Identification the Key Odorants in Different Parts of Hyla Rabbit Meat via Solid Phase Microextraction Using Gas Chromatography Mass Spectrometry

Hexanal (GC, >99.3 pure), heptanal (GC, >99.3 pure), octanal (GC, >99.3 pure), butanoic acid (GC, >99.3 pure), 2-nonenal (GC, >99.3 pure), dodecanoic acid (GC, >99.3 pure) and nonanal (GC, >99.3 pure) were supplied by Sigma-Aldrich (USA). C7-C30 Saturated Alkanes Standard (1000 mg/mL in hexane) was purchased from Supelco (USA). Saturated NaCl aqueous was prepared according to the following steps in the lab: 1. 36 g NaCl was weighted by electronic scales precisely; 2. Dissolved in beaker using 100 g water at room temperature; 3. Labeled and stored in narrow-necked bottles. 2, 4, 6-Trimethylpyridine (TMP) was obtained from J&K Scientific Ltd (China).

Both fifty 75-d old male and female Hyla rabbits were purchased and slaughtered in College of Animal Science and Technology, Southwest University, Chongqing, with the average slaughter weight of 2.53±0.09 kg. Table 1 shows the diet composition of Hyla rabbit (Xue et al., 2015). Foreleg, hind leg, abdomen and Longissimus dorsi of rabbit meat were segmented and deboned immediately after slaughtering and stored at −20℃ respectively until use. The animal experiment was followed by the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China.

Carboxen polydimethylsiloxane (CAR/PDMS) fiber and the SPME holder was purchased from Supelco (USA) and a 75 μm carboxen polydimethylsiloxane (CAR/PDMS) SPME fiber (Supelco, USA) was chosen for this experiment due to better extraction ability than others and lower polarity for volatile organic compounds (Kataoka et al., 2000).

Different parts of rabbit meat were well minced and homogenized during 1 min in a household blender. Three grams of the rabbit meat of each part and 3 mL saturated NaCl aqueous solution were placed in a 25-mL vial, sealed with a PTFE-silicone septum and screw cap. After shaking with a lab dance min vortexer for 1 min, the sample was equilibrated in the water bath at 45℃ for 15 min. Then fiber which was preconditioned at 250℃ for 60 min in the GC injector port was inserted into the vial. The fiber coating was exposed to the sample headspace and the sampling process last at 75℃ for 60 min in a thermostat controlled water bath (±1℃). The selection of 75℃ as the absorption temperature for HS-SPME was on the basis that requirement of central temperature for meat, poultry and aquatic dishes according to the critical control point in China, so as to simulate the volatiles of boiled rabbit meat. Finally, the fiber was retracted into a needle, transferred to an injection port of the GC system, and desorbed for 5 min at 250℃.

The volatile compounds in rabbit meat analyzed using an SHIMADZU QP2010 gas chromatograph tandem mass spectrometry. The sample was desorbed in the injection port at 250℃ for 5 min. Ultra-pure helium was used as the carrier gas at a flow of 1.1 mL/min. Volatile organic compounds of each sample were separated with DB-5 ms column (122-5532, 30 m length × 250 μm × 0.25 μm film thickness). Each extract was injected by manual with the splitless mode (injector temperature, 250℃). The GC oven temperature was initially held at 40℃ for 1 min, then to 180℃ at 6℃ /min holding for 3 min, to 230℃ at 10℃/min with a final hold of 1 min. MS parameters were as follows: ionization energy, 70 eV; ion source temperature, 230℃; quadrupole temperature, 280℃; mass range, m/z 35-350; detector interface temperature, 250℃.

The volatile compounds in rabbit meat were tentatively identified by comparing for mass spectra library to those found in data NIST11.L, by comparing of van den Dool and Kratz indices to those reported in the literature and by comparison of GC retention indices (RI). In this study, 100 μL diluted TMP as an internal standard substance, with a concentration of 0.92 g/mL, was added to 30 g sample. The identified compounds can be calculated by comparing the peak areas with standard substance and the calibration factors were all considered as 1.00. The content of volatile compounds is calculated as follows:

Where M_C is the content of compound, A_S is the peak area of single compound; A_I is the peak area of internal standard material; C_TMP is the concentration of TMP, g/mL; V_TMP is the volume of internal standard material, μL; M_S is the weight of meat sample, g.

A sensory test was carried out by a trained panel of 9 members, with 5 male and 4 female students, staffs and teachers. Panelists were asked to point out the intensity of rabbit meat odor of hind leg, fore leg, abdomen and Longissimus dorsi. Sensory attribute for rabbit meat odor was assessed with a 5 point intensity line scale, where 1 = no obvious odor and 5 = extremely intensity. All the samples were cooled to room temperature after boiling.

All statistical analyses were performed using SPSS 22.0 version. The significant differences (p<0.05) among concentrations of volatile compounds in four parts of male and female rabbit were analyzed by ANOVA. PCA was used to determine the key odorants of volatile compounds in rabbit meat, while CA was applied in this study based on OAV data to sort different patterns.

