Determination of Main Bitter Compounds in Soaked and Germinated Sesame Pastes

the flavor or taste of germinated sesame products. Olagunju and Ifesan （ 2013 ） reported the decreased sensory scores of wheat-sesame cookies with increased addition of germinated sesame seed flour 12 ） found previous experiment that the sesame paste prepared with the germinated sesame seeds became bitter, and the bitter taste was stron-ger with the longer duration of germination （ unpublished work ） . So far, the bitter components in the germinated sesame seeds haven ’ t been explored and identified. In the present study, the volatile flavors and the soluble taste components including oxalic acid, free amino acids, and sesaminoltriglucoside （ STG ） were thoroughly studied, and the bitter component of the germinated sesame pastes was identified for the first time. Abstract: The flavor and taste of the foods play an important or even a decisive role in the acceptance and preference of the consumers. It was found that the sesame paste prepared with the germinated sesame seeds was bitter in our previous experiment. In the study, the volatile and non-volatile bitter-taste components of the sesame paste samples were comprehensively analyzed. 2-methylbutanal, hexanal, acetic acid, and butyric acid were the predominant volatile compounds in the soaked and germinated sesame pastes. Oxalate was significantly reduced by the germination ( p < 0.05). The contents of sesaminoltriglucoside in sesame pastes ranged from 129.04 to 217.57 μg/g. Both total and individual free amino acid contents increased with the prolongation of the germinating time. The bitter-taste amino acid Arg had the highest score of Taste Activity Value for the bitterest sample made from the seeds germinated for 36 hours. The bitter-tasting Arg was first reported to impart a bitter taste to the germinated sesame paste.

richer in Vitamin E 10 .
The flavor and taste of the foods play an important or even decisive role in the acceptance and preference of the consumers. Generally, both the volatile flavor substance and soluble compounds present the consumers the sensory response, including olfactory and gustatory perceptions 11 . However, there is scarce literature about the flavor or taste of germinated sesame products. Olagunju and Ifesan 2013 reported the decreased sensory scores of wheatsesame cookies with increased addition of germinated sesame seed flour 12 . We found in our previous experiment that the sesame paste prepared with the germinated sesame seeds became bitter, and the bitter taste was stronger with the longer duration of germination unpublished work . So far, the bitter components in the germinated sesame seeds haven t been explored and identified. In the present study, the volatile flavors and the soluble taste components including oxalic acid, free amino acids, and sesaminoltriglucoside STG were thoroughly studied, and the bitter component of the germinated sesame pastes was identified for the first time.

Materials
The white sesame seeds cultivar: Yuzhi No.11 of about 50 kg were kindly provided by Henan Academy of Agricultural Sciences Zhengzhou, China . The year of harvest was 2014.
2.2 Preparation of the sesame pastes from the soaked and germinated seeds Sesame seeds 1 kg were cleaned, soaked in distilled water in the ratio of 1:2 w/v at room temperature for 2 h. The excess water was drained and the soaked sesame seeds were evenly put on the wet tray in the incubator Model WS250 , Shanghai Shuli instrument and Meter Co. Ltd., China for 0 h, 9 h, 19 h, 29 h, and 36 h, respectively at 28 and 95 relative humidity. The germination process was monitored to make humidity constant and prevent mould growth. The seeds without any treatment served as a control.
The sesame paste samples were prepared from the seeds after different germination duration. Germinated sesame seeds were baked to 20 -25 moisture, and then roasted at 130 using Automatic Roasting Machine Zhucheng Hong-nuo Mechanical Technology Co. Ltd., Weifang, China for about 40 minutes, until the sesame seeds were yellow and crisp. Sesame seeds were cooled to approximately 45 , then milled to paste using the colloid mill Model JM-L80, Wenzhou Longwan Huawei Machinery Factory, Wenzhou, China , and stored at 4 until use.

Analysis of the volatile compounds solid-phase mi-
croextraction GC-MS The volatiles of the sesame pastes was extracted using the solid-phase microextraction SPME and subjected to GC-MS analysis 13 15 . Briefly, each sesame paste sample of 5 0.1 g was put in a 10 mL headspace vial, and extracted with DVB/CAR/PDMS solid microextraction fiber 2 cm/50 μm, Supelco, Bellefonte at 40 in a thermostatic water bath for 60 min.
The extracted samples were desorbed at 250 for 0.5 h and analyzed on a GC-MS QP2010, Shimadzu, Japan with an Rtx-5MS 30 m 0.25 mm 0.25 μm chromatographic column Agilent Technologies, USA .
The helium gas purity ≥ 99.99 as a carrier was at a flow rate of 1.0 mL/min. The temperature was programmed at initial 40 for 1 min, then increased at 3 /min to 100 and held for 5 min, increased at 10 /min to 230 , finally increased at 15 /min to 290 , held for 5 min.
Mass spectrometry was performed in the ionizing mode, with electronic energy of 70 eV; ion source temperature of 230 , a port temperature of 250 , and a scanning range of 40 500 m/z.
The volatiles was identified by comparison with NIST14. lib and NIST14s.lib Mass Spectral Database Hewlett-Packard Co., Palo Alto, CA USA and their retention index. The retention index RI was calculated using a series of linear hydrocarbons C 6 -C 30 Sigma-Aldrich trading Co., LTD, Shanghai, China run at the same chromatographic conditions.
The relative amount of each volatile component was determined 16 , calculated by the area normalization method, and the results were expressed in percentage.

