Identification of characteristic aroma compounds in raw and thermally processed African giant snail (Achatina fulica)

Background Food flavor appreciation is one of the first signals along with food appearance and texture encountered by consumers during eating of food. Also, it is well known that flavor can strongly influence consumer’s acceptability judgment. The increase in the consumption of snail meat across the world calls for the need to research into the aroma compounds responsible for the distinctive aroma notes of processed snail meat. Results The odorants responsible for the unique aroma notes in thermally processed giant African snail meats were evaluated by means of aroma extract dilution analysis (AEDA), gas chromatography–olfactometry (GC–O) and odor activity values (OAVs) respectively. Results revealed significant differences in the aroma profiles of the raw and thermally processed snail meats. Whilst the aroma profile of the raw snail meat was dominated with the floral-like β-ionone and β-iso-methyl ionone, sweaty/cheesy-like butanoic acid, and the mushroom-like 1-octen-3-one, the boiled and fried samples were dominated with the thermally generated odorants like 2-methylpyrazine, 2,5-dimethylpyrazine, 2-acetylthiazole and 2-acetylpyridine. Conclusion Finally, results have shown that sotolon, 2-acetyl-1-pyrroline, 2-furanmethanethiol, 2-methylbutanal, 1-octen-3-one, octanal, furanone, 2-methoxyphenol, 2-acetylpyridine, 2-acetylthiazole, and 2-methylpyrazine contributed to the overall aroma of the thermally processed snail meat.


Background
The giant African snail (Achatina fulica Bowdich) belongs to the Achatinoidea family and its native to East Africa. However, it has been widely distributed to different parts of the world such as; China [1], Taiwan [2], India, West Indies and the United States [3]. The snail's habitat covers the dense tropical forest of West Africa, Pacific Islands, Southern and Eastern Asia, and the Caribbean [4]. Different breeds of land snails have been reported and the most common breeds in Africa are Achatina achatina, Achatina fulica, Achachatina marginata and Limocolaria species [5]. The giant African snail is considered as one of the worst invasive species, because of its impact on agricultural and horticultural crops [6].
In spite of its invasive activities, African giant snails have been reported to exhibit antimicrobial properties. For instance, snails produce mucin in abundance in their mucus secretion. The mucin also called slim contains a bactericidal glycoprotein known as 'achacin' [7]. Also, the use of snail mucin for wound healing has been reported [8]. The giant African snails are highly relished delicacy in some parts of Africa, Taiwan, and South Korea [9]. France is the world leading consumer of snails followed, in order by Italy, Spain and Germany [10]. The snails are excellent sources of nutrition, as they contain abundant levels of calcium, phosphorous, magnesium and protein [11]. In addition, the distinctive aroma of fried snails is very effective in enhancing the flavor of dishes.
From a consumer perspective, the most appealing features of a processed snail meat are its flavor and nutrition. Food flavor appreciation is one of the first evaluation signals along with food appearance and texture encountered by consumers during eating [19].
However, to the best of our knowledge, there has been no report on the odorants responsible for the typical flavor of processed giant African snail. The aim of this study was to evaluate the potent aroma-active compounds in thermally processed giant African snail.

Odorants in raw snail meat
The aroma-active compounds in raw and thermally processed African giant snail meat (A. fulica) were evaluated. The most aroma-active components identified in the raw snail meat are listed in Table 1 and Fig. 1 respectively. The application of aroma extract dilution analysis (AEDA) and gas chromatography olfactometry (GC-O) revealed 13 odor-active compounds with FD factors from 4 to 32. Of this number, 8 odorants were obtained in the neutral basic fractions (NBF), while 5 odorants were found in the acidic fraction (AF). The major odorants with flavor dilution (FD ≥ 8) in the raw snail meat were 1-octen-3one, benzaldehyde, octanal, β-ionone and β-iso-methyl ionone. Odorant with the least FD of 2 was identified as 2,3-pentanedione. 2,3-Pentanedione, 1-octen-3-one, benzaldehyde and octanal have been widely reported in different species of mollusks such as shellfish [20], squid [21] and steamed mangrove crab [22]. However, β-iso-methyl ionone (Apo-carotenoid) to the best of our knowledge has not previously been detected or described in snail meat or any other mollusks.
A comparative analysis of the aroma profiles of raw and boiled snail meats revealed a significant number of thermally generated odorants in the boiled snails. Some of the identified odorants were; 2-methylpyrazine, 2,5-dimethylpyrazine, 2-acetylthiazole and 2-acetylpyridine (Fig. 2). Whereas, the aroma profile of the raw snail meat was dominated by floral, faint fatty, mushroom and sweaty/cheesy notes, the boiled snail meat elicited malty, popcorn-like, seasoning and mushroom nuances (Fig. 3). While the aroma notes developed in the boiled snail meat strongly increased in the fried snail samples, the faint fatty and mushroom notes decreased significantly. In order to elucidate the reasons behind this observation, the fried snail meats were subjected to AEDA and GC-O as earlier describe for the boiled snails.
In addition, the presence of the coffee-like 2-furanmethanethiol and 2-acetylthiazole in the fried snail meat are of particular interest. While, majority of Sulphur compounds such as thiazoles, sulfides and thiophenes are chemically stable and can be extracted easily, thiols are very reactive and susceptible to oxidation, dimerization, and reacts with carbonyls. Hence they deserve special attention to ensure minimum losses during analysis. 2-Acetylthiazole and 2-furanmethanethiol have been reported as major odorants in coffee [26] and identified in cooked meat, popcorn and baguette bread [27]. Furthermore, 2-furanmethanethiol has been identified as a major aroma component of steamed mangrove crab (Scylla serrata) [28]. On the other hand, 2-acetylthiazole a product of non-enzymatic browning reactions between reducing sugars and amino acids in the presence of H 2 S

