Abstract
The content of a kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly), in several kinds of commercial fermented shrimp paste condiments was determined using a LC/MS/MS method after derivatization with 6-aminoquinoyl-N-hydroxy-succinimidyl-carbamate (AQC). Commercial fermented shrimp paste condiments from Indonesia (Terasi), the Philippines (Bagoong) and China (Xiajiang) were analyzed. The analyses revealed that the concentration of γ-Glu-Val-Gly in Terasi, Bagoong, and Xiajiang was 5.2 μg/g, 1.0 μg/g, and 0.9 μg/g, respectively. These results suggested that γ-Glu-Val-Gly is widely distributed in fermented shrimp paste condiments.
Introduction
It was recently reported that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR) in humans (Ohsu et al., 2010; Maruyama et al., 2012). These studies revealed that glutathione can activate human CaSR, and that several γ-glutamyl-peptides including γ-Glu-Ala, γ-Glu-Val, γ-Glu-Cys, γ-Glu-α-aminobutyryl-Gly (ophthalmic acid) and γ-Glu-Val-Gly can also activate the CaSR. These substances possess the characteristics of kokumi substances, being able to modify the five basic tastes, especially sweet, salty and umami, when they are added to basic taste solutions or food, even though these substances themselves have no taste at the concentrations tested (Ueda et al., 1990; Ueda et al., 1997; Dunkel et al., 2007; Toelstede et al., 2009; Toelstede and Hofmann, 2009). The CaSR activity of these γ-glutamyl-peptides has also been shown to be positively correlated with the sensory activity of kokumi substances, suggesting that kokumi substances are perceived through the CaSR in humans. Among kokumi peptides, γ-Glu-Val-Gly was reported to be a potent kokumi peptide, with a sensory activity 12.8-fold greater than that of glutathione (Ohsu et al., 2010). Nevertheless, few studies have been conducted to determine the contents of γ-Glu-Val-Gly in foods. A recent investigation revealed the presence of γ-Glu-Val-Gly in fishery products such as scallop products (Kuroda et al., 2012a) and various commercial fish sauces (Kuroda et al., 2012b), as well as in various commercial soy sauces (Kuroda et al., 2013). Despite these findings, no studies have been conducted to investigate the presence of this peptide in other foodstuffs. In particular, since fish sauces contain γ-Glu-Val-Gly, other fermented fish products such as fermented shrimp paste condiments, which are widely consumed in East Asian and Southeast Asian countries, were assumed to contain γ-Glu-Val-Gly.
In this study, the distribution of γ-Glu-Val-Gly in various foods was investigated. Because the contents of γ-Glu-Val-Gly were very low, a new method for the determination and quantification of this peptide using LC/MS/MS after derivatization with 6-aminoqui-noyl-N-hydroxysuccinimidyl-carbamate (AQC) reagent, which is a representative amino-group derivatizing reagent (Cornelius et al., 1995), was developed. In the present study, the presence and quantity of γ-Glu-Val-Gly in several kinds of fermented shrimp paste condiments was investigated.
Materials and Methods
Chemicals γ-Glutamyl-valyl-glycine was chemically synthesized as previously reported (Ohsu et al., 2010). The stable isotope of 15N-uniformly labeled L-Arg (Arg-UN) was purchased from Isotec (Tokyo, Japan). An AccQ Fluor reagent kit was acquired from Waters (Milford, MA, USA). HPLC grade acetonitrile (Junsei Chemicals Co., Ltd., Osaka, Japan) and formic acid (99%; Wako Pure Chemical Industries Ltd., Osaka, Japan) were used for the mobile phase. Deionized water was prepared using a Milli-Q system (Millipore, Billerica, MA, USA).
Fermented shrimp paste Filipino fermented shrimp paste condiment (Bagoong), Indonesian fermented shrimp paste (Terasi) and Chinese fermented shrimp paste(Xiajiang) were purchased at a local market in 2008. The raw materials of Terasi and Xiajiang were shrimp and salt, while those of Bagoong were shrimp, salt, palm oil, vinegar, onion, garlic and sugar. All samples were stored at 5°C until analysis.
