- Email: [email protected]
Comparison of umami taste peptides in water-soluble extractions of Jinhua and Parma hams
LWT - Food Science and Technology 60 (2015) 1179e1186
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Comparison of umami taste peptides in water-soluble extractions of Jinhua and Parma hams* Yali Dang a, Xinchang Gao b, Fumin Ma c, Xueqian Wu a, * a
Institute of Health Food of Zhejiang Academy of Medical Sciences, Hangzhou 310013, China ACEA Biosciences Inc., Hangzhou, China c Food Science and Engineering Teaching and Research Section, School of Traditional Medicine, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2013 Received in revised form 30 August 2014 Accepted 1 September 2014 Available online 18 September 2014
Peptides are critical to the taste of dry-cured ham. To isolate and identify umami peptide fractions from the water-soluble extractions (WSEs) of Jinhua and Parma hams, separation procedures utilizing Sephadex G-25 and reversed-phase high-performance liquid chromatography (RP-HPLC) were combined with sensory evaluations (taste dilution analysis and an electronic tongue). The amino acid sequences of the umami peptides, Cys-Cys-Asn-Lys-Ser-Val (CCNKSV) from Jinhua ham and Ala-His-Ser-Val-Arg-PheTyr (AHSVRFY) from Parma ham, were identified by MALDI-TOF-MS; subsequently, both were synthesized, and their umami taste properties were evaluated. The results indicate that the tastes of the umami peptides were similar to those of the hams' WSEs. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ham Water-soluble extraction Umami taste Peptide
1. Introduction “Umami taste” is a Japanese concept meaning “savory” or “delicious” and is defined by the taste of its prototypical stimulus, monosodium L-glutamate (MSG) (Sentandeu et al., 2003). It has been found that the characteristics of umami taste can be induced by monosodium L-glutamate, sodium lactate or peptides. A large number of umami peptides have been isolated and identified, particularly dipeptides and tripeptides from cheese, soy sauce and miso (Smit et al., 2000), including Asp-Asp, Asp-Glu, Glu-Asp, GluGlu, Glu-Leu, Glu-Lys, Glu-Ser, Glu-Thr, Lys-Gly, Thr-Glu, and AlaAsp-Ala, Ala-Glu-Ala, Asp-Glu-Leu, Glu-Asp-Phe and Glu-Asp-Val. Additionally, it has been reported that a fraction of an enzymatic fish protein hydrolyzate (such as Glu-Asp-Glu, Asp-Glu-Ser, Thr-Glu and Ser-Glu-Glu) has similar sensory properties to sodium glutamate (Martin, Grigorov, Affolter, & Kochha, 2003). Glutamic acids containing di- and tri-peptides have been of particular interest because they are related to the unique taste of umami. Most studies have concluded that the glutamate-like taste of peptides stems from the high molar contents of glutamic acid and hydrophilic
* This manuscript was presented in the international conference of “Food Innova2012” Hangzhou, China, December 12e14, 2012. * Corresponding author. Tel.: þ86 571 88215480. E-mail addresses: [email protected], [email protected] (Y. Dang), [email protected] (X. Wu).
http://dx.doi.org/10.1016/j.lwt.2014.09.014 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
amino acid residues (Aristoy & Toldra, 1995; Tamura, Nakatsuka, Tada, Kawasaki, & Kikuchiokai, 1988). Another specific aspect of umami taste is its enhancement by 5’ribonucleotides inosine 5'-monophosphate and guanosine 5’monophosphate (Sforza et al., 2001; Sforza et al., 2006; Zhou & Zhao, 2005). g-L-glutamyl dipeptides have been found to enhance the umami sensation of matured cheese. The candidates, g-Glu-Glu, g-Glu-Gly, g-Glu-Gln, g-Glu-Met, g-Glu-Leu and g-Glu-His, have been identified as the key kokumi molecules enhancing mouthfulness and the complex taste continuity of matured cheese. The strength of the umami solution was enhanced by a 500e1000 Da fraction of hydrolyzed wheat gluten. Peptides from cooked pork loins exert a sourness-suppression effect. In addition, some peptides and other taste-active components (salt, monosodium glutamate, and acidity), in appropriate concentrations, could mutually strengthen their tastes. Such peptides might have little or no taste, and more than half contain the acidic amino acids, Glu or Asp (Blenford, 1994; Sforza, Bonim, Ruozi, Virgili, & Marchelli, 2003; Virginia & Orazio, 2000). Oligopeptides have also been considered to contribute to the characteristic taste of dry-cured ham. It was reported that the amount of glutamyl dipeptides remarkably increased during the extended aging of ham, acting as permanent taste-active compounds (Sforza et al., 2001). The peptide Glu-Tyr has been identified to be positively related to aged taste, which is a major accepted trait of dry-cured hams. This finding was in agreement with the role postulated for short
1180
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
peptides containing at least one glutamic acid, which were reported to be responsible for the onset of the so-called “umami taste”. However, this property is still controversial (Van den Oord & Van Wassenar, 1997). Leticia (Leticia et al., 2009) found the contents of carosine and anserine gradually increased during the processing of Serrano ham. According to reports for aged hams, the peptides, Val-Glu, Pro-Glu, Pro-Ala-Gln and Asn-Gly-Gly, have been shown to elicit umami taste (Ishibashi, et al., 1987). Various umami peptides have been found in different types of dry-cured hams, however, very little is known about the differences between umami peptides generated in Chinese hams and those of western hams. Many researchers have used gel filtration chromatography, reverse phase high performance liquid chromatography, tandem mass spectrometry, sensory evaluation analysis and electronic tongue to isolate and identify a large number of flavor peptides (Zhang, Wang, Liu, Xu, & Zhou, 2012). Thus, one objective of this study was to reveal the differences between the umami peptides of Chinese Jinhua ham and western Parma ham. Furthermore, because the taste characteristics of peptides are very complicated, their precise involvement in producing an umami taste in drycured ham remains unclear. Thus, umami peptides were isolated from Chinese Jinhua ham and a well-known western dry-cured meat product, Parma ham. The results of this study are useful for the quality improvement and the scientific production of traditional dry-cured hams. 2. Materials and methods 2.1. Samples Jinhua ham was provided by Jinzi ham CO., LTD. Parma ham was purchased in the Shanghai Jiuguang Supermarket and was produced by Prosciuttifcio IL Conte Company. The hams, with bones and fat removed, were frozen at 20 C. The water-soluble extraction (WSE) of Jinhua ham was obtained according to the method of Engel, et al., 2002, with some modification. Jinhua ham (100 g) was homogenized with five different types of solvents in a Stomacher homogenizer for 8 min and centrifuged at 2000 g for 20 min. The supernatant was filtered through glass wool and centrifuged further at 10,000 g and 4 C for 30 min, freeze-dried, and frozen at 20 C until analysis. (Dang, Wang, & Xu, 2008). Then, the extract was vacuum freeze-dried using a Consol 24 dryer (American Consol Company) and ultrafiltrated using a Vivaflow 50 (purchased from the Sartorius Group, Germany). The MSG standard was purchased from the Sigma-Aldrich company. 2.2. Separation and purification of the taste-active peptides of ham by gel filtration chromatography Sephadex G-25 gel filtration chromatograms of WSE fractions with molecular weights (MW) less than 5000 Da were obtained from analyzing the ham. Fractionation was performed using a 2.6 100-cm glass column at 4 C under a flow rate of 40 ml/h, with Milli-Q water as the eluant. The sample was prepared by adding 10 g WSE powder to 50 ml Milli-Q water. The loading volume was 5 ml, and the sensitivity was 1.0, with a detection wavelength of 220 nm. 2.3. Separation and purification of the taste-active peptides of ham by reversed phase high performance liquid chromatography (RPHPLC) The taste-active fractions, separated by gel filtration chromatography, were dissolved in water to generate a solution with a
Fig. 1. Purification of crude peptide in hams by Sephadex G-25. (A) Jinhua ham (B) Parma ham.
