. The two ellipses in Fig. 1b represent the contribution rates of 70% and 100%, respectively. The aroma-active compounds between the two ellipses showed a higher correlation with the overall volatile flavor of the seven types of spiced beef. Fig. 1b shows that F14 (p-allyl-anisole), F15 (1-methoxy-4-(1-propenyl)- benzene), F17 (3-methyl-1-butanol), F20 (linalool), F28 (chavicol), F11(α-phellandrene), F16 (myristicin), F18 ((Z)-3-hexenol), F21(1-terpinen-4-ol), F23 (furfuryl alcohol), F24 (dimethyl pyrazine), and F27 (3-mercaptothiophene) contributed the most on PC1, and F7 (α-pinene), F9 (sabinene), F10 (β-pinene), F12 (d-limonene), and F25 (2-pentylfuran) contributed significantly on PC2, indicating that these were the important aroma-active compounds of the seven types of spiced beef. These compounds could be determined as the key flavor components that were widely accepted for spiced beef by customers. Among these compounds, F14 and F15 derived from star anise contributed the most on PC1 and might be the primary components of sauce-flavored spiced beef, which was consistent with previous research (Gong et al., 2017).
PCA results of the 30 aroma-active compounds
a, PCA score plot depicting the distribution of the samples for the first two principal components (PC1 and PC2); b, PCA loading plot showing the distribution of the 30 aroma-active compounds for the first two principal components (PC1 and PC2).
The 30 aroma-active compounds were clustered by HCA to further analyze the differences among the seven types of spiced beef; the cluster heat map is depicted in Fig. 2. The results demonstrated that the seven types of spiced beef could be divided into three categories. B1 and B2 were classified into one category, primarily due to the higher relative contents of F23 (furfuryl alcohol), F18 ((Z)-3-hexenol), F27 (3-mercaptothiophene), and F26 (dimethyl trisulfide) than those in others, indicating that B1 and B2 better presented the odors produced from Strecker degradation and thiamine degradation compared with other types of spiced beef, which were consistent with sensory description of meaty, sauce and nutty of the actual products (Qi et al., 2011). B4, B5, B6, and B7 were classified into one category, primarily due to the higher relative contents of F2 (pentanal), F3 (hexanal), and F4(heptanal), indicating that B4, B5, B6, and B7 better presented the odors produced from lipid oxidation. The actual products presented a grass and spicy flavor. B3 was classified into one category, primarily due to the higher relative contents of F14 (p-allyl-anisole), which was rich in anise, sweet, and mint odors, whereas the actual product presented a sweet and spicy flavor. The volatile compounds explained the main characteristics of the flavour of the actual products. The HCA results were essentially consistent with the results of PCA. The results also demonstrated that the seven types of spiced beef had certain geographical characteristics. B1 and B2 were the popular products in Beijing, whose characteristic flavors were significantly different from those of other samples. B4, B5, B6, and B7 consumption was prevalent in areas that were geographically close. The characteristic flavor of B3 was consistent with the typical sweet and light taste in southern China.
Cluster analysis heat map of the 30 aroma-active compounds.
Although spiced beef is a popular meat product in China, study on its flavor characteristics is still limited, which is not conducive to the establishment of its quality evaluation and standardized production of packaged products. This study revealed the key aroma compounds with widely accepted flavors in the seven types of spiced beef, including p-allyl-anisole, 1-methoxy-4-(1-propenyl)-benzene, 3-methyl-1- butanol, linalool, chavicol, α-phellandrene, myristicin, (Z)-3- hexenol, 1-terpinen-4-ol, furfuryl alcohol, dimethyl pyrazine, 3-mercaptothiophene, α-pinene, sabinene, β-pinene, d-limonene, and 2-pentylfuran. The aroma profile characteristics of the typical spiced beef had also been clarified. The characteristic aroma-active compounds of B1 and B2 were furfuryl alcohol, (Z)-3-hexenol, 3-mercaptothiophene, and dimethyl trisulfide, which had a rich odor of sulfur-containing compounds. The characteristic aroma compounds of B4, B5, B6, and B7 were pentanal, hexanal, and heptanal, which had a rich aldehyde odor. The characteristic aroma compound of B3 was p-allyl-anisole that had rich anise, sweet, and mint odors. The characteristic aroma reflects the geographical differences in consumption habits. The results of this study provided a theoretical reference for quality evaluation of packaged spiced beef aroma profile.
All authors certify that there is no conflict of interests.
Acknowledgments The authors gratefully acknowledge the financial support from the National Key R& D Program of China (Project No. 2016YFD0400403) and the Transformational Technologies for Clean Energy and Demonstration (Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDA21060300).