Sample preparation and derivatization procedure A sample of Bagoong (10.29 g) or Xiajiang (10.06 g) was added to 100 mL of deionized water, and the solution was filtered through a 0.45 μm membrane filter (GD/X Syringe Filters; Whatman PLC, Maidstone, UK), to remove any insoluble matter. The filtered solutions were further treated using an Amicon Ultra Centrifugal Filter Device (regenerated Cellulose 10,000 MWCO, Millipore) at 7,500g for 15 min at 4°C. A sample of Terasi (5.02 g) was added to 50 mL of 0.012 N hydrochloric acid aqueous solution, and stirred for 20 min on a magnetic stirrer. Next, a 50-mL aliquot was centrifuged at 5,000g for 15 min at 4°C, and the resulting supernatant was filtered through a 0.45 μm membrane filter (GD/X Syringe Filters). The filtrate was further treated using an Amicon Ultra Centrifugal Filter Device at 7,500g for 15 min at 4°C. The samples were stored at −20°C before the derivatization step. The naturally occurring γ-Glu-Val-Gly in shrimp paste products was determined by derivatization with AQC reagent (AccQ Fluor reagent kit, Millipore) coupled with LC/MS/MS analysis. The AQC powder was dissolved in acetonitrile according to the supplier's protocol. The derivatization procedure was as follows. A 10-μL aliquot from each shrimp paste sample (described above) was mixed with a 20-μL internal standard solution containing 0.089 mg/dL Arg-UN. Next, 10 μL of standard γ-Glu-Val-Gly solution (for spiked samples) or deionized water (for unspiked samples) was added and vortexed. Then, the 10-μL aliquots of the above mixture and 10 μL of AQC/ acetonitrile solution were added to 30 μL of borate buffer (included in the AccQ Fluor reagent kit). Finally, the resulting 50-μL solutions were subsequently transferred into 1.5-mL microtubes, vortexed and heated at 55°C for 10 min on a block-heater. After cooling to room temperature, 100 μL of 0.1% aqueous formic acid was added to the reaction mixture.
Apparatus The analysis of γ-Glu-Val-Gly after derivatization was performed using an LC/MS/MS system according to the method reported previously (Kuroda et al., 2013). An Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, a degasser, an auto-sampler, and a column compartment was used for the separation. An AB SCIEX 3200 QTrap LC/MS/MS system (AB Sciex, Framingham, MA, USA) was used for detection. The turbo ion spray interface was operated in positive mode at 5500 V and 650°C. The MRM (multiple reaction monitoring) method was performed with a dwell time of 170 ms. Operations were controlled using the Analyst software (AB Sciex, version 1.4.2).
Identification and quantification of AQC-derivatized γ-Glu-Val-Gly Peak separation of AQC-derivatized γ-Glu-Val-Gly was carried out by reversed-phase high-performance liquid chromatography using a CAPCELL PAK C18 MG II column (2.0 mm ID x 100 mm, 3 μm; Shiseido, Tokyo, Japan). The column temperature was maintained at 40°C. Mobile phase A (MP A) consisted of aqueous 25 mM formic acid (pH 6.0, adjusted with aqueous ammonia solution), while mobile phase B (MP B) consisted of water/acetonitrile (40/60). The elution conditions were as follows: 0 min (15% MP B), 12 min (25% MP B), 12.1 – 14 min (100% MP B), and 14.1 – 20 min (15% MP B). The flow rate was 0.25 mL/min. A 20-μL aliquot of each derivatized sample was injected for the analysis. Peak identification of AQC-derivatized γ-Glu-Val-Gly was achieved using the MRM method with six MRM transition channels monitoring. The combination of precursor/product ions (Q1/Q3) and the collision energies (CE (V)) were 474.2/171.2 (51V), 474.2/145.3 (30V), 474.2/300.3 (30V), 474.2/229.4 (20V), 474.2/304.0 (20V) and 474.2/72.1 (50V). The concentration of AQC-derivatized γ-Glu-Val-Gly in the samples was determined based on a single-point standard addition approach at the most sensitive channel (474.2/171.2; Q1/Q3). The internal standard of Arg-UN was monitored in the MRM transition channel at 349.0/171.1 (Q1/Q3) with collision energy at 30V. The equation used was X = S x Ix / (Is-Ix), where X is the calculated γ-Glu-Val-Gly concentration in the sample, S is the spiked standard concentration, and Is and Ix are the relative peak intensities calculated from peak area against the internal standard peak (Arg-UN) for the spiked and unspiked samples, respectively.
