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Food Chemistry

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Identification of a novel umami peptide in tempeh (Indonesian fermentedsoybean) and its binding mechanism to the umami receptor T1R

Muhamad Nur Ghoyatul Amina,b,1, Joni Kusnadib, Jue-Liang Hsua,⁎, Robert J. Doerksenc,⁎,Tzou-Chi Huanga,⁎

a Department of Biological Science and Technology, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, TaiwanbDepartment of Agricultural Product Technology, Faculty of Agricultural Technology, Brawijaya University, Veteran Street, Malang 65145, East Java, Indonesiac Department of BioMolecular Sciences and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, 209 Graduate House, University,MS, 38677, USA

A R T I C L E I N F O

Keywords:TempehUmamiPeptideT1R familyLC-MS/MSDocking

A B S T R A C T

Tempeh, a traditional Indonesian soybean product produced by fermentation, is especially popular because of itsumami taste. In this study, a novel umami peptide GENEEEDSGAIVTVK (GK-15) was identified in the smallpeptide (< 3 kDa) fraction of the water extract of tempeh using LC-MS/MS analysis and database-assistedidentification. The umami taste of GK-15 was further validated using sensory evaluation, which suggested thatGK-15 may be one of the key components contributing to the umami taste in tempeh. To rationalize the bio-logical effect of GK-15, molecular docking of GK-15 into the N-terminal extracellular ligand-binding domain ofthe umami (T1R) receptor was performed. ZDOCK data showed that GK-15 could perfectly bind either to theopen or closed conformation of T1R3. To the best of our knowledge, the present work is the first study to focuson the screening of umami peptides from tempeh.

1. Introduction

Tempeh is a soybean product which is produced by fermentationthat has been made for centuries in Indonesia, originally in Java(György, Murata, & Ikehata, 1964; Mo et al., 2013; Nakazawa &Takeda, 1928). In Indonesia, tempeh was consumed in 70% of house-holds in 2009, which was greater than the household consumptionpercentage for any other soy-based products such as tofu, sauce, oncom,tauco or soymilk. Other countries that have been identified as hightempeh consumers are China, Taiwan, Australia, Japan, as well as somecountries in Europe, the Americas and Africa. Several competitive ad-vantages of tempeh have been discovered, such as high content of crudeprotein, isoflavones, vitamin B12, folate, fat, and carbohydrates (Moet al., 2013), its antihypertensive activity, which is thought to derivefrom its small peptide composition (Gibbs, Zougman, Masse, &Mulligan, 2004), and its umami taste. Those competitive advantagesmay explain the high consumption percentages in some countries.

Tempeh is produced by the following process: whole soybeans areacidified, boiled, cooled to 30–38 °C, and inoculated with the fungusRhizopus microsporus var. oligosporus, which grows throughout the

boiled soybeans over 1–2 days and transforms them into a compactcake. The protein digestion occurs during fermentation, because of theproduction of proteases during the growth of Rhizopus microsporus var.oligosporus, which belongs to the zygomycete class. R. microsporus var.oligosporus secretes an aspartic acid protease abundantly. This protease,which is simply called acidic protease, a pepsin-like endopeptidase, cancleave soy proteins at particular aspartic acid residues, at low pH (pH 3to 4). Another endopeptidase also commonly found in R. microsporusvar. oligosporus is a serine protease which digests proteins by cleavingthem at amino acids with relatively small (or no) side chains, such asglycine or alanine. Serine proteases are generally active at neutral oralkaline pH, with an optimum activity between pH 7 and 11. In com-merical production, the mixed culture is inoculated using cooked soy-bean. There are some moulds, yeasts, and microflora which growduring fermentation, with Rhizopus as the dominant genus. Those spe-cies secrete particular enzymes to cause fermentation (Samson, 1987).