concentration of 1.0 mg/ml. This solution was injected into a RPHPLC (Waters Company, USA) with a preparative column (Hanbon Hedera ODS-2 C18, 10 250 mm). The separating conditions were as follows: injection volume, 10 ìL; flow rate, 1 ml/min; mobile phase, phase A: 0.05 ml/100 ml TFA aqueous solution, phase B: 0.05 ml/100 ml TFA methyl-cyanide solution; gradient elution procedure: 0e20 min, 10e20 ml/100 ml B, 20e25 min, 20e100 ml/ 100 ml B; column temperature, 30 C; and detection wavelength, 220 nm (using a Model Waters 2996 Diode Array Detector, Waters company, USA). Each fraction was collected and freeze-dried. Each analysis had three independent replicates (n ¼ 3). 2.4. Evaluation of the structure of the taste-active peptides by MALDI-TOF-MS analysis A sample of 0.5 mL was loaded on the MALDI stainless steel target plates of a MALDI-TOF-MS and an ESE-MS-MS (Japanese companies) and dried naturally; 0.5 mL a-L-4-hydroxycinnamic acid (CCA) solution (0.5 g/L, in 0.1 ml/100 ml TFA þ 50 ml/100 ml Table 1 Sensory evaluation of Sephadex G-25 elution in Jinhua ham and Parma ham (n ¼ 3). Ham
Sephadex G-25 elution
Taste dilution (TD) value
Taste description
Jinhua
I Ⅱ III IV V I Ⅱ III IV V Ⅵ
64 32 128 16 4 32 64 32 128 16 4
Strong sour and meaty Slight ham flavor Strong ham and umami Slight salty Slight sweet Slight ham and meaty flavor Strong ham taste and salty Slight salty flavor Strong ham taste, meaty and salty Slight salty and sweet flavor Slight flavor
Parma
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
1181
Fig. 2. Results of electronic tongue for Sephadex G-25 fraction of hams' WSE. (A) Jinhua ham (B) Parma ham.
methyl-cyanide) was added. The mixture was dried at room temperature. The control was 0.5 mL CCA solution (0.5 g/L) dripped on the plate (Hao & March, 2001). Mass spectrometry and sequence analysis: Using a 4700 tandem time-of-flight mass spectrometer for mass spectrometry (Japanese companies), the laser source was 355 nm Nd. For the YAG laser, the accelerating voltage was 20 kv, and the positive ion and automatic modes were adopted to collect the data. The instrument along with myoglobin protease solution was the peptide used for external standard calibration. The scanning range for the substrate and samples of the peptide mass fingerprint (PMF) was 700e3500 Da. For the MS/MS spectrum, the quality of the area under 150 Da was checked to determine the preliminary inference possible residue. The instrument software, 4700 Explorer's own analysis tools De Novo Explorer, was utilized for sequencing from scratch. After obtaining the sequence, the software Data Explorer was used on the MS/MS spectrum superscripts a, b, c, x, y, z and others, using the parent molecular ion after fracture. 2.5. Synthetic taste-active peptides The taste-active peptides were synthesized by Jier Biochemistry Corporation (Shanghai) with a purity of over 98 g/100 g. Solid phase peptide synthesis has evolved over three decades into a tremendously powerful method for preparing peptides and small proteins. An absolute prerequisite for successful syntheses in all solid phase
schemes is that reactions that accumulate solid supported products must proceed cleanly and efficiently because the very nature of the technique contaminates the by-products, (Steven, 2008). 2.6. Sensory evaluation The taste dilution analysis (TDA) was performed according to the method of reference (Frank, Ottinger, & Hofmann, 2001), with slight modification. Samples were diluted stepwise 1:1 with deodorized distilled water. These solutions were submitted to several trained inspectors to increase the concentration, and these solutions were evaluated at all concentration levels using the three-probe ultrasonic method. When the taste difference between a solution at a certain concentration level and two glasses of water could be distinguished, the dilution times or the dilution level, expressed as the taste dilution (TD) value, was recorded. The TD value was the average value of the evaluation results provided by the inspectors. The differences among the results should be less than or equal to one of the dilution levels (Kim & Lee, 2003). 2.7. Evaluation profile of the taste by the electronic tongue The measurement was performed in a three-electrode cell, located inside a Faraday cage composed of aluminum sheets. The standard three-electrode system consisted of a platinum electrode
1182
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
Fig. 3. Elution profile of G-25-J3 by RPeHPLC. (A) Jinhua ham G-25-J3. (B) Parma ham G-25-P4.
(F2 mm), gold electrode, palladium electrode, titanium electrode and wolfram electrode, which were used as the work electrodes, a platinum pole electrode (1 5 mm) as the auxiliary electrode, and Ag/AgCl as the reference electrode. The concentration of the KCl solution was 3 mol/L, and the conductivity of distilled water was above 18 MU. The reference solution consisted of 1.5 g/100 g monosodium glutamate (MSG) and 0.5 g/100 g NaCl (Bagnasco, et. al., 2014). Electrochemical measurements were performed in nondeoxygenated samples equilibrated at room temperature (23 ± 1 C). Cyclic voltammograms (CVs) were recorded by applying the following setup parameters: initial potential (Ei) ¼ 0.0 V; low potential E ¼ 1.0 V; high potential E ¼ þ 2.0 V; final potential (Ef) ¼ 0.0 V; initial scan polarity negative; and scan rate ¼ 50 mV. For each sample, a total of three replicate measurements were recorded non-consecutively, in a random order, and averaged. The taste-active fractions of the ham were tested at room temperature, 50 ml samples were taken and every portion was tested three times (n ¼ 3). The electronic tongue was rinsed with de-ionized water between measurements. Solutions with 80 ml of each of the nine peptide fractions (1 mg/ml) (including one ultrafiltration fraction that represented the taste of the synthesized peptides of Jinhua and Parma hams), 12 mM sodium chloride (NaCl), and 4 mM monosodium glutamate (MSG) combined with 12 mM sodium chloride (NaCl), were prepared four times in random order. Each of the samples was measured using seven replications by each of the seven sensors, and the last three replications were averaged into one point, totaling four points for each sample in the score plot (Zhang, et al, 2012).
