Food Chemistry 178 (2015) 122–127

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Judgment of pure fermented soy sauce by fluorescence resonance energy transfer of OPA-tryptophan adduct You-Syuan Gao, Bo-Chuan Hsieh, Tzong-Jih Cheng, Richie L.C. Chen ⇑ Department of Bio-Industrial Mechatronics Engineering, College of Bio-Resources and Agriculture, National Taiwan University, Taipei 10617, Taiwan

a r t i c l e

i n f o

Article history: Received 5 November 2013 Received in revised form 5 May 2014 Accepted 3 January 2015 Available online 22 January 2015 Keywords: Fluorescence resonance energy transfer (FRET) Tryptophan o-Phthalaldehyde (OPA) Soy sauce Flow-injection analysis (FIA)

a b s t r a c t Tryptophan was detected with a flow-injection manifold after reacting with mM order of fluorogenic o-phthalaldehyde (OPA)/thiol reagent (pH 10.0) in the carrier stream (0.63 mL/min). Based on the intra-molecular fluorescence resonance energy transfer of OPA-tryptophan adduct, the difference in fluorescence intensity obtained at 280 and 300 nm excitation was used to detect tryptophan content with satisfactory precision (CV < 6.5% for concentration higher than 0.5 lM), linearity (0.1–10 lM, R2 = 0.9893) and sensitivity (10 nM). Since tryptophan will decompose during manufacturing non-fermented soy sauce by acid-hydrolysis procedure, the method was used to discriminate pure fermented soy sauces, adulterated soy sauces and chemical soy sauces in less than 5 min. The ratio of tryptophan to total amino acid content served as the index for the judgment, and the results were validated by capillary electrophoresis. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Soy sauce was thought to be the most important seasoning in Asian food culture (Yokotsuka, 1986). Traditionally, it is brewed from mainly soybeans (sometimes fortified with wheat grains) for over 6 months after seeding with soy sauce koji (Aspergillus oryzae as usual). The prolonged fermentation process will gradually hydrolyze the vegetable proteins and release free amino acids as the chief ingredients of its umami (Kaneko, Kumazawa, Masuda, Henze, & Hofmann, 2006). However, the time-consuming procedure has become a problem in mass production to meet the increasing demand of daily use (Sasaki & Nunomura, 2003; Yang et al., 2011; Yongmei et al., 2009). From the early 20th century, acid-hydrolysis method was developed to shorten the lengthy brewing process. The proteins in soybeans and wheat are hydrolyzed by heating in hydrochloric acid for about 12–16 h until practically all the amino acids are liberated (Yong & Wood, 1974). Nevertheless, some side products such as levulinic acid (LV), 3-chloro-propane-1,2-diol (3-MCPD), formic acid and sulfur compounds are generated in the chemical process, which cause unnatural flavors. According to JAS (Japanese Agricultural Standards (JAS) Association, 1978) and CNS (Chinese ⇑ Corresponding author at: Department of Bio-Industrial Mechatronics Engineering, College of Bio-Resources and Agriculture, National Taiwan University, No.1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan. Fax: +886 2 23627620. E-mail address: [email protected] (R.L.C. Chen). http://dx.doi.org/10.1016/j.foodchem.2015.01.028 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