Umami is one of the basic tastes that can be detected by humans(Kurihara, 2015). Umami taste could be attributed to the free aminoacids aspartic acid and glutamic acid or other small molecule acids suchas succinic acid (Istiqamah, Lioe, & Adawiyah, 2019), to small peptides

https://doi.org/10.1016/j.foodchem.2020.127411Received 5 January 2020; Received in revised form 18 June 2020; Accepted 21 June 2020

Abbreviations: RP-HPLC, Reversed-phase high pressure liquid chromatograph; LC, Liquid chromatography; MS/MS, Tandem mass spectrometry⁎ Corresponding authors.E-mail addresses: jlhsu@mail.npust.edu.tw (J.-L. Hsu), rjd@olemiss.edu (R.J. Doerksen), tchuang@mail.npust.edu.tw (T.-C. Huang).

1 Present address: Department of Marine, Faculty of Fisheries and Marine, Campus C Universitas Airlangga, Surabaya, East Java 60115, Indonesia.

Food Chemistry 333 (2020) 127411

Available online 04 July 20200308-8146/ © 2020 Elsevier Ltd. All rights reserved.

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which result from protein digestion through fermentation with mole-cular weight< 3 kDa that contain one or more aspartic acids and/orglutamic acids (Dang, Gao, Ma, & Wu, 2015; Rhyu & Kim, 2011; Su,Cui, Zheng, Yang, Ren, & Zhao, 2012; Zhang et al., 2008; Zhuang et al.,2016), or to nucleotides such as ionosine monophospate (IMP) andguanosine monophospate (GMP) (Yamaguchi & Ninomiya, 2000). Theumami taste receptor is a class C G-protein coupled receptor (GPCR),which responds to L-glutamate and, to some extent, L-aspartate. Thereceptor belongs to the T1R family, which includes T1R1 and T1R3(Dang, Gao, Xie, Wu, & Ma, 2014; Yu, Zhang, Miao, Li, & Liu, 2017). Itmay function as a heterodimer or a homodimer. T1R family taste re-ceptors are related to the metabotropic glutamate receptors (mGluR),which also have been studied for their role in mediating umami taste(Kurihara, 2015).

In this work, umami peptides were isolated from water soluble ex-tracts from protein-based product and purified using RP-HPLC, afterwhich the amino acid sequences were identified using LC-MS/MS. Inorder to study the taste characteristics of peptides, sensory evaluationof the synthetic peptides has been suggested as a suitable technique (Suet al., 2012). There have been several studies of natural-product pep-tides as the source of umami taste in foods (Dang, Gao, Ma, & Wu, 2015;Su et al., 2012; Zhang, Venkitasamy, Pan, Liu, & Zhao, 2017; Zhuanget al., 2016) and reports on the interactions between umami peptidesfrom hams and the umami receptor (Dang et al., 2015). Also, otherumami taste agents have been evaluated such as L-glutamate(Gunawan-Puteri, Hassanein, Prabawati, Wijaya, & Mutukumira, 2015)and guanosine monophosphate (Zhang et al., 2008), and the interactionbetween the particular umami substances and the umami receptor hasbeen studied using molecular docking into homology models of the li-gand-binding domain of T1R1/T1R3 (Dang, Gao, Xie, Wu, & Ma, 2014;Dang et al., 2019; Liu, Da, & Liu, 2019). Our hypothesis was that wecould identify which peptides from tempeh contribute to its umamitaste, using LC-MS/MS; evaluate the taste profile of the umami pep-tides, using sensory evaluation; and explore the mechanism of thebinding of umami peptides to the umami receptor, using moleculardocking into published protein models.

2. Materials and methods

2.1. Materials

Soybeans were purchased commercially in a traditional market inNeipu, Pingtung, Taiwan; commercial tempeh mould was purchasedfrom a cottage industry in Malang, Indonesia; acetonitrile (ACN), tri-fluoroacetic acid (TFA), citric acid, caffeine, and sucrose were pur-chased from Sigma Chemical Co. (St. Louis, MO, USA); sodium chloridewas purchased from Taiyen Biotech Co., Ltd. (Miaoli, Taiwan); mono-sodium glutamate was purchased from Ajinomoto Co. (Bangkok,Thailand), and formic acid (FA) was purchased from J. T. Baker Co.(Phillipsburg, NJ, USA). Molecular weight cut-off (MWCO) ultra-fil-tration membranes with a 3 kDa cut-off were procured from MilliporeCorp. (Bedford, MA, USA). The synthetic peptide (GENEEEDSGAIV-TVK) was obtained from MDBio, Inc. (Taipei, Taiwan). The water usedin this study was obtained using a Milli-Q® water purification systemfrom Millipore (Billerica, MA, USA). All other chemicals used were ofanalytical grade.