3. Results and discussion 3.1. The separation of the taste-active peptide of ham WSE using the Sephadex G-25 In Fig. 1A, five peaks were isolated by the Sephadex G-25 from the WSE of Jinhua ham and named G-25-J1, G-25-J2, G-25-J3, G-25J4, G-25-J5, corresponding to peaks IeV. The relative proportion was 11.2∶36.5∶22.4∶6.3∶9.6, and the crude peptide recovery was 94 g/100 g. In Fig. 1B, six peaks were isolated by Sephadex G-25 from the WSE of Parma ham and named G-25-P1, G-25-P2, G-25-P3, G-25P4, G-25-P5, G-25-P6, corresponding to peaks I-VI. The relative proportion was 7.2∶3.5∶9.8∶30.7∶39.2∶9.6, and the crude peptide recovery was 92 g/100 g. All elutions profiles fraction peaks were collected to determine their taste profiles. Fractions referred to the elutions profiles in Fig. 1. 3.2. The taste characteristics of the fraction peaks in ham WSE by Sephadex G-25 The results of sensory evaluation of the fraction peaks in the ham WSEs are shown in Table 1. The results of electronic tongue analysis are shown in Fig. 2. As shown in Table 1, elution III had the strongest umami taste for Jinhua ham, with a TD value up to 128. The taste of elution III was similar to the WSE (Fig. 2A). Combined with the results of the sensory evaluation, elution III (G-25-J3) in the WSE of Jinhua ham was selected and further purified.
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
1183
Fig. 4. Spectrum of the precursor ion spectrum of the WSE in Jinhua ham and Parma ham. (A) MS of G-25-J3-r1 (m/z 653.4), (B) MS/MS of G-25-J3-r1 (m/z 653.4), (C) MS of G-25-P4r1 (m/z 879.4914), (D) MS/MS of G-25-P4-r1 (m/z 879.4914).
1184
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
Fig. 5. Taste profile of synthetic peptides by the electronic tongue. (A) peptide (concentration: 1 mg/ml) and MSG, (B) peptide solution containing 0.6 g/100 g NaCl and MSG, (C) peptide (concentration: 1 mg/ml) and the WSE from Jinhua ham and Parm ham.
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
As shown in Table 1, the elution IV had the strongest umami taste for Parma ham, with a TD value up to 128. The elution IV was closest to the Parma ham WSE (Fig. 2B). Combining these data with the results of the sensory evaluation, elution IV (G-25-P4) was selected and purified for the further studies. 3.3. The taste-active peptide in ham WSE purified by RP-HPLC As shown in Fig. 3A, peak r1 was the maximum peak of G-25-J3 of the Jinhua ham, and the retention time was 5.25 min. As shown in Fig. 3B, peak1 was the maximum peak of G-25-P4 of the Parma ham, and the retention time was 4.95 min. Both were collected and lyophilized for MS analysis because the other peaks were too difficult to collect. 3.4. The structure of the taste-active peptide in ham WSEs The fingerprints of G-25-J3-r1 and G-25-P4-r1 were determined by MALDI-TOF-MS (Fig. 4A and C). The strongest intensity ions were selected for MS/MS analysis, as shown in Fig. 4 (B, D). As shown Figs. 4 and 1, the short peptides had molecular weights less than 1000 Da. The main ion m/z of the peptide from G-25-J3-r1 was 653.4, and the main ion m/z of the peptide from G-25-P4-r1 was 879.5. Their amino acid sequences were Cys-Cys-Asn-Lys-Ser-Val (CCNKSV), Ala-His-Ser-Val-Arg-Phe-Tyr (AHSVRFY), respectively, in the MS/MS analysis. 3.5. The taste characteristics of synthetic peptides in ham WSEs MSG is considered to be representative of umami taste, so we employed an MSG solution as the reference for the umami evaluation. To verify the taste profile of G-25-J3-r1 and G-25-P4-r1, the peptides CCNKSV and AHSVRFY were synthesized using the solid phase method (GL Biochem. Comp., Shanghai) and then determined using an electronic tongue (Alpha MOS company, France). The taste differences between CCNKSV, AHSVRFY and MSG are shown in Fig. 5A, which might be why the umami taste of the peptides differs from that of MSG's umami. The umami taste was shown to be closest to the MSG's umami taste in the presence of 0.6 g/100 g salt solution (Fig. 5B). The tastes of CCNKSV and AHSVRFY were shown to be very similar to that of the ham WSEs (Fig. 5C). The main difference of isolated peptides from Jinhu ham and Parma ham on umami tase is the peptides' primary structure, so as to the length and amino acid sequence of peptides. The hydrolysis of meat proteins generates polypeptides that can be further degraded to smaller peptides. This proteolytic activity can affect the final flavor of the product (Santos, Sanz, Bolumar, Aristoy, & Toldra, 2001). The endogenous cleavage sites of a bioactive peptide depend on proteolysis. It also addresses the subsequent biochemical purification of a candidate peptidase on the basis of these cleavage sites and the validation of the candidate peptidase's role in the physiological regulation of the bioactive peptide. The taste from peptide CCNKSV (Cys-Cys-Asn-Lys-Ser-Val) was similar to that of umami based on the analysis of the electronic tongue, which might be attributed to the Asn residue presented in the sequence. The result was in agreement with published reports that peptides containing Glu or Asp residues are essential in most umami peptide fractions, such as low-molecular-weight fractions (less than 500 Da) in two types of traditional Japanese soy sauce called Koikuchi and tamari shoyu (Hanifah, Takara, & Yasuda, 2006). Furthermore, the peptide AHSVRFY had a stronger umami taste than did the peptide CCNKSV, according to the electronic tongue analysis. Wang (Wang et al., 2012) reported that
1185
nine dipeptides and ten tripeptides had been identified. Among them, Val-Glu, Pro-Glu, Pro-Ala-Gln and Asn-Gly-Gly showed a umami taste, whereas Ala-His, His-Pro, Val-Tyr and Leu-His (consisting of hydrophobic amino acid and alkaline amino acid) might have a umami taste. For AHSVRFY (Ala-His-Ser-Val-ArgPhe-Tyr) contains Ala-His, Val, His and Tyr, which might contribute to its umami taste. The molecular mechanism of the umami taste was studied by Li and colleagues, who examined the molecular mechanism of the synergy using chimeric T1R receptors, site-directed mutagenesis, and molecular modeling (Zhang, Klebansky, Fine, & Xu, 2008). They proposed a cooperative ligand-binding model involving the Venus flytrap domain of T1R1, where L-glutamate binds close to the hinge region and 50 ribonucleotides bind to an adjacent site close to the opening of the flytrap, to further stabilize the closed conformation. Further investigation into the relationship between the structure of peptides and umami taste is required. Endogenous exo- and endo-peptidase activities, together with processing parameters (salt amount, aging temperature and length of the aging phase), played an important role in the degree of proteolysis, yielding different amounts, and in the patterns of peptides (Pearson, Wolzak, & Gray, 1983). Chinese Jinhua ham is produced under high salt and high temperature conditions. In contrast, western Parma ham is produced under low salt and low temperature conditions. Although the processing conditions of these two types of the ham are different, the key technologies employed during the entire process are similar, including curing, fermentation, drying and ripening. The enzymatic reaction occurring during the processing of dry-cured hams provided a thorough understanding of the peptide generation within the process. This reaction involved the degradation of both sarcoplasmic and myofibrillar proteins by muscle endopeptidases (mainly by cathepsins and calpains) and the further degradation of the generated polypeptides through the action of certain groups of exopeptidases, such as dipeptidyl peptidases. Both of the peptides that we found contained six or seven amino acids, which suggested that peptidyl peptidases might be a key enzyme responsible for the generation of oligopeptides, which in turn contribute to the characteristic taste associated with Jinhua and Parma hams. It has been reported that dipeptidyl peptidase I (DPPI) might be a key enzyme responsible for the generation of dipeptides in Jinhua ham. Experiments in an in vitro environment showed that both DPPI and dipeptidyl peptidase IV (DPPIV) possessed significant activities in Chinese Jinhua ham during processing. Better knowledge regarding the enzymatic reactions is necessary for a better understanding of how the main enzymes are activated and how these reactions influence the taste generated during processing.