Measurement of oxalic acid
The content of oxalic acid was performed according to Sá et al. 17 , and Ran and Zhou 18 . Briefly, 1.5 g of sesame paste was weighed, transferred into 250 mL flask, and hydrolyzed with 50 mL of 0.1 N hydrochloric acid. The mixture was filtered through Whatman No. 4 filter paper, and the filtrate was precipitated by adding 25 calcium chloride w/v . The precipitant was dissolved using 1 N sulfuric acid, and titrated by 0.02 N potassium permanganate solution until the color of the solution turned to be pink and kept for more than 30 s.

Sesaminoltriglucoside analysis
The sesaminoltriglucoside STG was extracted and analyzed with HPLC and further identified with LC-MS according to Moazzami et al. 19 . Briefly, the sesame pastes were defatted and extracted with 85 ethanol for 5 h followed by 70 ethanol. The supernatants obtained after centrifugation were filtered with the 0.45 μm filter membrane. The extract was analyzed using a Waters LC-MS E2695, Waters Co., Ltd., Milford, USA equipped with the AQ-C18 column 46 mm 150 mm, 5 μm . The chromatographic condition was as follows: mobile phase A was methanol, and water as mobile phase B, linear-gradient elution of 30 60 methanol for 45 min, a flow rate of 1.0 mL/min, detection wavelength of 290 nm; column temperature of 30 , and injection volume of 20 μL. MS was performed in the ESI ionizing mode, the drying gas was 500 L/h, the drying gas temperature was 400 , capillary voltage: 3.0 kV and the scanning range was 100 1500 m/z. Naringenin 4 ,5,7-trihydroxyflavanone, purity ≥ 98 was bought from Nanjing Guangrun Biotechnology Co. Ltd.
Nanning, China . The standard solution 80 μg/mL was added to the samples for internal calibration. According to the corresponding chromatographic peak area and relative correction factor 0.345 19 , the contents of STG in the germinated sesame pastes were calculated.

Determination of free amino acids
The measurement of free amino acids was performed according to Song et al. 20 . Briefly, the samples were treated with 1 sulfosalicylic acid, and the supernatant was filtered with a 0.22 μm membrane. Free amino acids were analyzed using the Automatic Amino Acid Analyzer Model L-8900, Hitachi Co. Ltd., Tokyo, Japan equipped with an-ion-exchanged column 4.6 mm 150 mm . The detection wavelength was 570 nm and 440 nm, and the flow rate of the eluent was 0.4 mL/min.

Taste activity value TAV
TAV was determined according to Rotzoll, Dunkel and Hofmann 21 , and calculated by dividing the determined concentration of the flavor compound by its threshold value. The compounds with TAV greater than one were considered as active taste ingredients in the food.

Statistical analysis
All measurements were carried out in triplicate. The data were evaluated by one-way analysis of variance ANOVA and Fisher s least significant differences LSD at p 0.05. ANOVA and LSD tests were carried out using statistical software SPSS version 16.0, SPSS, Chicago, IL .

Results and Discussion
3.1 The volatiles of the sesame pastes prepared from the soaked and germinated sesame seeds The volatile components were analyzed using solidphase microextraction GC-MS and their relative contents were listed in Table 1 Table 1 The volatiles identified and quantified in the sesame pastes prepared from the soaked and germinated sesame seeds . SG2-0: the sesame paste prepared from the seed subjected to soaking of 2h and germination of 0h; SG2-9: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 9h; SG2-19: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 19h; SG2-29: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 29h; SG2-36: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 36h; CK: the control, the sesame paste prepared from the seeds subjected to nor soaking or germination.
were identified by comparing their spectra and retention indices with those in mass spectra database and authentic standards. The main classes of the volatile compounds included pyrazines, aldehydes, and acids. The contents of pyrazines showed a vast variation among the samples and were significantly increased by solely soaking the sesame seeds for 2 hours p 0.05 . The results may be ascribed to that during the soaking process, the sesame seeds absorbed the water. With the increasing activities of the amylase and protease, polysaccharides and proteins decomposed; reducing sugars and amino acids increased, and thus, more pyrazines can be formed from the Maillard reaction.
2-methylbutanal and hexanal were the most abundant aldehydes in the soaked and germinated seeds sesame pastes, and they were also reported to be the important aroma ingredients in the roasted almonds, 2-methylbutanal is formed via Strecker degradation of isoleucine 22 , while hexanal by the lipids autoxidation 21 .
The relative contents of the acids increased through germination. The most abundant acid in the germinated samples was acetic acid and butyric acid, which are formed by carbohydrate degradation 23 .

