Influence of Roasting Condition on Flavor Profile of Sunflower Seeds: A flavoromics approach

Sunflower see/ds (Helianthus annuus L.) were roasted in an electric forced air oven for 15, 30, 45, and 60 min at 125, 135 and 145 °C. The effect of temperature and time on the flavor profile of the samples were evaluated by headspace solid-phase microextraction coupled with gas chromatography-mass spectroscopy (HS-SPME-GC-MS). Unsupervised Principle Component Analysis (PCA) and Agglomerative Hierarchical Clustering (AHC) multivariate statistical methods were used to visualize, group and classify the samples. 114 volatiles were identified in the roasted sunflower seeds (RSF), with terpenes (α-pinene, β-pinene), heterocyclic compounds (2-ethyl-3-methylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, pyridine), aldehydes (2-methylbutanal, furfural, hexanal, phenylacetaldehyde), hydrocarbons (octane, 2-isobutyl-1,4-dimethylcyclohexane, 6,6-dimethylundecane), alcohol (3-methyl-2-propyl-1-pentanol), and γ-butyrolactone being dominant compounds. The content of most volatile compounds increased with increase in roasting temperature and time, such as esters, terpenes, pyrazines, aldehydes, ketones, and alcohols. 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-3-methylpyrazine, and 2-ethyl-3,5-dimethylpyrazine contributed to be the major role in roast and nutty flavor of the roasted sunflower seeds. Roasting at 125 °C for 45 min was found to be the better condition for roasted sunflower seeds, which gave the lowest off-flavor and burnt tastes.

www.nature.com/scientificreports www.nature.com/scientificreports/ (P < 0.05) with the increase of roasting temperature and time, the highest concentration was observed at 125 °C. Meanwhile, the amount of the corresponding hexanal increased significantly (P < 0.05), its probable mechanism may be the result of oxidation of the alcohol to the corresponding aldehyde. Whereas the amount of 3-methyl-2-propyl-1-pentanol increased up to 60 min at three temperature.
Toluene decreased up to 30 min, after that increased during roasting with the amount of 0.24 to 0.63 μg/g, the decrease may be due to the original toluene exist in the raw seed volatilized during thermal treatment. It was previously identified to have a negative effect on roasted and peanut aromas 20,21 ; as it is responsible for a paint aroma.
Generally, furans are normally responsible for the caramel-like odor of heated carbohydrates 22 . With the exception of furfural, There were other furan compounds found in sunflower seeds including 2-acetylfuran, 2,5-dimethyltetrahydrofuran, and 2-pentylfuran. These compounds represented from 0 up to 0.91, 0 up to 0.51, and 0.33 up to 1.53 μg/g in the roasted sunflower seeds with the increasing time.
Most of the volatiles increased in the roasted sunflower seeds were already present in raw seeds. 41 volatiles were not present in the raw seed that formed during thermal treatment, including 20 hydrocarbons, 6 easters, 5 ketones, 3 furans, 2 pyrazines, 2 aldehydes, 1 phenol, 1 pyrrole, 1 ether. Based on comparing the key odor compounds in roasted and raw samples, only a quite limited number of important odor compounds in roasted sunflower seed, such as 2-ethyl-3,5-dimethyl-pyrazine, 2,3-dimethylpyrazine, 2-acetylpyrrole, 2,5-dimethyltetrahydrofuran, 5-methyl-2-furfural were clearly formed during roasting. On the other side, 2-methylbutanal, 3-methylbutanal, furfuran, 2,5-dimethylpyrazine, α-pipene, 1-octen-3-ol, benzaldehyde were already present in considerable contents in the raw samples. The results showed that α-pinene was by far the most abundant odorant (7.05 to 21.85 μg/g in roasted seed and 7.57 μg/g in the raw seed) followed by 2,5-dimethylpyrazine, hexanal, and furfural in the roasted seed with amounts of 0.20 to 10.19, 1.35 to 8.49 and 0.95 to 8.18 μg/g, respectively. Furfural and hexanal in the raw seed with amounts of 1.35 and 0.95 μg/g, respectively (Table 1). β-Pinene is an organic compound of the terpene class which is one of the two isomers of pinene, it has a woody-green pine-like smell. β-Pinene increased with the