General composition analysis Moisture levels were analyzed by measuring the change in weight after drying at 105°C for 5 h. Crude protein content was calculated by multiplying the total nitrogen content by 6.25. Total nitrogen content was determined by the micro-Kjeldahl method, while the crude fat content was determined by the Soxhlet extraction method using diethyl ether as the solvent. Ash levels were determined from the weight after heating at 550°C for 16 h. Sodium content was determined through atomic absorption spectrochemical analysis using a Spectro AA240FS spectrometer (Varian Technologies Japan Ltd., Tokyo, Japan).
Results and Discussion
Table 1 shows the general composition of various fermented shrimp paste condiments. Except for the Filipino shrimp paste condiment (Bagoong), the major component of the commercial fermented shrimp paste, besides water and salt, was crude protein. These results were consistent with the results reported previously (Mizutani et al., 1992; Surono and Hosono, 1994; Sumino et al., 1999; Montano et al., 2001). The major component of Filipino shrimp paste condiment (Bagoong) was crude fat. It was considered that the high content of fat was derived from the palm oil added as an ingredient. Since the Filipino fermented shrimp paste condiment (Bagoong) contained several other ingredients, its general composition differed from that of other samples.
Table 1.
Characteristics and general composition of various fermented shrimp paste condiments.
| Samples |
Country of Origin |
Contents of general components (%, w/w) |
| Moisture |
Crude protein |
Crude fat |
Ash |
Sodium |
NaCl* |
| Terasi |
Indonesia |
26.7 |
31.2 |
3.7 |
34.5 |
9.94 |
26.2 |
| Bagoong |
Philippines |
30.9 |
8.9 |
28.8 |
19.7 |
3.54 |
9.4 |
| Xiajiang |
China |
64.5 |
11.5 |
1.9 |
20.8 |
7.23 |
18.8 |
* NaCl content was calculated from the sodum content.
In our previous study, we established the method for γ-Glu-Val-Gly identification in food samples, and confirmed the presence of this peptide in raw and processed scallop products (Kuroda et al., 2012a), fish sauces (Kuroda et al., 2012b), and soy sauces (Kuroda et al., 2013). AQC-derivatization and multichannel MRM monitoring were adopted to increase retention capacity and separation ability for the highly hydrophilic γ-Glu-Val-Gly, as well as to increase sensitivity and selectivity for mass spectrometric detection. In this study, the same analytical approach was successfully implemented. Figure 1 (A) shows the mass chromatograms for the AQC-derivatized standard γ-Glu-Val-Gly, simultaneously monitored in six MRM transition channels, with the peak at 5.04 min corresponding to the AQC-derivatized γ-Glu-Val-Gly standard. Figure 1 (B) shows the typical chromatograms for fermented shrimp paste (Terasi). As was observed in the reported analyses of scallop and fish sauces, the complexity of the chromatograms made it difficult to confirm the existence of AQC-derivatized γ-Glu-Val-Gly. Hence, to confirm the existence of this peptide, the relative peak intensities at 5.04 min, obtained from six MRM transition channels, and corresponding to the standard γ-Glu-Val-Gly and Terasi analysis were compared. The relative peak intensities from Figure 1 (A) were 1.00/0.40/0.12/0.08/0.23/0.22, while those from Figure 1 (B) were 1.00/0.44/0.11/0.07/0.26/0.22. This good agreement indicates that the peak at 5.04 min in Figure 1 (B) was γ-Glu-Val-Gly. Figure 2 displays a summary of the results of the comparison of relative peak intensities from the analyses of the four fermented shrimp pastes and the standard γ-Glu-Val-Gly, and strongly supports that all the tested samples contained γ-Glu-Val-Gly.