National Standard (CNS), 1993), LV (Sano et al., 2007) and the carcinogenic 3-MCPD (Hamlet, Sadd, Crews, VelõÂsÏek, & Baxter, 2002; Xing & Cao, 2007) were selected as the official markers for unnatural soy sauces. However, the official limitation for LV (e.g. 1 mg/mL in Taiwan standard) is ambiguous and LV may also originate from the caramel colorant conventionally added in some types of soy sauce products (Li & Geng, 2005; Zhu et al., 2010). On the other hand, the residual 3-MCPD in chemical soy sauce can be reduced to a non-detectable level by using defatted soybeans. Recently, tryptophan was considered to be a practical index of naturally brewed soy sauce (Zhu et al., 2010) because acidhydrolysis process degrades several amino acids especially tryptophan (>50% will be degraded) that is costly and hard to be recovered (Pickering & Newton, 1990; Yano, Aso, & Tsugita, 1990; Yokotsuka, 1986). In addition, high tryptophan content may reflect the quality of soy sauce for containing a higher level of other savory amino acids. The data were obtained by HPLC (Zhu et al., 2010) which is not practical for routine analysis of this popular seasoning. Most methods for tryptophan analysis depend on time-consuming separation process such as chromatography or electrophoresis (Alagendran et al., 2009; Delgado-Andrade, Rufián-Hanares, Jiménez-Pérez, & Morales, 2006; Sugimoto, Wong, Hirayama, Soga, & Tomita, 2010; Yust et al., 2004; Zhao et al., 2011; Zhu et al., 2010). Direct analytical methods such as a colorimetric (Basha & Robert, 1977) and electrochemical methods (Liu & Xu, 2007; Moreno, Merkoçi, Alegret, Hernández-Cassou, & Saurina,

Y.-S. Gao et al. / Food Chemistry 178 (2015) 122–127

2004) were also investigated, but neither the sensitivity nor the selectivity was sufficient for biological samples. Vincke, Vire, and Patriarche (1986) measured ammonia evolved from tryptophanase-catalyzed deamination reaction of Escherichia coli, but the slowness of the reaction limited the sensitivity. Simonian utilized the oxidative decarboxylation reaction of tryptophan-2-monooxygenase (TMO) to develop an amperometric tryptophan biosensor (Simonian, Rainina, Fitzpatrick, & Wild, 1995, 1997, 1999) based on oxygen consumption, but the detection limit was only 25 lM with the selectivity affected by co-existing phenylalanine. Sensitive and selective fluorometric methods are useful in biomedical applications, and tryptophan molecules possess a strong native fluorescence from its indole moiety. Yoshitake, Sejima, et al. (2007), Yoshitake, Nohta, et al. (2007) labeled the bioamines in urine by a pre-column derivatization reaction with OPA, and practically all primary amino groups will become isoindoles and fluoresce under UV illumination (Roth, 1971). In the case of tryptophan, an intra-molecular FRET phenomenon between the inherent indole moiety and the newly formed isoindole group on derivatized tryptophan molecule occurred efficiently, which made the fluorescent detection sensitive and selective for tryptophan. In this study, a similar FRET approach was adopted in a flow-injection manner (Hung, Chen, Chen, & Cheng, 2010); both the contents of tryptophan and total amino acids in different kinds of soy sauce were analyzed to evaluate the quality. The ‘‘chemical’’, purely fermented and blended soy sauces can be easily distinguished in minutes. The analytical results were validated by a simple capillary electrophoresis protocol proposed also by this study. 2. Materials and methods

123

was stored at 4 °C over night before use. Amino acid stock solutions were prepared with ultrapure water as 1 mM solutions and stored at 4 °C. 2.3. Samples and the pretreatment Totally 13 kinds of soy sauces were purchased from local markets, 10 of which are labeled with total nitrogen and amino acid nitrogen contents higher than 1.2 g/100 mL and 0.48 g/100 mL, respectively. Among the 10 soy sauces with nitrogen content labels, 8 are from trustful makers and were claimed as pure fermented. The remaining three are cheap and without information regarding fermentation and nitrogen contents. The samples were kept at 4 °C after the first use. Before the measurements, sample solutions were deproteinized either by salting out or by alcohol precipitation. For the salting out process, 10 mL of sample solution was added with 3.5 g of NaCl and heated at 80 °C for at least 3 h. The solution was filtered through 0.22 lm membrane filter (MCE membrane, Merck Millipore, USA) to remove the precipitation. For alcohol precipitation, 1 mL of sample solution was added with 4 mL of absolute alcohol and then centrifuged (4000 rpm  5 min at room temperature). The supernatant was filtered through the aforementioned 0.22 lm MCE membrane, and the filtrate was served as the deproteinized sample solution. 2.4. Fluorescence spectrometer Fluorescence spectra were measured with a commercialized spectrofluorometer (F4500, Philips); the bandwidths of the excitation and emission slits were 5 and 10 nm, respectively. The scan speed was 240 nm/min with the response of 0.004 s.