4. Conclusion In this study, two umami peptides from Jinhua ham and Parma ham were isolated, purified and identified by gel filtration chromatography, RP-HPLC chromatography and MALDI-TOF/TOF MS/ MS. The amino acid sequence of the G-25-J3-r1 was Cys-Cys-AsnLys-Ser-Val (CCNKSV), and the amino acid sequence of G-25-P4r1 was Ala-His-Ser-Val-Arg-Phe-Tyr (AHSVRFY). The two peptides were further synthesized, and the synthesized peptides were found to have a similar taste to the ham WSEs because they were synthesized based on the sequence of the amino acid from the ham WSEs. We found that both had a similar taste to the ham WSE, whereas the umami taste was not similar to the MSG standard using electronic tongue analysis.
1186
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
Acknowledgments This work was supported by the National Natural Science Foundation of China under Grant No.31101344 and by Zhejiang Provincial Natural Science Foundation of China under Grant No.Y3100320. References Aristoy, M. C., & Toldra, F. (1995). Isolation of flavour peptides from raw pork meat and dry-cured ham. Developments in Food Science, 37, 1323e1344. Bagnasco, L., Cosulich, M., Speranza, G., Medini, L., Oliveri, P., & Lanteri, S. (2014). Application of a voltammetric electronic tongue and near infrared spectroscopy for a rapid umami taste assessment. Food Chemistry, 157, 421e428. Blenford, D. (1994). Protein WSEs-functionalitied and uses in nutritional products. International Food Ingredients, 3, 45e49. Dang, Y. L., Wang, Z., & Xu, S. Y. (2008). Methods for extracting the taste compounds from water soluble extract of Jinhua ham. European Food Research and Technology, 228, 93e102. Engel, E., Lombardot, J. B., Garem, A., Leconte, N., Septier, C., LeQuere, J. L., et al. (2002). Fractionation of the water-soluble extract of a cheese made from goats' milk by filtration methods: behavior of fat and volatile compounds. International Dairy Journal, 12, 609e619. Frank, O., Ottinger, H., & Hofmann, T. (2001). Fraternization of an intense bittertasting 1H,4H-Quinolizinium-7-olate by application of the taste dilution analysis, a novel bioassay for the screening and identification of taste-active compounds in foods. Journal of Agriculture and Food Chemistry, 49, 231e238. Hanifah, N. L., Takara, K., & Yasuda, M. (2006). Evaluation of peptide contribution to the intense umami taste of Japanese soy sauces. Journal of Food Science, 17(3), 277e283. Hao, C. Y., & March, R. E. (2001). A survery of recent research activity in quadrupole ion trap mass spectrometry. International Journal of Mass Spectrum, 212, 337e357. Ishibashi, N., Sadamori, K., Yamamoto, O., Kanehisa, H., Kouge, K., Kikuchi, E., et al. (1987). Bitterness of phenylalanine and tyrosine-containing peptides. Agricultural and Biological Chemistry, 51(12), 3309e3313. Kim, S. H., & Lee, K. A. (2003). Evaluation of taste compounds in water-soluble extract of a doenjang (soybean paste). Food Chemistry, 83, 339e342. Leticia, M. S., Sentandreu, M. A., Koistinen, K. M., Fraser, P. D., Toldr a, F., & Bramley, P. M. (2009). Naturally generated small peptides derived from myofibrillar proteins in Serrano dry-cured ham. Journal of Agricultural and Food Chemistry, 57(8), 3228e3234. Martin, G., Grigorov, H. S. C., Affolter, M., & Kochha, S. (2003). Design of virtual libraries of umami-tasting molecules. Jounal of Chemistry and Information Computer Science, 43, 1248e1258.
Pearson, A. M., Wolzak, A. M., & Gray, J. I. (1983). Possible role of muscle protein in flavor and tenderness of meat. Journal of Food Biochemistry, 7, 189e210. Santos, N. N., Sanz, Y., Bolumar, T., Aristoy, M. C., & Toldra, F. (2001). Hydrolysis of pork muscle sarcoplasmic proteins by Debaryomyces hansenii. International Journal of Food Microbiology, 68(3), 199e206. , F. (2003). Sentandeu, M. A., Stoeva, S., Aristoy, M. C., Laib, K., Voelter, W., & Toldra Identification of small peptides generated in Spanish dry-cured ham. Journal of Food Science, 68, 64e69. Sforza, S., Bonim, Ruozi, R., Virgili, R., & Marchelli, R. (2003). Identification and significance of the N-terminal part of swine pyruvate kinase in aged Jinhua hams. Meat Science, 63, 57e61. Sforza, S., Galaverna, G., Schivazappa, C., Marchelli, R., Dossena, A., & Virgili, R. (2006). Effect of extended aging of Parma dry-cured ham on the content of oligopeptides and free amino acids. Journal of Agriculture and Food Chemistry, 54, 9422e9429. Sforza, S., Pigazzani, A., Motti, M., Porta, C., Virgili, R., & Galaverna, G. (2001). Oligopeptides and free amino acids in Jinhua hams of known cathepsin B activity. Food Chemistry, 75, 267e273. Smit, G., Verheul, A., Kranenburg, R., Ayad, E., Siezen, R., & Engels, W. (2000). Cheese flavor development by enzymatic conversions of peptides and amino acids. Food Research International, 33, 153e160. Steven, K. (2008). Development of new tags for solid-phase peptide synthesis. Basel: University of Basel. PhD Thesis. Tamura, M., Nakatsuka, T., Tada, M., Kawasaki, Y., & Kikuchiokai, H. (1988). The relationship between taste and primary structure of “delicious” peptide (LysGly-Asp-Glu-Glu-Ser-Leu-Ala) from beef soup. Agriculture and Biology Chemistry, 53, 319e325. Van den Oord, A. H. A., & Van Wassenar, P. D. (1997). Umami peptides: assessment of their alleged taste properties. Zeitschrift fur Lebensmittel Untersuchung und Forschung-A, 205, 125e130. Virginia, L., & Orazio, T. S. (Eds.). (2000). Flavor fragrance and chemistry [M] (p. 272). Newtherlands: Kluwer Academic publishers. Wang, J., Zhao, G. M., Zhang, J. W., Liu, Y. X., Li, M. Y., & Hu, D. H. (2012). Separation, purification and structural identification of small peptides from Jinhua Ham. Food Science, 33(20), 15e20 (Chinese). Zhang, F., Klebansky, B., Fine, R. M., & Xu, H. (2008). Molecular mechanism for the umami taste synergism. Proceedings of the National Academy of Sciences, 105(52), 20930e20934. Zhang, M. X., Wang, X. C., Liu, Y., Xu, X. L., & Zhou, G. H. (2012). Isolation and identification of flavour peptides from Puffer fish (Takifugu obscurus) muscle using an electronic tongue and MALDI-TOF/TOF MS/MS. Food Chemistry, 135, 1463e1470. Zhou, X. S., & Zhao, M. M. (2005). Study of the peptide's taste. China Condiment, 6, 38e42.