increasing temperature and time with the amount of 0.76 to 3.00 μg/g. Table 2 showed the key odorants content obtained from RSF at 125, 135 and 145 °C of roasting for 15, 30, 45 and 60 min. When the concentration of the volatile compound is higher than its threshold, it is accepted that the volatile compound is perceived. The concentration of 2,5 dimeththyl-pyrazine, 2,3-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 3-ethyl-2,5-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine detected in the RSF were superior to their threshold, those compounds were considered representative of the active compound, suggesting that nutty, roast characteristics might be responsible for the aroma of roasted sunflower seeds. 2,5 dimeththylpyrazine showed enough concentration to be detected at 135 °C for 30 and 60 min and 145 °C for 45 and 60 min in the RSF. 2-Ethyl-3-methylpyrazine showed the high concentration in all treatments except 145 °C for 60 min in the RSF, and it was thus significantly responsible for the characteristic nutty aroma of roasted sunflower seed. As a result of the low threshold value of 2-methylbutanal, it showed the major potential to be responsible for almond and malt aroma in the RSF. Phenylacetaldehyde had lower threshold value, the concentrations in all treatment of RSF were superior to its threshold, it suggested to be the major role in the flowery and sweet flavor. Hexanal and nonanal were responsible for the off-flavor because the concentrations of those compounds in the RSF were superior to its threshold, hexanal can be detected at all the treatments in RSF.   www.nature.com/scientificreports www.nature.com/scientificreports/ Nonanal was not detected at 125 °C for 30 and 45 min, 135 °C for 30 and 60 min, 145 °C for 60 min. Heptanal was not detected at all treatment. Furfuryl alcohol along with furfural presented in many fruits, tea, coffee 23 , and their flavor characteristics was known as sweet, bread-like and caramellic. They are formed during the acid hydrolysis or heating of polysaccharides containing hexoses or pentoses 23 . Based on the results, the major contributors to bread, almond, sweet aroma came from furfuryl alcohol and furfural. The high content of pyridine was observed at a higher temperature, as the concentration superior to its odor threshold, it was possibly responsible for the burnt aroma. Moon and Shibamoto 24 reported that γ-butyrolactone generated from chlorogenic acid degradation formed more at high roasting temperature. γ-Butyrolactone had higher concentration superior to the odor threshold value, which was responsible for creamy odor. a-Pinene was the major volatile compound detected in the RSF, the concentration was far in excess of the odor threshold, and possibly be responsible for the pine aroma.
The score plot of volatiles generated from the comparison of the first two PCs (Fig. 2b). Sunflower samples were separated based on roasting temperatures and times. Volatile profiles in the highest temperature and late roasting times (145 °C, 45 and 60 min) separated from early roasting times (15 and 30 min). Along the PC1, late roasting times clustered on the right side while early roasting times clustered on the left side. PC1 and PC2 clearly separated treatments into three groups. Cluster I consisted of four treatments (125 °C for 60 min, 135 °C for 60 min, 145 °C for 45 min, 145 °C for 60 min) due to its positive correlation of volatile compounds on PC1. Cluster II including a single treatment 125 °C for 15 min, cluster III consisted eight treatments (0, 125 °C for 30 and 45 min, 135 °C for 15, 30 and 45 min, 145 °C for 15 and 30 min). Figure 2c showed PCA biplot which combined PCA score plot and loading plot.
AHC also gave an overview of similarities and differences among the treatments. These results were following the PCA analysis. AHC dendrogram of 114 volatile compounds was shown in Fig. 3. Dendrogram generated from hierarchical clustering was to assess the relationship between treatments 28 . The data sets were grouped into three clusters, whereby all the treatments of the close similarity were grouped together.