Table 1 Most aroma-active components (FD ≥ 4) in raw and boiled giant snail meat (A. fulica)
AF acidic fraction, NBF neutral and basic fraction, FD flavour dilution a Compounds were identified by comparing their retention indices on DB-5 and FFAP columns, mass spectra, and their aroma impressions were compared with the respective reference compounds b Fractions in which the odorants were detected by GC-O after fractionation  [28], has been identified in nearly all cooked or roasted food aromas [28]. For instance, 2-acetylthiazole was reported as important odorant in steamed squid [21] and fried prawn meat [29]. Other thermally induced carbohydrate or protein degradation compounds such as 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF, furanone ® ), and 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolon) were detected with higher FD factors in the fried and boiled snail meats respectively (Tables 1, 2). Furanone and sotolon are important aroma compounds and are considered key flavor odorants in many food products. They are also highly appreciated in the food industry. Furanone and sotolon are products of the Maillard reaction and numerous methods for their synthesis have been published [30,31].
Additionally, the identification of β-ionone and β-isomethyl ionone for the first time in the snail meat was of interest. Although these aroma compounds exhibited low FD factors in the snail samples, they are known for their significant contribution to the aroma of flowers and foods [32]. In nature, β-ionone an example of Apo carotenoid is obtained by specific cleavage of β-carotenoid. This reaction is often catalyzed by the action of carotenoid cleavage deoxygenase 1 (CCD 1), which cleaves carotenoids at the 9, 10 position and 9′, 10′ position in the presence of oxygen [33]. However, Baldermann et al. [34] have shown that β-ionone can also be produced through carotenoid-cleavage like enzymes in Enteromorpha compressa (L.) Nees. Thus, the formation of this compound by carotenoid-cleavage like enzymes in raw snail meat seems likely.

Contribution of aroma compounds to the overall aroma quality of the raw and thermally processed snail meats
Finally, to have an idea of the contribution of the odorants to the aroma characteristics of the raw and thermally processed snail meats exhibited in Fig. 3, the 13 odorants detected through AEDA as the key odorants (FD factors ≥ 8) (Table 3) were quantified. Results of the aroma potencies showed that fried snail meat exhibited greater potency for 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolon), 2-acetyl-1-pyrroline, 2-furanmethanethiol and 2-methylbutanal as revealed by their high odor activity values (OAVs) ( Table 3). Again, boiled snail meat exhibited similar but lower potency for the same aroma compounds as those of the fried snail meat. Moreover, the raw snail showed stronger potencies for 1-octen-3one, β-ionone and octanal respectively. While, the OAVs indicated that 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furanone), octanal, 1-octen-3-one, 2-acetylpyridine, 2-methoxyphenol and 2-methylpyrazine contributed to the seasoning, popcorn and coffee-like aroma of the  thermally processed African giant snail meat. 1-Octen-3-one, octanal and β-ionone were the major contributors to the mushroom, sweaty/cheesy notes of the raw snail meat. A detailed analysis on aroma recombination experiments will be needed to determine the contribution of single odorant to the overall aroma of the snail meat.

Sensory evaluation
To corroborate the analytical data, sensory evaluations were performed on the snail samples by trained panelists. Sensory evaluation of the raw, boiled and fried snail meats revealed distinct aroma characteristics (Fig. 3). While the raw snail meat exhibited sweaty/cheesy, mushroom and faint-fatty notes, the boiled snail meat was characterized by popcorn, seasoning-like, malty and mushroom notes. The fried snail elicited similar but stronger aroma notes as the boiled snail meat. In addition, the fried snail meat also had strong coffee-like nuance.