2.1. Chemicals 2.5. Flow-injection system o-Phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) were purchased from Wako Ltd., Japan. Sodium tetraborate, sodium hydroxide, sodium chloride and L-phenylalanine were obtained from Nacalai Tesque, Ltd., Japan. L-Tyrosine, L-glutamic acid, L-tryptophan, L-histidine, N-acetyl-L-cysteine (NAC) were from Sigma–Aldrich, Inc., USA. Albumin from egg (OVA) was from Acros Organics, USA. Alcohol (99.5%) was analytical grade and purchased from Shimakyu’s Pure Chemicals, Osaka, Japan. All chemicals were of analytical-reagent grade and used as received without further purification. Deionized water (conductivity < 1 lS/cm) was obtained with RDI pure water system (Sunway Scientific, Co., Taiwan) and used for buffer preparation and sample dilution. Ultrapure water was prepared with Direct-Q gradient system (Millipore, Milford, MA, USA) and with a resistivity higher than 18.2 MX cm. 2.2. Buffers and reagents Borate buffer (0.1 M, pH 10.0 with NaOH) was prepared with deionized water and was stored at room temperature as the stock. OPA reagent A was prepared by mixing 30 mg of o-phthalaldehyde (OPA) and 20 lL of 2-mercaptoethanol in 4.5 mL of ethanol and then diluted to 100 mL with borate buffer (0.01 M, pH 10.0); the reagent (containing 2.2 mM OPA) was stored at 4 °C over night before use. OPA reagent B (containing 1 mM OPA) was freshly prepared by dissolving 13 mg of OPA and 16 mg of NAC with 3 mL of ethanol and then diluted to 100 mL with borate buffer (0.01 M, pH 10). OPA reagent C (containing 1 mM OPA) was prepared by mixing 6.7 mg of OPA and 3.5 lL of 2-ME in 3 mL of ethanol and then diluted to 50 mL with borate buffer (0.01 M, pH 10.0); the reagent

Conventional flow-injection tubing (silicon tubes with 1 mm i.d.) and connectors were used. The carrier solution was driven with a peristaltic pump (SMP23S, Tokyo Rikakikai Ltd., Japan) and the sample solution was injected via a 6-port medium pressure injection valve (V450, IDEX Co., USA). The injected sample plug (90 lL) passed through a 126 cm mixing coil before reaching the fluorometric detector (FP-1520, Jasco Inc., Japan). The bandwidths for both excitation and emission are 18 nm. The fluorescence intensity was converted to voltage signal and recorded/monitored using a 32-bit DAQ card (NI cDAQ 9172, NI Co., Texas) interfaced to a personal computer, and a control program was written under LabVIEW (ver. 10.0 Development System, NI Co., Texas) environment to handle the data acquisition and signal processing. The FIAgrams were smoothed by averaging 10 adjacent points (by moving average algorithm) to remove the noise, and the peak heights were taken for quantification. 2.6. Capillary electrophoresis Capillary electrophoresis was performed with a commercialized system (G1600A CE, Agilent Technologies, USA) and a bare fusedsilica capillary (50 lm i.d.  375 lm o.d. with 61.5 cm of effective length, Beckman coulter, Inc., USA). Before separation, the capillary was rinsed by purging (940 mbar) sequentially with 1 M NaOH for 5 min, deionized water for 5 min and then the running buffer (10 mM borate buffer, pH 10) for 5 min. After injecting (50 mbar  10 s) the sample solution into the capillary, 25 kV was imposed for typically 20 min of migration. Tryptophan and tyrosine were detected by A280.