A total of 63 volatile compounds, including 23 aldehydes, 4 alcohols, 5 ketones, 11 esters, 5 aromatics, 8 acids and 7 hydrocarbons, were identified in four different parts of both male and female rabbits (Table 2). Among the 63 volatile compounds, 33 and 42 were found in the foreleg of male and female rabbit meat respectively, 31 and 34 were in the hind leg, 33 and 48 were in the abdomen, 27 and 33 were in the Longissimus dorsi (Fig. 1). Comparing with other parts, abdomen in both male and female rabbits owned all seven types volatile compounds, and the number of volatile compounds in abdomen was more than that of other three parts. This indicated that the stronger odor may generate in abdomen than in other parts of rabbit meat. On the contrary, the volatiles numbers in Longissimus dorsi were the least among all four parts, which means weak odor intensity. Aldehydes, of all detected volatile compounds, owned largest amount in four parts of both male and female rabbits. Esters and hydrocarbon owned the second and third largest amount in male rabbit meat, while esters and acids took up the second and third largest quantity in female rabbit meat. As we all know that aldehydes play a significant role in overall flavor of meat, due to its very low threshold. For example, saturated aldehydes have the second important relation with lamb’s overall flavor (Bueno et al., 2014). Pentanal, hexanal, heptanal, 2-heptenal, octanal, 2-octenal, nonanal, 2-nonenal, 2-decenal, undecanal, 2, 4-decadienal, decanal, butanoic acid, dodecanoic acid, hexadecanoic acid methyl ester, octadecanoic acid methyl ester and 9,12-octadecadienoic acid methyl ester were detected in all four parts of both male and female rabbit meat. It was shown that no unique substance was found exclusively in both male and female rabbit meat, but the concentrations of those substances were significantly different (p<0.05). This distinction may elucidate the strength of the odor in male rabbits was stronger than that of female rabbits. From Fig. 2, significant difference of detected volatile compounds between genders was found in FL, AB and LD, except HL (p<0.05). Thus, we confirmed that the odor of rabbit meat had closely relationship with genders. These significant differences may relate with the amount of intramuscular lipids in different parts and between genders (Neethling et al., 2016). Moreover, the peak areas of male rabbit meat are larger than those of female one, representing that the odor of male rabbit meat is stronger than female one (Fig. 3). However, the reason is yet unknown and is worth further studying.

Fig. 1. Number differences of volatile compounds among four parts of both male (A) and female (B) Hyla rabbit meat. FL, foreleg; HL, hindleg; AB, abdomen; LD, Longissimus dorsi.

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Fig. 2. Concentration of detected volatile compounds of different parts in both male and female Hyla rabbit meat. FL, foreleg; HL, hindleg; AB, abdomen; LD, Longissimus dorsi.

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Fig. 3. Comparisons of volatile compounds in four edible parts of both male (A) and female (B) Hyla rabbit meat. FL, black line; HL, pink line; AB, blue line; LD, brown line.

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Eight volatile compounds, with OAV greater than one, were found among the whole 63 volatile compounds. All eight volatile compounds were aldehydes. Decanal owns the highest OAV (77.7) among the aldehydes, and 2, 4-decadienal (35.8) is the second. Although the concentration of hexanal is larger than other seven aldehydes, the OAV is the smallest in all eight volatile compounds. Pentanal and hexanal were positively related with liver and rancid off-flavor in various beef muscles (Stetzer et al., 2008). Both pentanal and hexanal showed closely relationship with TBARS in meat volatile flavor. However, only the female rabbit foreleg’s concentration of pentanal was larger than its threshold. This indicated that pentenal made no contribution to the overall flavor of rabbit meat. Hexanal represents the the lipid oxidation status of the meat better than any other volatile component (Brunton et al., 2000), and it was regarded as an indicator of flavor deterioration in various meat volatile compounds (Goodridge et al., 2003; Shahidi and Pegg, 1994). However, the OAV of hexanal in rabbit meat is the lowest of all eight aldehydes, which means it is just one of the key odorants. The concentration of hexanal in abdomen of both male and female rabbits showed highest, with lowest in both male and female Longissimus dorsi. The odor description of octanal is solvent, lemon and bitter. It can be produced during oxidation of saturated or unsaturated fatty acids from tallow, and also had relevance with off-flavors in cooked chicken (Kang et al., 2013; Shi et al., 2013). The concentration distribution of octanal showed that hind leg and abdomen were higher than foreleg and Longissimus dorsi. Nonanal which is one of the major aldehydes found in boiled beef, was also found in rabbit meat (Ruan et al., 2015). According to the research on the “Hanwoo” beef, what is different is that octanal and nonanal derived from oleic acid shows pleasant flavor (Hoa et al., 2013). The concentration of nonanal in foreleg was lowest in both male and female rabbit meat among four parts. The OAV of 2-nonenal (15.9) which smells like cardboard in rabbit meat is third highest in our research, and it was one of the main contributors to the overall off-flavor of porcine liver (Im et al., 2004). Like octanal, 2-nonenal was also common product generated from thermal oxidation of tallow, and the concentration of 2-nonenal in male rabbits was higher than in female rabbits. From aspect of distribution in different parts, the content of 2-nonenal in Longissimus dorsi of male rabbits was highest, whereas that in abdomen of female rabbits was lowest. 2, 4-Decadienal was detected as one of the key volatile compounds in many kinds of meat (Chen et al., 2009; Christlbauer and Schieberle, 2009; Madruga et al., 2009). In our study, the concentration of 2, 4-decadienal in hind leg was highest for both male and female rabbit, and they were lowest in Longissimus dorsi of both male and female rabbits. Moreover, decanal was not detected in hind leg while its concentration was highest in part of Longissimus dorsi. As compounds of decanal and 2, 4-decadienal has first two highest OAV, it means that the flavor pattern of hind leg and Longissimus dorsi were obviously different.