2.2. Collection of small peptides from water extract of tempeh

The soybeans were dehulled, soaked in acidic water which con-tained citric acid (pH: 5) for 6 h, then boiled, and cooled in an openenvironment under ambient temperature before inoculation. 0.2% (w/w) inoculum (Rhizopus microsporus var. oligosporus) was mixed thor-oughly into the boiled soybean. Tempeh was fermented in a plasticpolyethylene bag with 5 holes in it so that the fermentation would occurunder aerobic conditions; the incubation was performed in an incubator

at 30 °C for 48 h. Next the tempeh was pulverized with mortar andpestle, thoroughly dried, and milled into powder form. Dried tempehwas dissolved and extracted in water (tempeh:water = 1:4), and heatedin a thermostat-controlled water bath at 40 °C for 1 h. The mixture wascentrifuged (3.600 g, 20 °C, 30 min), after which the supernatant wasultracentrifuged (10.000 g; 20 °C; 30 min). The supernatant was re-moved and freeze-dried to give the crude extract (Gómez-Ruiz,Taborda, Amigo, Ramos, & Molina, 2007).

2.3. Collection of small peptides using 3 kDa cutoff membrane

The dried crude extract of tempeh was pulverized, dissolved inwater, and passed through an ultrafiltration membrane (3 kDa cutoff)using ultracentrifugation (14.000 g; 40 min, 20 °C). The filtrate waslyophilized to give a small peptide mixture which was kept at −20 °Cprior to LC-MS/MS analysis and sensory evaluation (Gómez-Ruiz et al.,2007).

2.4. Peptide identification using ESI LC-MS/MS

LC-MS/MS has been commonly used to discover compounds’structures and molecular weights. Some novel compounds have beendiscovered by using this method. This method of umami peptide pur-ification and identification was similar to the method reported by Guand Wu (2013) for the discovery of some novel angiotensin I convertingenzyme inhibitory peptides from the crude extract of soy protein hy-drolysate.

In this work, the dried water soluble extract (crude extract) derivedfrom tempeh was diluted in water and passed through 3 kDa MWCOultrafiltration membrane to give a mixture of small peptides (< 3 kDa).The freeze-dried filtrate containing small peptides (< 3 kDa) was dis-solved in 5% ACN and 0.1% FA in deionized water for LC-MS/MSanalysis. LC-MS/MS analysis was performed using a Thermo LCQ DECAXP MAX system with an electrospray ionization (ESI) source (ThermoScientific, Inc., USA). Samples were loaded onto C18 columns(150 mm × 2.1 mm, particle size: 5 μm). The samples were elutedusing a gradient from 5% to 70% acetonitrile in 0.1% formic acid over75 min, with a flow rate of 200 μl/min. MS/MS spectra were acquiredusing Thermo Xcalibur™ (Thermo-Scientific) data acquisition. Thesheath gas flow rate was 50 arbitrary units, the spray voltage appliedfor the full mass scan was 4 kV and the capillary voltage was 20 V withcapillary temperature of 300 °C. The MS scan was carried out from m/z100 to m/z 1000. The MS/MS raw data were converted into MGF filesusing Mascot Distiller v 2.3.2.0 (Matrix Science, London, UK). The re-sulting MGF files were searched using the Mascot search engine v 2.3(Matrix Science) with the following search parameters: 1. The proteindatabase was set to be a home-made database ‘Soybean’ which wasestablished from the combined FASTA files of soybean proteins in NCBI;2. the enzyme was set as ‘no enzyme’; 3. the precursor and product ionmass tolerance were set to be 2 Da and 1 Da, respectively; 4. the sig-nificance threshold was set to p < 0.05. The peptide sequences wereidentified through database matching as well as using the manual in-terpretation of their MS/MS spectra. Peptides with ion scores more thanthe identity threshold (score > 65) were regarded as identified pep-tides. The identified peptide sequences were pursued by comparing theretention time, m/z, and MS/MS spectra of synthetic peptides withidentified peptides in the sample (Rawendra et al., 2013). Enzymessecreted from Rhizopus microsporus var. oligosporus that contributed toprotein digestion in this study were predicted using a proteomics toolcalled PeptideCutter on the ExPASy molecular biology server at http://web.expasy.org.