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Comparison of umami taste peptides in water-soluble extractions of Jinhua and Parma hams* Yali Dang a, Xinchang Gao b, Fumin Ma c, Xueqian Wu a, * a
Institute of Health Food of Zhejiang Academy of Medical Sciences, Hangzhou 310013, China ACEA Biosciences Inc., Hangzhou, China c Food Science and Engineering Teaching and Research Section, School of Traditional Medicine, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2013 Received in revised form 30 August 2014 Accepted 1 September 2014 Available online 18 September 2014
Peptides are critical to the taste of dry-cured ham. To isolate and identify umami peptide fractions from the water-soluble extractions (WSEs) of Jinhua and Parma hams, separation procedures utilizing Sephadex G-25 and reversed-phase high-performance liquid chromatography (RP-HPLC) were combined with sensory evaluations (taste dilution analysis and an electronic tongue). The amino acid sequences of the umami peptides, Cys-Cys-Asn-Lys-Ser-Val (CCNKSV) from Jinhua ham and Ala-His-Ser-Val-Arg-PheTyr (AHSVRFY) from Parma ham, were identified by MALDI-TOF-MS; subsequently, both were synthesized, and their umami taste properties were evaluated. The results indicate that the tastes of the umami peptides were similar to those of the hams' WSEs. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ham Water-soluble extraction Umami taste Peptide
1. Introduction “Umami taste” is a Japanese concept meaning “savory” or “delicious” and is defined by the taste of its prototypical stimulus, monosodium L-glutamate (MSG) (Sentandeu et al., 2003). It has been found that the characteristics of umami taste can be induced by monosodium L-glutamate, sodium lactate or peptides. A large number of umami peptides have been isolated and identified, particularly dipeptides and tripeptides from cheese, soy sauce and miso (Smit et al., 2000), including Asp-Asp, Asp-Glu, Glu-Asp, GluGlu, Glu-Leu, Glu-Lys, Glu-Ser, Glu-Thr, Lys-Gly, Thr-Glu, and AlaAsp-Ala, Ala-Glu-Ala, Asp-Glu-Leu, Glu-Asp-Phe and Glu-Asp-Val. Additionally, it has been reported that a fraction of an enzymatic fish protein hydrolyzate (such as Glu-Asp-Glu, Asp-Glu-Ser, Thr-Glu and Ser-Glu-Glu) has similar sensory properties to sodium glutamate (Martin, Grigorov, Affolter, & Kochha, 2003). Glutamic acids containing di- and tri-peptides have been of particular interest because they are related to the unique taste of umami. Most studies have concluded that the glutamate-like taste of peptides stems from the high molar contents of glutamic acid and hydrophilic
* This manuscript was presented in the international conference of “Food Innova2012” Hangzhou, China, December 12e14, 2012. * Corresponding author. Tel.: þ86 571 88215480. E-mail addresses: [email protected], [email protected] (Y. Dang), [email protected] (X. Wu).
http://dx.doi.org/10.1016/j.lwt.2014.09.014 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
amino acid residues (Aristoy & Toldra, 1995; Tamura, Nakatsuka, Tada, Kawasaki, & Kikuchiokai, 1988). Another specific aspect of umami taste is its enhancement by 5’ribonucleotides inosine 5'-monophosphate and guanosine 5’monophosphate (Sforza et al., 2001; Sforza et al., 2006; Zhou & Zhao, 2005). g-L-glutamyl dipeptides have been found to enhance the umami sensation of matured cheese. The candidates, g-Glu-Glu, g-Glu-Gly, g-Glu-Gln, g-Glu-Met, g-Glu-Leu and g-Glu-His, have been identified as the key kokumi molecules enhancing mouthfulness and the complex taste continuity of matured cheese. The strength of the umami solution was enhanced by a 500e1000 Da fraction of hydrolyzed wheat gluten. Peptides from cooked pork loins exert a sourness-suppression effect. In addition, some peptides and other taste-active components (salt, monosodium glutamate, and acidity), in appropriate concentrations, could mutually strengthen their tastes. Such peptides might have little or no taste, and more than half contain the acidic amino acids, Glu or Asp (Blenford, 1994; Sforza, Bonim, Ruozi, Virgili, & Marchelli, 2003; Virginia & Orazio, 2000). Oligopeptides have also been considered to contribute to the characteristic taste of dry-cured ham. It was reported that the amount of glutamyl dipeptides remarkably increased during the extended aging of ham, acting as permanent taste-active compounds (Sforza et al., 2001). The peptide Glu-Tyr has been identified to be positively related to aged taste, which is a major accepted trait of dry-cured hams. This finding was in agreement with the role postulated for short
1180
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
peptides containing at least one glutamic acid, which were reported to be responsible for the onset of the so-called “umami taste”. However, this property is still controversial (Van den Oord & Van Wassenar, 1997). Leticia (Leticia et al., 2009) found the contents of carosine and anserine gradually increased during the processing of Serrano ham. According to reports for aged hams, the peptides, Val-Glu, Pro-Glu, Pro-Ala-Gln and Asn-Gly-Gly, have been shown to elicit umami taste (Ishibashi, et al., 1987). Various umami peptides have been found in different types of dry-cured hams, however, very little is known about the differences between umami peptides generated in Chinese hams and those of western hams. Many researchers have used gel filtration chromatography, reverse phase high performance liquid chromatography, tandem mass spectrometry, sensory evaluation analysis and electronic tongue to isolate and identify a large number of flavor peptides (Zhang, Wang, Liu, Xu, & Zhou, 2012). Thus, one objective of this study was to reveal the differences between the umami peptides of Chinese Jinhua ham and western Parma ham. Furthermore, because the taste characteristics of peptides are very complicated, their precise involvement in producing an umami taste in drycured ham remains unclear. Thus, umami peptides were isolated from Chinese Jinhua ham and a well-known western dry-cured meat product, Parma ham. The results of this study are useful for the quality improvement and the scientific production of traditional dry-cured hams. 2. Materials and methods 2.1. Samples Jinhua ham was provided by Jinzi ham CO., LTD. Parma ham was purchased in the Shanghai Jiuguang Supermarket and was produced by Prosciuttifcio IL Conte Company. The hams, with bones and fat removed, were frozen at 20 C. The water-soluble extraction (WSE) of Jinhua ham was obtained according to the method of Engel, et al., 2002, with some modification. Jinhua ham (100 g) was homogenized with five different types of solvents in a Stomacher homogenizer for 8 min and centrifuged at 2000 g for 20 min. The supernatant was filtered through glass wool and centrifuged further at 10,000 g and 4 C for 30 min, freeze-dried, and frozen at 20 C until analysis. (Dang, Wang, & Xu, 2008). Then, the extract was vacuum freeze-dried using a Consol 24 dryer (American Consol Company) and ultrafiltrated using a Vivaflow 50 (purchased from the Sartorius Group, Germany). The MSG standard was purchased from the Sigma-Aldrich company. 2.2. Separation and purification of the taste-active peptides of ham by gel filtration chromatography Sephadex G-25 gel filtration chromatograms of WSE fractions with molecular weights (MW) less than 5000 Da were obtained from analyzing the ham. Fractionation was performed using a 2.6 100-cm glass column at 4 C under a flow rate of 40 ml/h, with Milli-Q water as the eluant. The sample was prepared by adding 10 g WSE powder to 50 ml Milli-Q water. The loading volume was 5 ml, and the sensitivity was 1.0, with a detection wavelength of 220 nm. 2.3. Separation and purification of the taste-active peptides of ham by reversed phase high performance liquid chromatography (RPHPLC) The taste-active fractions, separated by gel filtration chromatography, were dissolved in water to generate a solution with a
Fig. 1. Purification of crude peptide in hams by Sephadex G-25. (A) Jinhua ham (B) Parma ham.