124

Y.-S. Gao et al. / Food Chemistry 178 (2015) 122–127

3. Results and discussion 3.1. Emission and excitation spectra of OPA-tryptophan adduct As Fig. 1a, the emission spectrum of tryptophan at 280 nm excitation showed a maximum at 360 nm due to the intense native fluorescence of the indole-moiety of tryptophan. After reacting with OPA, the emission maximum shifted to 455 nm, which revealed the fluorescence resonance energy transfer (FRET) from the intrinsic indole moiety to the isoindole formed with OPA. In addition to the 340 nm peak of primary amines, the excitation spectra of OPA-tryptophan adduct (Fig. 1b) exhibit another dosedependent emission maximum at 280 nm, which can be used to selectively quantify tryptophan. Moreover, the emission spectrum of tryptophan shows a broad overlap (65%) with the excitation

Fig. 1. Comparison of fluorescence spectra of tryptophan and the OPA-adduct: (a) emission spectra (kex = 280 nm) of 10 lM tryptophan before (solid curve) and 5 min after (dashed curve) reacting with OPA reagent C; (b) dose-dependent excitation spectra (kem = 445 nm) of 5 lM (solid curve) and 10 lM (dashed line) tryptophan 5 min after reacting with OPA reagent C; (c) the emission spectrum (kex = 280 nm) of 10 lM tryptophan (solid curve) and the excitation spectrum (kem = 445 nm) of 10 lM tryptophan 5 min after reacting with OPA reagent C (dashed curve).

spectrum of its OPA-adduct (Fig. 1c), which leads to a high energy transfer efficiency and therefore a high sensitivity in tryptophan measurement based on FRET.

3.2. Interference from other amino acids Though the emission maximum under 280 nm excitation is unique for OPA-tryptophan adduct, the shoulder peaks of other co-existing amino acid derivatives are still problematic (Fig. 2a). Since the excitation spectra of OPA-amino acid adducts generally have plateaus around 290 nm, the differences of fluorescence

Fig. 2. (a) Excitation spectra (kem = 445 nm) of 10 lM glutamic acid (dashed curve), 10 lM tryptophan (solid curve), 10 lM histidine (dotted curve), and 10 lM phenylalanine (dash-dotted curve) 5 min after reacting with OPA reagent C; (b) comparison of the fluorescence differences (DF) obtained by subtracting the florescence intensity at k280 with that of the indicated excitation wavelength. Each datum was the average of nine DF obtained from the excitation spectra (kem = 445 nm) of amino acid-OPA adducts generated by mixing 1, 5, and 10 lM glutamate, histidine or phenylalanine with OPA reagent C for 5 min; (c) dosedependent excitation spectra (kem = 445 nm) of 10 lM tyrosine (solid curve) and 5 lM tyrosine (dashed curve) 5 min after reacting with OPA reagent C.

Y.-S. Gao et al. / Food Chemistry 178 (2015) 122–127

125

intensities (DF) between the tryptophan-specific 280 nm excitation (F280) and 290, 295, 300, 305, 310 nm excitation (Fk) were compared (Fig. 2b: DF(k) = F280  Fk). The DF between 280 and 300 nm were practically equal to zero with a negligible standard deviation, the interferences of the co-existing amino acid derivatives can therefore be effectively removed as Eq. (1). 445 DF 445 ¼ F 445 280  F 300

ð1Þ

where F 445 280 represents the fluorescence intensity at 445 nm under 280 nm excitation, F 445 300 is the fluorescence intensity at 445 nm under 300 nm excitation. DF445 is contributed mainly by the intra-molecular energy resonance between OPA and the aromatic side chain of amino acids. Basically all amino acids except tyrosine and tryptophan show an emission plateau around 290 nm (Fig. 2c), so DF445 can be considered as a combined FRET signal from tyrosine and tryptophan for an amino acid mixture. Although the contribution of tyrosine on DF445 is ca. 37% of the same concentration of tryptophan (data from Fig. 2), the natural abundance of tyrosine is at least 3 times higher than tryptophan, which may cause problems if tryptophan content is the major concern of our study. Although somewhat decreased in fluorescence intensity, the interference from co-existing tyrosine can be further reduced by red-shifting the emission wavelength from 445 to 475 nm if necessary (Table 1). 475 DF 475 ¼ F 475 280  F 300