2.5. Sensory evaluation

The sensory evaluation was carried out based on the protocol re-ported by Su et al. (2012). Panelists with Food Technology background

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were chosen from the Department of Biological Science and Tech-nology, National Pingtung University of Science and Technology, andthe panelists’ ages were from 23 to 26 years old. The panelists weretrained to recognize the five basic tastes, including sweet, bitter, sour,salty, and umami. Taste profiles for the crude extract of tempeh wereevaluated using a 10-point intensity scale (1, no taste; 10, very strongtaste). Taste reference samples for sweet, bitter, sour, salty and umamiwere solutions of sucrose (1%), caffeine (0.08%), citric acid (0.08%),sodium chloride (0.35%), and MSG–salt (0.35%–0.35%), respectively(Su et al., 2012). Descriptive analysis was performed to determine thedifferences of taste characteristics between the crude extract, the 3 kDaMWCO ultrafiltration fraction and the synthesized peptide (GK-15).First, 1 mg of crude water soluble extract was tested to determine eachof its basic taste intensities based on referenced samples used as stan-dards. Furthermore, the crude extract and the 3 kDa MWCO ultra-filtration fraction were used as standards to determine the intensity ofeach taste for the 3 kDa MWCO ultrafiltration fraction and peptide GK-15, respectively. All standards used in this study were ranked as 10.

2.6. Statistical analysis

Statistical calculations were performed using the statistical packageSPSS 22.0 (IBM SPSS Statistics 22) for one-way ANOVA. TheStudent–Newman–Keuls (SeNeK) test was used for comparison ofmean values among treatments, and to identify significant differences(p < 0.05) among treatments.

2.7. Molecular docking of identified peptide to umami receptor

The protein receptor is a heterodimer T1R1/T1R3, and the bestavailable structure was established by homology modeling (LópezCascales, Oliveira Costa, de Groot, & Walters, 2010) (no X-ray structureis available). The homology modeling was carried out using the close-d–open state of mGluR1 as the template (PDB code 1EWK). The ligandbinding domain of mGluR1 has 26.8% sequence identity with humanT1R1 and 24.1% identity with human T1R3. Two models were gener-ated by López Cascales et al. (2010) for the umami receptor, includingform 1 which has T1R1 in the closed conformation and T1R3 in theopen conformation, and form 2 which has T1R1 open and T1R3 closed.In our research, the heterodimer T1R1/T1R3 for each form was sepa-rated into the monomers T1R1 and T1R3 before calculations were done.Therefore, we had four protein conformations available as input proteinstructures for molecular docking (Fig. S1).

The preparation of the peptide GK-15 was done by submitting thesequence to the protein modeling web server (PS)2-v2: ProteinStructure Prediction Server (http://ps2v2.life.nctu.edu.tw/), whichuses a knowledge-based method to build peptide conformations (Chen,Hwang, & Yang, 2009). For each conformation of peptide GK-15 theelectrostatic solvation energy was calculated using the CHARMm GUIPBEQ-Solver web server (Jo, Vargyas, Vasko-Szedlar, Roux, & Im,2008). The generated conformations were visualized using RASMOLversion 2.7.2.5 (Sayle & Milner-White, 1995).

The molecular docking was performed using the reliable onlinedocking server ZDOCK (http://zdock.umassmed.edu/) (Pierce, Hourai,& Weng, 2011) and the binding site to be used in docking for eachprotein conformation was selected according to López Cascales et al.

(2010). The amino acid positions in the reference were matched withthe amino acid positions in the current models before specifying thebinding pocket amino acids in the model, because the monomers T1R1and T1R3 were separated from the heterodimer T1R1/T1R3, and aminoacid renumbering was needed. The binding sites for each of the currentmodels are given in Table S1. This system was confirmed by successfuldocking of L-Glu (glutamic acid) into each protein receptor conforma-tion in its specified binding pocket.