Conclusions
DVB/CAR/PDMS absorbed a large number of volatile compounds in roasted sunflower seeds, HS-SPME combined with GC-MS was used to evaluate the dynamic change of flavor profile during roasting sunflower seed. 114 volatiles were identified and quantified in the roasted sunflower seeds. The influence of roasting on flavor development varies depending on the temperature and time. The typical aroma compounds from Maillard reaction were formed at higher temperature and time, such as terpenes, pyrazines, and aldehydes. 2-Ethyl-3-methylpyrazine, 2,5-dimethylpyrazine, 2,3-dimethylpyrazine, and 2-ethyl-3,5-dimethylpyrazine which contribute to roasty and nutty flavor were found in higher concentration in the roasted sunflower seeds. The undesirable flavor notes, such as hexanal, nonanal, and pyridine were produced more in the sunflower seed roasted at three temperature

Materials and Methods
Material. Sunflower seeds harvested in 2017 were purchased from the local market, Thailand. The seeds were collected in aluminum foil bags and stored at 4 °C. By removing small, shriveled and broken seeds, good quality sunflower seeds were selected for further use.
Roasting process. Sunflower seeds were roasted by an electric forced air oven (Model UF55, Memmert, Thailand) at 125, 135 and 145 °C for 15, 30, 45, 60 min. The roasted sunflower seeds were cooled to ambient temperature, shelled and ground by an electric grinder (Panasonic, Japan) and stored in a sealed aluminum foil bag at −20 °C for further analysis.
Flavor extraction. For extraction of volatiles, the ground roasted sunflower seeds (2 g) was placed into headspace extraction vial, with 100 μg/2 g ethyl decanoate of sunflower seed (ethyl decanoate 1 mg/ml in 10% methanol) internal standard, prior to sealing with caps. 1 μl C6-C26 n-alkanes mixture (100 μg/ml each in methanol) were analyzed under the same condition. The sample was equilibrated for 20 min at 60 °C in the HS of the vial. After the equilibration, a 50/30 μm DVB/CAR/PDMS SPME fiber (57348-U, Supelco) was exposed to the HS for 30 min at 60 °C. PDMS is used for non-polar analytes, DVB is for polar analytes, especially useful for pyrazines. The application of this fiber successfully identified the aroma compounds of roasted almond 29 and roasted plantains 30 .
Identification of the volatile flavor compounds. GC-MS system Agilent 7890A gas chromatograph (Agilent Technologies, USA) with a 5975C mass spectrometer was used for analysis. A 60 m × 0.25 mm × 0.25 μm DB-1 ms column was used for analytes separation. The analytes were desorbed to the hot injection port of GC for 20 min at 250 °C in a splitless mode. Helium was operated at a constant flow rate of 1.5 ml/min. The temperature program was 50 °C for 1 min, followed by 5 °C/min to 100 °C (5 min), 4 °C/min to 140 °C (5 min), 5 °C/min to 180 °C (2 min), and 10 °C/min to 250 °C (7 min). The MS source temperature was 230 °C, transfer line temperature was 225 °C, quadrupole temperature was 150 °C. The electron ionization energy was set at 70 eV, scan range, m/z 50-550. The reference standards were operated under the same GC-MS condition described previously, an injection volume of 0.2 μl reference standard mixtures was employed in split mode (split ratio 100:1). The temperature program for n-alkane mixture was 5 °C/min to 100 °C (5 min), 4 °C/min to 140 °C (5 min), 5 °C/min to 180 °C (2 min), 10 °C/min to 250 °C (7 min), 10 °C/min to 280 °C (5 min), and 5 °C/min to 300 °C (10 min).
The identification of volatile compounds was comparison mass spectra with reference standards. Volatile compounds without authentic standards were identified by comparing retention indexes and/or mass spectrum based on the NIST library (NIST 11, Version 2.0, Gaithersburg, USA). Retention index of each compound was calculated by the retention time of a series of C6-C26 n-alkanes. The relative concentration of each compound was calculated based on the area of the internal standard.