# Comparative Analysis of Volatile Flavor Compounds in Seven Mustard Pastes via HS-SPME-GC-MS
## 2 Results and Discussion
### Identification of Volatiles in MPs  
As shown in Table 1, a total of 27 volatiles were isolated and detected from 7 MP samples, with 19, 16, 14, 12, 14, 12, and 10 compounds in MPs 1, 2, 3, 4, 5, 6, and 7, respectively. Among the 27 volatiles isolated, 12 compounds were positively identified and 15 compounds were tentatively identified. In addition, out of 27 compounds, 17 volatiles had matching degrees greater than 900, indicating that these compounds had excellent matching with the database. Figure 2 indicates that six components were common to all seven samples, including AITC, phenethyl isothianate, allyl thiocyanate, benzenepropanitrile, carbon disulfide, and allyl chloride. In addition, five compounds were unique to MP 1, including \(\alpha\)-turnerone, \(\beta\)-turnerone, ar-turnerone, \((-)\beta\)-sesquiphellandrene, and curcuminene. The number of volatiles detected in the seven MPs were more than those previously reported in the literature [5]. This could be attributed to the use of a 2 cm-long extractive fiber composed of three materials, which had a better extraction efficiency for the volatiles with different polarities. Furthermore, the raw materials of MP samples used in this experiment might be different from those used in previous studies. Nine esters were detected in the seven MP samples, the main ones of which were ITCs, which was consistent with the results reported previously [10]. Additionally, three sulfur-containing compounds, three ketones, three alkenes, two nitriles, and seven other compounds were detected in the samples. Among the 27 volatile components found in the 7 MPs, 3 ketones (\(\alpha\)-turnerone, \(\beta\)-turnerone, and ar-turnerone) and 3 alkenes (\(\alpha\)-zingiberene, \((-)\beta\)-sesquiphellandrene, and curcuminene) were detected for the first time, which might be related to the turmeric used in the samples. Since turmeric contains the natural pigment curcumin, the mixture of turmeric, lemon yellow, and bright blue pigment could yield the light green color of MP. A previous study reported that six compounds mentioned above (three ketones and three alkenes) were the volatiles of turmeric oil [11], and the use of turmeric in MP could not only play a role in the color but also might have had a certain contribution to the overall aroma profile. Turmeric was used in the MP in samples 1, 2, 3, and 5 as a raw material (Table 2), and six turmeric components mentioned above were all detected in MP 1; only \(\alpha\)-zingiberene was found in MP 5. However, the turmeric components were not detected in MP 2 and MP 3, possibly due to the low amount of turmeric used in these samples, which primarily served as a toning agent. AITC, commonly referred to as artificial mustard oil, exhibits a strong characteristic aroma similar to that of mustard. It is produced by the degradation of black mustard glucoside (sinigrin, a kind of thioglucide) catalyzed by the mustard enzyme [12], and has a threshold value of 0.046 mg/kg in water [13]. AITC plays a crucial role in the aroma of MP products, and it might have originated from various raw materials used in MP, such as mustard oil, wasabi, horseradish powder, fresh wasabi, edible spices, and flavor compounds. Of the seven MPs analyzed, six samples contained horseradish or horseradish powder as a raw material, while mustard oil was used in MP 1. Horseradish powder contained 0.85% volatile oil, which comprised 31.8% AITC [14]. Therefore, the amount of AITC provided by horseradish or horseradish powder was limited, and most of the AITC in MP was likely from the added flavor compounds or food flavoring. Both allyl thiocyanate and AITC are isomers (C\({}_{4}\)H\({}_{5}\)NS) with different functional groups. Allyl thiocyanate has two main formation pathways, as depicted in Figure 3. One is that it can be produced by the degradation of black mustard glucoside in the presence of mustard enzyme [15], while the other pathway involves the rearrangement of AITC via a six-membered ring transition state [16].