ð2Þ

where F 475 280 represents the fluorescence intensity at 475 nm under 280 nm excitation, F 475 300 is the fluorescence intensity at 475 nm under 300 nm excitation. As revealed by Table 1, the contribution of 50 lM tyrosine on DF445 was ca. 120% of 10 lM tryptophan, the influence of co-existing tyrosine was significantly reduced for DF475. Red-shift to 475 nm may be helpful if the sample contains a high concentration of tyrosine. 3.3. Flow-injection determination of tryptophan Since the fluorescent OPA-derivatives are not stable enough for a batch-by-batch quantification approach (Lochmann, Stadlhofer, Weyermann, & Zimmer, 2004; Molna´r-Perl, 2001) especially when 2-mercaptoethanol (OPA reagent A) is used, flow-injection approach was adopted in this study. The flow-injection manifold is similar to the previous documents (Hung et al., 2010; Molna´rPerl, 2001) and was optimized as the following. The flow rate was 0.63 mL/min; the pH of the carrier solution was controlled at 10 by 10 mM borate buffer. When 2-ME was used as the thiol agent, the preferred OPA and 2-ME concentrations were 2.2 and 3.0 mM, respectively (OPA reagent A). When NAC was used as the thiol agent, the preferred OPA and NAC concentrations were 1 and 1 mM, respectively (OPA reagent B). Typical FIAgrams were obtained with a high reproducibility and sample throughput

Fig. 3. Typical FIAgram for obtaining DF445. Each flow-injection signal was obtained by injecting an amino acid mixture containing 10 lM tryptophan, 5 lM histidine, 5 lM glutamate, 2.5 lM phenylalanine, 1 lM tyrosine, 10 lM threonine and 1 lM valine. The peak heights were taken for calculating DF445 as Eq. (1).

(Fig. 3). The difference in peak height at two excitation wavelengths (280 and 300 nm) was used to estimate tryptophan contents. For quantifying tryptophan by DF445, OPA reagent A was used as the carrier solution of the FIA system. The calibration curve was linear up to tens of lM (R2 > 0.989) with satisfactory detection limit ( 0.9917; n = 3). 3.4. Effect of sample pretreatment Since soy sauce is basically the protein lysate of soy bean, the sample has to be deproteinized before a measurement based on primary amino group reaction. Nylon membranes were tried to bind coexisting proteins (Tovey & Brian, 1989), but the sample recovery for amino acids was poor. Therefore, a salting out and ethanol precipitation protocols were performed for removing the undesired proteins or large polypeptides, which were reported to conserve practically all amino acids in the samples (Al-Jumaily & Al-Safar, 2012; van Wuyckhuyse et al., 1995). After the pretreatments, the calibration curve was not significantly altered by co-existing proteins (Fig. 4) that may exert intra-molecular FRET effect if the N-terminal amino acid residue happens to be tryptophan (Yoshitake, Sejima, et al., 2007; Yoshitake, Nohta, et al., 2007).

Table 1 Effect of different emission wavelengths on the fluorescent responses of tryptophan and tyrosine.

DF445 10 lM Trp 50 lM Tyr Ratiob

a

0.0306 0.0378 1.2353

DF455

DF465

DF475

0.0312 0.0362 1.1603

0.0270 0.0297 1.1000

0.0188 0.0180 0.9574

a Data were obtained by injecting either 10 mM tryptophan or 50 mM tyrosine to the flow-injection system, and the difference in peak heights at 280 and 300 nm excitation wavelengths (as in Fig. 3, n = 3) were tabulated. OPA reagent B was used as the carrier solution. b Ratio = 50 mM Tyr/10 mM Trp.

Fig. 4. Effect of sample pretreatment on protein removal. Calibration curves of tryptophan (solid circles) and tryptophan with 0.1 mg/mL oval albumin (OVA) added (open circles) were compared. The sample solutions were deproteinized by the salting out process before the measurement.