Evaluation of the contents of oxalic acids
Oxalic acid is a major bitter ingredient in the sesame pastes. We found that it was significantly reduced by the germination treatment, and reached the lowest level at the germination for 29 h 0.65 , which was 51.13 lower than that of the control sample p 0.05 , while the content of oxalic acid just after soaking was as high as that in the control Table 2 . The results indicated that soaking did not reduce the level of the oxalic acid, and the oxalate in the sesame might be present mainly in the form of the insoluble oxalic acid-mineral complex. The oxalate content for wheat bran was 0.46 24 . The total oxalate contents were in the range of 5.45-5.92 in the leaflets of Averrhoabilimbi and Averrhoa carambola, and the oxalates were present in the form of the calcium oxalate crystals which may impair the health of the patients with reduced renal activity 24 . Our results indicated that germination could reduce the oxalate content, and thus reduce the astringency of the sesame paste and decrease its risk to individuals with renal disorders.

3.2
The effect of soaking and germinating on the contents of STG Sesaminoltriglucosides STG are the main lignan-glucosides in the sesame seeds 25,26 . Although possessing the strong antioxidant activity, the glycosides usually have a bitter taste 27 , and thus the content of STG was determined Table 2 .
The contents of STG in sesame pastes ranged from 129.04 to 217.57 μg/g. Samples SG2-0 and SG2-9 had higher content than the control group p 0.05 . The measured values of the STG content were lower than those reported by Moazzami and Kamal-Eldin 25 . The variation may Data were expressed as the means±SD (n=3). The values with the different letters are significantly different (p < 0.05). SG2-0: the sesame paste prepared from the seed subjected to soaking of 2h and germination of 0h; SG2-9: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 9h; SG2-19: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 19h; SG2-29: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 29h; SG2-36: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 36h; CK: the control, the sesame paste prepared from the seeds subjected to nor soaking or germination.
be due to the different sesame material used as well as the partial loss of the seed coat during the roasting of the sesame seeds.

The pro ling of the free amino acids
Free amino acids contribute to the taste directly and participate in the Maillard reaction and Strecker degradation to form the volatile flavor compounds. Phe, Val, Met, Ile, Leu, and Arg are bitter, Glu, Asp, and His have sour taste. Thr, Ser, Gly, Ala, and Pro are sweet 28 . Free amino acids of the sesame pastes prepared from the treated sesame seeds were shown in Table 3. The results showed that total and individual free amino acid contents were increased by germinating, which was in accord with those reported by Liu et al. 8 .
The sample SG2-36 had the highest content of both the total and the individual free amino acids except for Phe, Met, Glu, Asp, His, Gly, and Cys. The contents of free amino acids positively correlated with the activities of the protease and the endopeptidase 29 . This may be due to the fact that during the process of the germination, the proteases including the endopeptidase, carboxypeptidase and aminopeptidase have enhanced activity, and hydrolyzed the proteins to the small peptides and free amino acids.
In Table 4, free amino acids of the sesame paste samples were classified into four taste groups, i.e., bitter, acid, sweet, and aged. And TAVs were calculated to reveal the main amino acids contributing to the taste of the sesame paste samples. The sweet-taste amino acid Ala had the TAV of 1.33 for the sample SG2-36. The bitter-taste amino acid Arg had the highest score of TAV, i.e. 1.82, for the sample SG2-36 which tasted bitterest. The compounds with TAV greater than 1 were considered as active taste ingredients. The larger the TAVs, the greater impacts on the taste. Table 3 The levels of free amino acids in the sesame pastes prepared from the soaked and germinated sesame seeds. Data were expressed as the means±SD (n=3). SG2-0: the sesame paste prepared from the seed subjected to soaking of 2h and germination of 0h; SG2-9: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 9h; SG2-19: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 19h; SG2-29: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 29h; SG2-36: the sesame paste prepared from the seeds subjected to soaking of 2h and germination of 36h; CK: the control, the sesame paste prepared from the seeds subjected to nor soaking or germination.
Thus, Arg was considered to be the main compound contributing to the bitter taste of the soaked and germinated sesame pastes.

Conclusions
This study provides the first comprehensive characterization of the volatile and non-volatile bitter-taste components of the soaked and germinated sesame paste. Strecker degradation product 2-methylbutanal, the lipids autoxidation product hexanal, and acids from carbohydrate degradation were the predominant volatile compounds after germination. The bitter-taste amino acid Arg had the highest score of TAV for the sample SG2-36 which tasted bitterest, which imparted a bitter taste to the germinated sesame paste.