concentration of 1.0 mg/ml. This solution was injected into a RPHPLC (Waters Company, USA) with a preparative column (Hanbon Hedera ODS-2 C18, 10 250 mm). The separating conditions were as follows: injection volume, 10 ìL; flow rate, 1 ml/min; mobile phase, phase A: 0.05 ml/100 ml TFA aqueous solution, phase B: 0.05 ml/100 ml TFA methyl-cyanide solution; gradient elution procedure: 0e20 min, 10e20 ml/100 ml B, 20e25 min, 20e100 ml/ 100 ml B; column temperature, 30 C; and detection wavelength, 220 nm (using a Model Waters 2996 Diode Array Detector, Waters company, USA). Each fraction was collected and freeze-dried. Each analysis had three independent replicates (n ¼ 3). 2.4. Evaluation of the structure of the taste-active peptides by MALDI-TOF-MS analysis A sample of 0.5 mL was loaded on the MALDI stainless steel target plates of a MALDI-TOF-MS and an ESE-MS-MS (Japanese companies) and dried naturally; 0.5 mL a-L-4-hydroxycinnamic acid (CCA) solution (0.5 g/L, in 0.1 ml/100 ml TFA þ 50 ml/100 ml Table 1 Sensory evaluation of Sephadex G-25 elution in Jinhua ham and Parma ham (n ¼ 3). Ham
Sephadex G-25 elution
Taste dilution (TD) value
Taste description
Jinhua
I Ⅱ III IV V I Ⅱ III IV V Ⅵ
64 32 128 16 4 32 64 32 128 16 4
Strong sour and meaty Slight ham flavor Strong ham and umami Slight salty Slight sweet Slight ham and meaty flavor Strong ham taste and salty Slight salty flavor Strong ham taste, meaty and salty Slight salty and sweet flavor Slight flavor
Parma
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
1181
Fig. 2. Results of electronic tongue for Sephadex G-25 fraction of hams' WSE. (A) Jinhua ham (B) Parma ham.
methyl-cyanide) was added. The mixture was dried at room temperature. The control was 0.5 mL CCA solution (0.5 g/L) dripped on the plate (Hao & March, 2001). Mass spectrometry and sequence analysis: Using a 4700 tandem time-of-flight mass spectrometer for mass spectrometry (Japanese companies), the laser source was 355 nm Nd. For the YAG laser, the accelerating voltage was 20 kv, and the positive ion and automatic modes were adopted to collect the data. The instrument along with myoglobin protease solution was the peptide used for external standard calibration. The scanning range for the substrate and samples of the peptide mass fingerprint (PMF) was 700e3500 Da. For the MS/MS spectrum, the quality of the area under 150 Da was checked to determine the preliminary inference possible residue. The instrument software, 4700 Explorer's own analysis tools De Novo Explorer, was utilized for sequencing from scratch. After obtaining the sequence, the software Data Explorer was used on the MS/MS spectrum superscripts a, b, c, x, y, z and others, using the parent molecular ion after fracture. 2.5. Synthetic taste-active peptides The taste-active peptides were synthesized by Jier Biochemistry Corporation (Shanghai) with a purity of over 98 g/100 g. Solid phase peptide synthesis has evolved over three decades into a tremendously powerful method for preparing peptides and small proteins. An absolute prerequisite for successful syntheses in all solid phase
schemes is that reactions that accumulate solid supported products must proceed cleanly and efficiently because the very nature of the technique contaminates the by-products, (Steven, 2008). 2.6. Sensory evaluation The taste dilution analysis (TDA) was performed according to the method of reference (Frank, Ottinger, & Hofmann, 2001), with slight modification. Samples were diluted stepwise 1:1 with deodorized distilled water. These solutions were submitted to several trained inspectors to increase the concentration, and these solutions were evaluated at all concentration levels using the three-probe ultrasonic method. When the taste difference between a solution at a certain concentration level and two glasses of water could be distinguished, the dilution times or the dilution level, expressed as the taste dilution (TD) value, was recorded. The TD value was the average value of the evaluation results provided by the inspectors. The differences among the results should be less than or equal to one of the dilution levels (Kim & Lee, 2003). 2.7. Evaluation profile of the taste by the electronic tongue The measurement was performed in a three-electrode cell, located inside a Faraday cage composed of aluminum sheets. The standard three-electrode system consisted of a platinum electrode
1182
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
Fig. 3. Elution profile of G-25-J3 by RPeHPLC. (A) Jinhua ham G-25-J3. (B) Parma ham G-25-P4.
(F2 mm), gold electrode, palladium electrode, titanium electrode and wolfram electrode, which were used as the work electrodes, a platinum pole electrode (1 5 mm) as the auxiliary electrode, and Ag/AgCl as the reference electrode. The concentration of the KCl solution was 3 mol/L, and the conductivity of distilled water was above 18 MU. The reference solution consisted of 1.5 g/100 g monosodium glutamate (MSG) and 0.5 g/100 g NaCl (Bagnasco, et. al., 2014). Electrochemical measurements were performed in nondeoxygenated samples equilibrated at room temperature (23 ± 1 C). Cyclic voltammograms (CVs) were recorded by applying the following setup parameters: initial potential (Ei) ¼ 0.0 V; low potential E ¼ 1.0 V; high potential E ¼ þ 2.0 V; final potential (Ef) ¼ 0.0 V; initial scan polarity negative; and scan rate ¼ 50 mV. For each sample, a total of three replicate measurements were recorded non-consecutively, in a random order, and averaged. The taste-active fractions of the ham were tested at room temperature, 50 ml samples were taken and every portion was tested three times (n ¼ 3). The electronic tongue was rinsed with de-ionized water between measurements. Solutions with 80 ml of each of the nine peptide fractions (1 mg/ml) (including one ultrafiltration fraction that represented the taste of the synthesized peptides of Jinhua and Parma hams), 12 mM sodium chloride (NaCl), and 4 mM monosodium glutamate (MSG) combined with 12 mM sodium chloride (NaCl), were prepared four times in random order. Each of the samples was measured using seven replications by each of the seven sensors, and the last three replications were averaged into one point, totaling four points for each sample in the score plot (Zhang, et al, 2012).