126

Y.-S. Gao et al. / Food Chemistry 178 (2015) 122–127

3.5. Classification of soy sauces All deproteinized samples were diluted 1000-fold with deionized water before the measurements. Fig. 5a plots the relationship of DF475 to F 475 300 for 8 different pure fermented soy sauces from trustful makers, 2 soy sauces with nitrogen content labels and 3 cheap soy sauces of unknown quality. DF475 is positively related to the content of tryptophan and tyrosine (to a lesser extent), and F 475 300 reflects the content of total amino acids. For the pure fermented soy sauces, the absolute values of DF475 and F 475 300 varied but highly related to each other in a linear manner (R2 = 0.9678), which may be due to the different fermentation or blending (or dilution) extent for commercialization. The regression coefficient for DF445 2 and F 445 300 of pure fermented soy sauces was lower (R = 0.8145) 445 since DF is prone to be affected by tyrosine. Blended soy sauces were adulterated with savory amino acids such as glutamate, so the data points (the 2 solid triangles in Fig. 5a) deviated from the regression line and appeared in the upper-left of the plots. The cheap ‘‘chemical’’ soy sauces were low in both total amino acids and tryptophan contents since the additives of these seasonings are caramel colorants but not savory amino acids (the 3 solid squares in the lower left of Fig. 5a). The analysis showed that pure fermented soy sauces contained certain proportion of tryptophan against total amino acids (DF475/F 475 300  0.11–0.12), the ratio of adulterated soy sauces were about 0.09 and chemical soy sauces

were 0.06–0.08. The fluorometric methods was proven to be useful in categorizing soy sauces based on their amino acid composition, 475 475 and the ratio of DF445/F 445 /F 300 may become an index for 300 or DF estimating the degree of fermentation or judging if there is any adulteration in manufacturing process. Capillary electrophoresis for tryptophan and tyrosine was conducted to validate if the index is correlated with the relative tryptophan or tyrosine contents. For CE separation, all soy sauces were subjected to ethanol precipitation instead of the salting out process since the residual high salt content hampered the electrophoretic separation process. Tryptophan and tyrosine were separated and detected by A280 at 5.85 and 6.65 min, respectively. Since glutamic acid is the representative amino acids in soy sauce (Yokotsuka, 1961), the F 445 300 signal of the FIA system was calibrated with glutamate to calculate the total amino acids content as its glutamate equivalent. The ratios of tryptophan content (obtained by CE) against the total amino acid content (obtained by FIA as the glutamate equivalent) were compared with the aforementioned index, DF445/F 445 300 (Fig. 5b), and the results show a high correlation (R2 > 0.8595) between this two parameters. The samples with higher DF445/F 445 300 values conserved more tryptophan during the manufacturing process, which implied that these soy sauces were made by traditional fermentation but not acidic hydrolysis. Tyrosine was reported to be also labile (>20%) in acidic hydrolysis (Pickering & Newton, 1990), but the regression coefficient between DF445/F 445 300 and tyrosine/total amino acids was only 0.1551. The correlation shown in Fig. 5b was chiefly due to the influence of tryptophan contents, so DF445 was with sufficient selectivity in this case. 4. Conclusions The FRET-based analytical method is sensitive and selective for tryptophan with its reproducibility and rapidity greatly improved after being incorporated in a flow-injection system. The system can obtain both the information of tryptophan and total amino acid content in less than 5 min, which was used to rapidly discriminate soy sauces made by traditional fermentation process, acidhydrolysis process or adulteration. The results showed a high correlation with those obtained by a rapid capillary electrophoresis protocol developed also in this work. Due to its selectivity and high sensitivity, the present system may be used to rapidly detect tryptophan content in urine, serum and other biomedical samples. References

475 Fig. 5. Soy sauce quality as expressed by comparing the data of F 475 and 300 and DF 475 475 the ratio (DF445/F 445 were obtained by FIAgrams as Fig. 3, each 300 ). (a) F 300 and DF datum is the average of three measurements. The regression line was from eight pure fermented soy sauces (solid circles). On the lower left (solid squares) are three chemical soy sauces of poor quality. Two blended or adulterated samples were shown in solid triangles. (b) Ratio obtained by FIA (DF445/F 445 300 ) was positively related to the relative tryptophan content. For ratio obtained by CE, F 445 300 obtained by FIAgrams) was first converted to its glutamate equivalent after calibrating with glutamate, then the tryptophan content obtained by CEgrams was used to calculate the ratio for comparison. The symbols for the soy sauce samples are the same as in (a).