Materials
Thirty adult giant snails (A. marginata and A. achatina) weighing between 82.10 and 96.40 g were collected after rainfall from three different gardens located in Port Klang, Malaysia. The shells of the snails were removed

Table 3 Concentrations (µg Kg −1 fresh weight) and odour activity values (OAVs) of aroma-active odorants (FD ≥ 8) in raw, boiled and fried giant snail (A. fulica)
Mean ± SD OAVs odour activity value was calculated by dividing the concentration with the threshold value of compound in water, nd not determined a Rychlik et al. [35] b Tressl [36] c Silva et al. [37] No. DB-5

Thermal processing
Thawed snail meats were divided into three batches of 200 g each. A batch was cooked in unsalted boiling water (100 °C) [20] for 15 min. After boiling, the snail was frozen with liquid nitrogen and ground into powder. A second batch was pan-fried at 160 °C without using fat as described earlier by Mall and Schieberle [20]. The frying protocol was carried out in an open pan heated with cooking gas as is done in domestic uses. The frying was continued for 8 min. The snail meat was stirred and reversed every minute for uniform cooking. After frying, the snail was cooled and frozen with liquid nitrogen before milling into powder. The third batch was used as the control.

Sample preparation
Powdered snail meat (100 g) was blended with anhydrous sodium sulphate (50 g) and diethyl ether (300 mL) followed by continuous stirring (2 h). The obtained mixture was filtered and subjected to solvent assisted flavor evaporation (SAFE) [22]. The obtained distillate was dried over anhydrous sodium sulphate and concentrated to approximately 50 mL [23].

Fractionation of volatiles
The SAFE distillate was treated with 150 mL of aqueous sodium bicarbonate (0.5 mol L −1 ) to yield an organic and aqueous layer respectively. The organic layer was washed twice with 75 mL of brine and dried over anhydrous sodium sulphate to produce the neutral/basic fraction (NBF). The aqueous layers were combined and acidified (pH 2.5) with HCl (16%) and extracted with diethyl ether (200 mL). The extract was subsequently dried over anhydrous sodium sulphate to yield the acidic fraction (AF). Both NBF and AF were concentrated to 100 µL each as described by Lasekan et al. [23] the resulted fractions were subjected to GC-O and GC-MS.

Extraction of raw snail meat
Minced raw snail (200 g) was extracted as described for the thermally processed samples above. The obtained mixture was subjected to SAFE distillation [22] and extracted with dichloromethane (2 × 200 mL). The extract was dried over anhydrous sodium sulphate and the organic phase was subsequently concentrated as described above. The concentrated extract was subjected to GC-O and GC-MS.

GC-MS and GC-FID analyses
A Shimadzu (Kyoto, Japan) QP-5050A GC-MS equipped with a GC-17 A Ver.3, a flame ionization detector (FID) and fitted differently with columns DB-FFAP and DB-5 (each, 30 m × 0.32 mm i.d., film thickness 0.25 µm; Scientific, Inc., Ringoes, NJ) was employed [24]. The gas chromatographic and mass spectrometric conditions were the same as described previously by Lasekan and Ng [26]. The HP Chemstation Software was employed for the data acquisition and mass spectra were identified using the NIST/NB575K database.

GC-O analysis
A Trace Ultra 1300 gas chromatograph (Thermo Scientific, Waltham, MA, USA) fitted with either a DB-FFAP or DB-5 column 1:(30 m × 0.32 mm i.d., film thickness, 0.25 µm, Scientific Instrument Services, Inc., Ringoes, NJ) and an ODP 3 olfactory Detector Port (Gerstel, Mulheim, Germany), with additional supply of humidified purge air, was operated as earlier reported by Lasekan et al. [21]. The split ratio between the sniffing port and the FID detector was 1:1. Two replicate samples were sniffed by three trained panelists who presented normalized responses, with strong agreement with one another.

Identification and quantification
The linear retention indices were calculated according to Kovats method using a mixture of normal paraffin C 6 -C 28 as external references [24]. The identification of compounds was as described earlier by Lasekan [24]. Quantitative data were obtained by relating the peak area of each compound to that of the corresponding external standard and were expressed as µg kg −1 .

Aroma extracts dilution analysis (AEDA)
The extracts of snail meat were diluted step wise twofold with dichloromethane by volume to obtain dilutions of 1:2, 1:4, 1:8, 1:16 and so on [24]. Each of the obtained dilution was injected into the GC-O. The highest dilution in which an aroma compound was observed is referred to as the flavor dilution (FD) factor of that compound [25].