3. Results and discussion 3.1. The separation of the taste-active peptide of ham WSE using the Sephadex G-25 In Fig. 1A, five peaks were isolated by the Sephadex G-25 from the WSE of Jinhua ham and named G-25-J1, G-25-J2, G-25-J3, G-25J4, G-25-J5, corresponding to peaks IeV. The relative proportion was 11.2∶36.5∶22.4∶6.3∶9.6, and the crude peptide recovery was 94 g/100 g. In Fig. 1B, six peaks were isolated by Sephadex G-25 from the WSE of Parma ham and named G-25-P1, G-25-P2, G-25-P3, G-25P4, G-25-P5, G-25-P6, corresponding to peaks I-VI. The relative proportion was 7.2∶3.5∶9.8∶30.7∶39.2∶9.6, and the crude peptide recovery was 92 g/100 g. All elutions profiles fraction peaks were collected to determine their taste profiles. Fractions referred to the elutions profiles in Fig. 1. 3.2. The taste characteristics of the fraction peaks in ham WSE by Sephadex G-25 The results of sensory evaluation of the fraction peaks in the ham WSEs are shown in Table 1. The results of electronic tongue analysis are shown in Fig. 2. As shown in Table 1, elution III had the strongest umami taste for Jinhua ham, with a TD value up to 128. The taste of elution III was similar to the WSE (Fig. 2A). Combined with the results of the sensory evaluation, elution III (G-25-J3) in the WSE of Jinhua ham was selected and further purified.
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
1183
Fig. 4. Spectrum of the precursor ion spectrum of the WSE in Jinhua ham and Parma ham. (A) MS of G-25-J3-r1 (m/z 653.4), (B) MS/MS of G-25-J3-r1 (m/z 653.4), (C) MS of G-25-P4r1 (m/z 879.4914), (D) MS/MS of G-25-P4-r1 (m/z 879.4914).
1184
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
Fig. 5. Taste profile of synthetic peptides by the electronic tongue. (A) peptide (concentration: 1 mg/ml) and MSG, (B) peptide solution containing 0.6 g/100 g NaCl and MSG, (C) peptide (concentration: 1 mg/ml) and the WSE from Jinhua ham and Parm ham.
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
As shown in Table 1, the elution IV had the strongest umami taste for Parma ham, with a TD value up to 128. The elution IV was closest to the Parma ham WSE (Fig. 2B). Combining these data with the results of the sensory evaluation, elution IV (G-25-P4) was selected and purified for the further studies. 3.3. The taste-active peptide in ham WSE purified by RP-HPLC As shown in Fig. 3A, peak r1 was the maximum peak of G-25-J3 of the Jinhua ham, and the retention time was 5.25 min. As shown in Fig. 3B, peak1 was the maximum peak of G-25-P4 of the Parma ham, and the retention time was 4.95 min. Both were collected and lyophilized for MS analysis because the other peaks were too difficult to collect. 3.4. The structure of the taste-active peptide in ham WSEs The fingerprints of G-25-J3-r1 and G-25-P4-r1 were determined by MALDI-TOF-MS (Fig. 4A and C). The strongest intensity ions were selected for MS/MS analysis, as shown in Fig. 4 (B, D). As shown Figs. 4 and 1, the short peptides had molecular weights less than 1000 Da. The main ion m/z of the peptide from G-25-J3-r1 was 653.4, and the main ion m/z of the peptide from G-25-P4-r1 was 879.5. Their amino acid sequences were Cys-Cys-Asn-Lys-Ser-Val (CCNKSV), Ala-His-Ser-Val-Arg-Phe-Tyr (AHSVRFY), respectively, in the MS/MS analysis. 3.5. The taste characteristics of synthetic peptides in ham WSEs MSG is considered to be representative of umami taste, so we employed an MSG solution as the reference for the umami evaluation. To verify the taste profile of G-25-J3-r1 and G-25-P4-r1, the peptides CCNKSV and AHSVRFY were synthesized using the solid phase method (GL Biochem. Comp., Shanghai) and then determined using an electronic tongue (Alpha MOS company, France). The taste differences between CCNKSV, AHSVRFY and MSG are shown in Fig. 5A, which might be why the umami taste of the peptides differs from that of MSG's umami. The umami taste was shown to be closest to the MSG's umami taste in the presence of 0.6 g/100 g salt solution (Fig. 5B). The tastes of CCNKSV and AHSVRFY were shown to be very similar to that of the ham WSEs (Fig. 5C). The main difference of isolated peptides from Jinhu ham and Parma ham on umami tase is the peptides' primary structure, so as to the length and amino acid sequence of peptides. The hydrolysis of meat proteins generates polypeptides that can be further degraded to smaller peptides. This proteolytic activity can affect the final flavor of the product (Santos, Sanz, Bolumar, Aristoy, & Toldra, 2001). The endogenous cleavage sites of a bioactive peptide depend on proteolysis. It also addresses the subsequent biochemical purification of a candidate peptidase on the basis of these cleavage sites and the validation of the candidate peptidase's role in the physiological regulation of the bioactive peptide. The taste from peptide CCNKSV (Cys-Cys-Asn-Lys-Ser-Val) was similar to that of umami based on the analysis of the electronic tongue, which might be attributed to the Asn residue presented in the sequence. The result was in agreement with published reports that peptides containing Glu or Asp residues are essential in most umami peptide fractions, such as low-molecular-weight fractions (less than 500 Da) in two types of traditional Japanese soy sauce called Koikuchi and tamari shoyu (Hanifah, Takara, & Yasuda, 2006). Furthermore, the peptide AHSVRFY had a stronger umami taste than did the peptide CCNKSV, according to the electronic tongue analysis. Wang (Wang et al., 2012) reported that
1185
nine dipeptides and ten tripeptides had been identified. Among them, Val-Glu, Pro-Glu, Pro-Ala-Gln and Asn-Gly-Gly showed a umami taste, whereas Ala-His, His-Pro, Val-Tyr and Leu-His (consisting of hydrophobic amino acid and alkaline amino acid) might have a umami taste. For AHSVRFY (Ala-His-Ser-Val-ArgPhe-Tyr) contains Ala-His, Val, His and Tyr, which might contribute to its umami taste. The molecular mechanism of the umami taste was studied by Li and colleagues, who examined the molecular mechanism of the synergy using chimeric T1R receptors, site-directed mutagenesis, and molecular modeling (Zhang, Klebansky, Fine, & Xu, 2008). They proposed a cooperative ligand-binding model involving the Venus flytrap domain of T1R1, where L-glutamate binds close to the hinge region and 50 ribonucleotides bind to an adjacent site close to the opening of the flytrap, to further stabilize the closed conformation. Further investigation into the relationship between the structure of peptides and umami taste is required. Endogenous exo- and endo-peptidase activities, together with processing parameters (salt amount, aging temperature and length of the aging phase), played an important role in the degree of proteolysis, yielding different amounts, and in the patterns of peptides (Pearson, Wolzak, & Gray, 1983). Chinese Jinhua ham is produced under high salt and high temperature conditions. In contrast, western Parma ham is produced under low salt and low temperature conditions. Although the processing conditions of these two types of the ham are different, the key technologies employed during the entire process are similar, including curing, fermentation, drying and ripening. The enzymatic reaction occurring during the processing of dry-cured hams provided a thorough understanding of the peptide generation within the process. This reaction involved the degradation of both sarcoplasmic and myofibrillar proteins by muscle endopeptidases (mainly by cathepsins and calpains) and the further degradation of the generated polypeptides through the action of certain groups of exopeptidases, such as dipeptidyl peptidases. Both of the peptides that we found contained six or seven amino acids, which suggested that peptidyl peptidases might be a key enzyme responsible for the generation of oligopeptides, which in turn contribute to the characteristic taste associated with Jinhua and Parma hams. It has been reported that dipeptidyl peptidase I (DPPI) might be a key enzyme responsible for the generation of dipeptides in Jinhua ham. Experiments in an in vitro environment showed that both DPPI and dipeptidyl peptidase IV (DPPIV) possessed significant activities in Chinese Jinhua ham during processing. Better knowledge regarding the enzymatic reactions is necessary for a better understanding of how the main enzymes are activated and how these reactions influence the taste generated during processing.