The concentrations of butanoic acid, decanoic acid and dodecanoic acid in all four parts of rabbit meat were lower than their odor thresholds, so acids were not responsible for the odor of rabbits here. The same situation was also found in ketones, esters, alcohols, aromatics and hydrocarbon. It can be seen that aldehydes were the main characterization odor of rabbit meat. So, this means that the oxidation of lipid may be most responsible for the odor of rabbit meat.

From Fig. 4, the number of acids in female rabbit meat is more than male one. Apart from Longissimus dorsi, three other parts owned various kinds of volatile compounds between male and female rabbit meat. In order to find out the key odorants target of rabbit meat, all detected 16 chemical compounds were choose to analyze via PCA with software of SPSS22.0. The results of two-axis analysis were PC1 being 54.35% and PC2 being 32.41%, respectively. The first and second principal component can explain 86.76% of the total variance. From PC1 (Fig. 4), dodecanoic acid (13), hexanal (2), decanal (12) and octanal (4) represented the rabbit meat flavor, meanwhile heptanal (3), 2-heptenal (7), 2-nonenal (8) and 2-decenal (9) were the main effective volatile compounds in PC2. This means those eight chemical volatile compounds seriously affected the flavor of rabbit meat, and represented the key odorants of rabbit meat flavor. Combining with method of OAV, hexanal (2), octanal (4), 2-nonenal (8), 2-decenal (9) and decanal (12) were seen as key odorants, with OAV>1. The odor description and origination of those five odorants can be seen from Table 3.

Fig. 4. Principal component analysis of all 16 detected volatile compounds in Hyla rabbit meat. x1-x16: pentanal, hexanal, heptanal, 2-heptenal, octanal, 2-octenal, nonanal, 2-nonenal, 2-decenal, undecanal, 2, 4-decadienal, decanal, butanoic acid, dodecanoic acid, hexadecanoic acid methyl ester, octadecanoic acid methyl ester, 9,12-octadecadienoic acid methyl ester.

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Table 3. Potential key odorants in Hyla rabbit meat

Compounds	CAS	Molecular formula	Odor description13,20	Origination16,21,23,26
Hexanal	66-25-1		Green, grassy, fatty	Lipid degradation or decarboxylation
2-Nonenal	18829-56-6		Fatty, waxy	Oxidation of arachidonic acid
Octanal	124-13-0		Pungent, orange	Oxidation of fatty acids
2-Decenal	3913-81-3		Metallic, soap	Thermal oxidation of tallow
Decanal	112-31-2		Green, oily	Autoxidation of unsaturated fatty acids

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Four different parts of both male and female rabbit meat were divided into two groups according to CA of the key odorants (Fig. 5). The first group contained fore leg, hind leg and abdomen of both male and female rabbit, and the second group included both the male and female Longissimus dorsi of Hyla rabbit meat. This indicated that the flavor of Longissimus dorsi significantly different from other three parts. It was accordance with sensory analysis results which showed that the intensity of rabbit meat odor of Longissimus dorsi was obviously less than other three parts (Table 4). And it was clear that the content of intramuscular phospholipids in Longissimus dorsi was lower than both abdominal muscle and hind leg. Hence, this may relate with the content of intramuscular lipid and the profile of fatty acid composition (Xue et al., 2015). In the first sub cluster, case 3 was close to case 4 and case 5 was close to case 6, suggesting that flavor of hind leg and abdomen in both male and female rabbit meat were the same. In the second sub cluster, case 1 was separated from other cases, manifesting that flavor of male fore leg was greatly different from other parts. Overall, cluster analysis results provide reliable information for rabbit processing industry and for possible future sale.

Fig. 5. Clustering results of four edible parts based on the key odorants in both male and female Hyla rabbit meat. case 1, 3, 5 and 7: sample of FL, HL, AB and LD of male Hyla rabbit respectively; case 2, 4, 6 and 8: sample of FL, HL, AB and LD of female Hyla rabbit respectively.

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