The coefficient of variation (CV) of γ-Glu-Val-Gly contents in Bagoong (n = 3), which contained the most contaminant peaks of the samples tested, were determined across the six different MRM channels to identify the most appropriate channel for the quantification of this substance. CVs of 6.2%, 15.2%, 17.7%, 25.6%, 6.4%, and 34.5% were obtained for the Q1/Q3 MRM transition channels 474.2/171.2, 474.2/145.3, 474.2/300.3, 474.2/229.4, 474.3/304.0, and 474.2/72.1, respectively. Thus, the Q1/Q3 MRM transition channel of 474.2/171.2 was found to be the most sensitive of all of the channels tested in this study, and was consequently used to determine the concentration of γ-Glu-Val-Gly. In this method, the linearity of the peak-area ratio (γ-Glu-Val-Gly/Arg-UN) to the γ-Glu-Val-Gly concentration was verified at concentrations ranging from 0.003 to 1.5 mg/dL. The squared correlation coefficient (r2) was greater than 0.999. To overcome the matrix effect that sometimes affects analyses, the γ-Glu-Val-Gly concentration in the samples was calculated using a single-point standard addition approach (Kuroda et al., 2012a; Kuroda et al., 2012b; Kuroda et al., 2013), and the results are listed in Table 2. The recovery rates for Bagoong, Terasi and Xiajiang were 106.2%, 99.3%, and 111.2%, respectively
Table 2.
γ-Glu-Val-Gly content in the various fermented shrimp paste condiments.
| Samples |
Country of Origin |
Contents of γ-Glu-Val-Gly* (μg/g) |
| Terasi |
Indonesia |
5.2 ± 0.33 |
| Bagoong |
Philippines |
1.0 ± 0.06 |
| Xiajiang |
China |
0.9 ± 0.05 |
* Mean ± standard deviation (n=3)
The contents of γ-Glu-Val-Gly in various fermented shrimp paste condiments are indicated in Table 2. γ-Glu-Val-Gly was contained in all fermented shrimp paste condiments as follows: 5.2 μg/g, Indonesian fermented shrimp paste(Terasi); 1.0 μg/g, Filipino fermented shrimp paste condiment (Bagoong); and 0.9 μg/g, Chinese fermented shrimp paste (Xiajiang). These results indicate that γ-Glu-Val-Gly was most abundant in Terasi. The differences in the contents of this peptide likely reflect differences in shrimp species, microbial flora, and fermentation conditions during production. However, more investigations will be needed to clarify the reason for the variation in the γ-Glu-Val-Gly contents, especially the reason for the high content in Terasi.
In the present study, the kokumi peptide, γ-Glu-Val-Gly, was identified and quantified in several kinds of fermented shrimp paste condiments. Evaluation of the contribution of this peptide to the flavor of fermented shrimp paste is now in progress in our laboratory. The distribution of γ-Glu-Val-Gly in various foods is also currently being investigated.
Acknowledgements
We sincerely thank Dr. Kiyoshi Miwa, Dr. Tohru Kouda, and Mr. Hiroaki Takino of Ajinomoto Co., Inc., for their encouragement and continued support of this work. We are grateful to Ms. Yuko Iida, Dr. Yasuhisa Manabe, Dr. Seiichi Sato, Dr. Yutaka Maruyama, Dr. Yutaka Ishiwatari, and Dr. Takami Maekawa of Ajinomoto Co., Inc., for their valuable discussions and assistance.
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