4. Conclusion In this study, two umami peptides from Jinhua ham and Parma ham were isolated, purified and identified by gel filtration chromatography, RP-HPLC chromatography and MALDI-TOF/TOF MS/ MS. The amino acid sequence of the G-25-J3-r1 was Cys-Cys-AsnLys-Ser-Val (CCNKSV), and the amino acid sequence of G-25-P4r1 was Ala-His-Ser-Val-Arg-Phe-Tyr (AHSVRFY). The two peptides were further synthesized, and the synthesized peptides were found to have a similar taste to the ham WSEs because they were synthesized based on the sequence of the amino acid from the ham WSEs. We found that both had a similar taste to the ham WSE, whereas the umami taste was not similar to the MSG standard using electronic tongue analysis.
1186
Y. Dang et al. / LWT - Food Science and Technology 60 (2015) 1179e1186
Acknowledgments This work was supported by the National Natural Science Foundation of China under Grant No.31101344 and by Zhejiang Provincial Natural Science Foundation of China under Grant No.Y3100320. References Aristoy, M. C., & Toldra, F. (1995). Isolation of flavour peptides from raw pork meat and dry-cured ham. Developments in Food Science, 37, 1323e1344. Bagnasco, L., Cosulich, M., Speranza, G., Medini, L., Oliveri, P., & Lanteri, S. (2014). Application of a voltammetric electronic tongue and near infrared spectroscopy for a rapid umami taste assessment. Food Chemistry, 157, 421e428. Blenford, D. (1994). Protein WSEs-functionalitied and uses in nutritional products. International Food Ingredients, 3, 45e49. Dang, Y. L., Wang, Z., & Xu, S. Y. (2008). Methods for extracting the taste compounds from water soluble extract of Jinhua ham. European Food Research and Technology, 228, 93e102. Engel, E., Lombardot, J. B., Garem, A., Leconte, N., Septier, C., LeQuere, J. L., et al. (2002). Fractionation of the water-soluble extract of a cheese made from goats' milk by filtration methods: behavior of fat and volatile compounds. International Dairy Journal, 12, 609e619. Frank, O., Ottinger, H., & Hofmann, T. (2001). Fraternization of an intense bittertasting 1H,4H-Quinolizinium-7-olate by application of the taste dilution analysis, a novel bioassay for the screening and identification of taste-active compounds in foods. Journal of Agriculture and Food Chemistry, 49, 231e238. Hanifah, N. L., Takara, K., & Yasuda, M. (2006). Evaluation of peptide contribution to the intense umami taste of Japanese soy sauces. Journal of Food Science, 17(3), 277e283. Hao, C. Y., & March, R. E. (2001). A survery of recent research activity in quadrupole ion trap mass spectrometry. International Journal of Mass Spectrum, 212, 337e357. Ishibashi, N., Sadamori, K., Yamamoto, O., Kanehisa, H., Kouge, K., Kikuchi, E., et al. (1987). Bitterness of phenylalanine and tyrosine-containing peptides. Agricultural and Biological Chemistry, 51(12), 3309e3313. Kim, S. H., & Lee, K. A. (2003). Evaluation of taste compounds in water-soluble extract of a doenjang (soybean paste). Food Chemistry, 83, 339e342. Leticia, M. S., Sentandreu, M. A., Koistinen, K. M., Fraser, P. D., Toldr a, F., & Bramley, P. M. (2009). Naturally generated small peptides derived from myofibrillar proteins in Serrano dry-cured ham. Journal of Agricultural and Food Chemistry, 57(8), 3228e3234. Martin, G., Grigorov, H. S. C., Affolter, M., & Kochha, S. (2003). Design of virtual libraries of umami-tasting molecules. Jounal of Chemistry and Information Computer Science, 43, 1248e1258.
Pearson, A. M., Wolzak, A. M., & Gray, J. I. (1983). Possible role of muscle protein in flavor and tenderness of meat. Journal of Food Biochemistry, 7, 189e210. Santos, N. N., Sanz, Y., Bolumar, T., Aristoy, M. C., & Toldra, F. (2001). Hydrolysis of pork muscle sarcoplasmic proteins by Debaryomyces hansenii. International Journal of Food Microbiology, 68(3), 199e206. , F. (2003). Sentandeu, M. A., Stoeva, S., Aristoy, M. C., Laib, K., Voelter, W., & Toldra Identification of small peptides generated in Spanish dry-cured ham. Journal of Food Science, 68, 64e69. Sforza, S., Bonim, Ruozi, R., Virgili, R., & Marchelli, R. (2003). Identification and significance of the N-terminal part of swine pyruvate kinase in aged Jinhua hams. Meat Science, 63, 57e61. Sforza, S., Galaverna, G., Schivazappa, C., Marchelli, R., Dossena, A., & Virgili, R. (2006). Effect of extended aging of Parma dry-cured ham on the content of oligopeptides and free amino acids. Journal of Agriculture and Food Chemistry, 54, 9422e9429. Sforza, S., Pigazzani, A., Motti, M., Porta, C., Virgili, R., & Galaverna, G. (2001). Oligopeptides and free amino acids in Jinhua hams of known cathepsin B activity. Food Chemistry, 75, 267e273. Smit, G., Verheul, A., Kranenburg, R., Ayad, E., Siezen, R., & Engels, W. (2000). Cheese flavor development by enzymatic conversions of peptides and amino acids. Food Research International, 33, 153e160. Steven, K. (2008). Development of new tags for solid-phase peptide synthesis. Basel: University of Basel. PhD Thesis. Tamura, M., Nakatsuka, T., Tada, M., Kawasaki, Y., & Kikuchiokai, H. (1988). The relationship between taste and primary structure of “delicious” peptide (LysGly-Asp-Glu-Glu-Ser-Leu-Ala) from beef soup. Agriculture and Biology Chemistry, 53, 319e325. Van den Oord, A. H. A., & Van Wassenar, P. D. (1997). Umami peptides: assessment of their alleged taste properties. Zeitschrift fur Lebensmittel Untersuchung und Forschung-A, 205, 125e130. Virginia, L., & Orazio, T. S. (Eds.). (2000). Flavor fragrance and chemistry [M] (p. 272). Newtherlands: Kluwer Academic publishers. Wang, J., Zhao, G. M., Zhang, J. W., Liu, Y. X., Li, M. Y., & Hu, D. H. (2012). Separation, purification and structural identification of small peptides from Jinhua Ham. Food Science, 33(20), 15e20 (Chinese). Zhang, F., Klebansky, B., Fine, R. M., & Xu, H. (2008). Molecular mechanism for the umami taste synergism. Proceedings of the National Academy of Sciences, 105(52), 20930e20934. Zhang, M. X., Wang, X. C., Liu, Y., Xu, X. L., & Zhou, G. H. (2012). Isolation and identification of flavour peptides from Puffer fish (Takifugu obscurus) muscle using an electronic tongue and MALDI-TOF/TOF MS/MS. Food Chemistry, 135, 1463e1470. Zhou, X. S., & Zhao, M. M. (2005). Study of the peptide's taste. China Condiment, 6, 38e42.