Alagendran, S., Rameshkumar, K., Palanivelu, K., Puspha, N., Ranjani, M., Rulmozhi, N., et al. (2009). Salivary amino acids quantification using RP-HPLC during normal menstrual cycle. African Journal of Biochemistry Research, 3(5), 185–189. Al-Jumaily, E. F. A., & Al-Safar, M. A. (2012). The free amino acids (proline and arginine) concentration from human whole unstimulated saliva patients. International Journal of Advanced Biological Research, 2, 328–334. Basha, S. M. M., & Robert, R. M. (1977). A simple colorimetric method for the determination of tryptophan. Analytical Biochemistry, 77, 378–386. Chinese National Standard (CNS), (1993). Soy sauce. 423, N5006. Delgado-Andrade, C., Rufián-Hanares, J. A., Jiménez-Pérez, S., & Morales, F. J. (2006). Tryptophan determination in milk-based ingredients and dried sport supplements by liquid chromatography with fluorescence detection. Food Chemistry, 98, 580–585. Hamlet, C. G., Sadd, P. A., Crews, C., VelõÂsÏek, J., & Baxter, D. E. (2002). Occurrence of 3-chloro-propane-1,2-diol (3-MCPD) and related compounds in foods: A review. Food Additives and Contaminants, 19(7), 619–631. Hung, Y. T., Chen, P. C., Chen, R. L. C., & Cheng, T. J. (2010). Sequential determination of tannin and total amino acid contents in tea for taste assessment by a fluorescent flow-injection analytical system. Food Chemistry, 118, 876–881. Japanese Agricultural Standards (JAS) Association, (1978). Index of inspection on food sanitation II category of food. 67–68. Kaneko, S., Kumazawa, K., Masuda, H., Henze, A., & Hofmann, T. (2006). Molecular and sensory studies on the umami taste of Japanese green tea. Journal of Agriculture and Food Chemistry, 54(7), 2688–2694. Li, G. J., & Geng, Y. H. (2005). Determination of levulinic acid in soy sauce, hydrolyzed vegetable protein and caramel. Analysis and Examination, 9, 51–52.

Y.-S. Gao et al. / Food Chemistry 178 (2015) 122–127 Liu, Y., & Xu, L. (2007). Electrochemical sensor for tryptophan determination based on copper-cobalt hexacyanoferrate film modified graphite electrode. Sensors, 7, 2446–2457. Lochmann, D., Stadlhofer, S., Weyermann, J., & Zimmer, A. (2004). New protamine quantification method in microtiter plates using o-phthaldialdehyde/N-acetyll-cysteine reagent. International Journal of Pharmaceutics, 283, 11–17. Molna´r-Perl, I. (2001). Derivatization and chromatographic behavior of the ophthaldialdehyde amino acid derivatives obtained with various SH-groupcontaining additives. Journal of Chromatography A, 913, 283–302. Moreno, L., Merkoçi, A., Alegret, S., Hernández-Cassou, S., & Saurina, J. (2004). Analysis of amino acids in complex samples by using voltammetry and multivariate calibration methods. Analytica Chimica Acta, 507, 247–253. Pickering, M. V., & Newton, P. (1990). Amino acid hydrolysis: Old problems, new solutions. LG/GC, 8, 778–781. Roth, M. (1971). Fluorescence reaction for amino acids. Analytical Chemistry, 43(7), 880–882. Sano, A., Satoh, T., Oguma, T., Nakatoh, A., Satoh, J. I., & Ohgawara, T. (2007). Determination of levulinic acid in soy sauce by liquid chromatography with mass spectrometric detection. Food Chemistry, 105, 1242–1247. Sasaki, M., & Nunomura, N. (2003). Fermented foods/soy(soya) sauce. In B. Caballero, L. Trugo, & P. M. Finglas (Eds.), Encyclopedia of food sciences and nutrition (2nd ed., pp. 2359–2369). London: Academic Press. Simonian, A. L., Rainina, E. I., Fitzpatrick, P. F., & Wild, J. R. (1995). A biosensor for Ltryptophan determination based on recombinant pseudomonas savastanoi tryptophan-2-monooxygenase. Analytical Letters, 28(10), 1751–1761. Simonian, A. L., Rainina, E. I., Fitzpatrick, P. F., & Wild, J. R. (1997). A tryptophan-2monooxygenase based amperometric biosensor for tryptophan determination: Use of a competitive inhibitor as a tool for selectivity increase. Biosensors & Bioelectronics, 12(5), 363–371. Simonian, A. L., Rainina, E. I., Fitzpatrick, P. F., & Wild, J. R. (1999). Enhancement of the specificity of an enzyme-based biosensor for L-tryptophan. Advances in Experimental Medicine and Biology, 467, 833–840. Sugimoto, M., Wong, D. T., Hirayama, A., Soga, T., & Tomita, M. (2010). Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles. Metabolomics, 6, 78–95. Tovey, E. R., & Brian, B. A. (1989). Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting. Journal of Biochemical and Biophysical Methods, 19, 169–184. Vincke, B. J., Vire, J. C., & Patriarche, G. J. (1986). Potentiometric determinations of amino acids using enzymatic and bacterial electrodes. Electrochemistry Sensors and Analysis, 147–154. van Wuyckhuyse, B. C., Perinpanayagam, H. E. R., Bevacqua, D., Raubertas, R. F., Billings, R. J., Bowen, W. H., et al. (1995). Association of free arginine and lysine

127

concentrations in human parotid saliva with caries experience. Journal of Dental Research, 74, 686–690. Xing, X., & Cao, Y. (2007). Determination of 3-chloro-1,2-propanediol in soy sauces by capillary electrophoresis with electrochemical detection. Food Control, 18, 167–172. Yang, H. J., Park, S., Pak, V., Chung, K. R., & Kwon, D. Y. (2011). Fermented soybean products and their bioactive compounds. In: Hany El-Shemy (Ed.), Soybean and health. InTech, ISBN: 978-953-307-535-8, available from: . Yano, H., Aso, K., & Tsugita, A. (1990). Further study on gas phase acid hydrolysis of protein: Improvement of recoveries for tryptophan, tyrosine, and methionine. Journal of Biochemistry, 108, 579–582. Yokotsuka, T. (1986). Soy sauce biochemistry. Advanced in Food Research, 30, 195–329. Yokotsuka, T. (1961). Aroma and flavor of Japanese soy sauce. Advances in Food Research, 10, 75–134. Yong, F. M., & Wood, B. J. B. (1974). Microbiology and biochemistry of soy sauce fermentation. Advances in Applied Microbiology, 17, 157–194. Yongmei, L., Xiaohong, C., Mei, J., Xin, L., Rahman, N., Mingsheng, D., et al. (2009). Biogenic amines in Chinese soy sauce. Food Control, 20(6), 593–597. Yoshitake, M., Sejima, N., Yoshida, H., Todoroki, K., Nohta, H., & Yamaguchi, M. (2007). Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection. Analytical Sciences, 23, 949–953. Yoshitake, M., Nohta, H., Ogata, S., Todoroki, K., Yoshida, H., Yoshitake, T., et al. (2007). Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection. Journal of Chromatography B, 858, 307–312. Yust, M. M., Pedroche, J., Calle, J. G., Vioque, J., Millán, F., & Alaiz, M. (2004). Determination of tryptophan by high-performance liquid chromatography of alkaline hydrolysates with spectrophotometric detection. Food Chemistry, 85, 317–320. Zhao, J., Chen, H., Ni, P., Xu, B., Luo, B., Zhan, Y., et al. (2011). Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection. Journal of Chromatography B, 879, 2720–2725. Zhu, Y., Yang, Y., Zhou, Z., Li, G., Jiang, M., Zhang, C., et al. (2010). Direct determination of free tryptophan contents in soy sauces and its application as an index of soy sauce adulteration. Food Chemistry, 118, 159–162.