The best docked pose from ZDOCK was selected and minimizedusing CHARMm, after which the ΔG of the complex was calculated. Theinteraction between the receptor binding pocket and peptide GK-15 wasvisualized using PYMOL version 1.7.0.3, and Accelrys Discovery Studioversion 4.0.

3. Results and discussion

3.1. Identification of umami peptide in tempeh using ESI LC-MS/MS

Umami peptides from protein hydrolysate have been discovered,including those having some glutamic acid or asparatic acid residues intheir sequences (Gómez-Ruiz, et al., 2007; Maehashi, Matsuzaki,Yamamoto, & Udaka, 1999; Rhyu & Kim, 2011; Su et al., 2012; Zhang,Wang, Liu, Xu, & Zhou, 2012). In this study, four peptides having morethan two glutamic acid residues were identified, as shown in Table 1.Among the identified peptides, GENEEEDSGAIVTVK (GK-15) which isderived from glycinin subunit G2, was regarded as a candidate umamipeptide because it bears the most Glu residues. The retention time ofthis peptide is 10.7 min and the m/z and molecular weight of thisidentified peptide are 789.6 Da (doubly-charged ion) and 1577.2 Da,respectively (Fig. 1A and Table S2). The MS/MS spectrum suggestedthat the sequence of ion m/z 789.6 Da is GENEEEDSGAIVTVK based onb- and y-series ion matching in the database, as shown in Fig. 1B. Theidentity of GK-15 was further confirmed using a synthetic peptide withthe corresponding sequence by comparing their retention time, m/z andMS/MS fragments. The protein origin of GK-15 is glycinin subunit G2[Glycine max], a major soy protein which has sometimes been found tobe as much as 85% of the protein content in soy protein samples(Keshun, 1997).

To determine which protease can hydrolyse glycinin subunit G2 toproduce GK-15, Peptide Cutter in the ExPASy Molecular Biology Server(http://kr.exp-asy.org/) was used. The pattern of protein digestion wasmodeled from the original sequence (Keil, 1992) (Fig. S2). The proteasein Peptide Cutter was adjusted and mapped into glycinin subunit G2,based on cleavage site. From calculations using the Proteomics tools(Peptide Cutter) on the ExPASy Molecular Biology Server, no proteasewas found to be available for specific cleavage at this particular posi-tion. A similar phenomena has been reported (Su et al., 2012). In thatwork, the authors used Aspergillus oryzae to digest defatted peanut. Theresults showed that no single enzyme can produce the umami tastepeptide from peanut proteins, but Aspergillus oryzae could secrete acomplex array of enzymes, such as proteases, lipases, and cellulolyticenzymes, which could hydrolyze the peanut protein to peptides si-multaneously. In this work, the moulds in cooked soybeans may secretemultiple enzymes such as proteases, lipases, α-amylases, and glutami-nases (Han, Ma, Rombouts & Robert Nout, 2003) and they may digestsoy proteins simultaneously to form the peptides.

Table 1Identified small peptides from the water-soluble extract of tempeh.

Identified protein Identified peptide Position Start–end m/z MW (Da) Mascot score

Glycinin subunit G2 [Glycine max] GENEEEDSGAIVTVK (GK-15) 242–256 789.64 1577.25 104Alpha' subunit of beta-conglycinin [Glycine max] DEGEQPRPFPFP 33–44 708.89 1414.65 85Mutant glycinin subunit A1aB1b [Glycine max] GENEGEDKGAIVTVK 245–259 773.99 1544.77 70

NLQGENEGEDKGAIVTVK 242–259 951.16 1899.95 78

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3.2. Sensory evaluation of umami taste in tempeh

According to Maehashi, Matsuzaki, Yamamoto and Udaka (1999),the sensory properties of proteins can be increased through hydrolysisby certain enzymes. In this study, the five basic tastes of a crude extractof tempeh were evaluated and recognized by panelists, as shown inFig. 2A. The results showed that bitter taste had the highest rating(6.83) among the five basic tastes, followed by umami and salty, forwhich the ratings were 5.50 and 5.17 respectively, while sweet and sourwere given rather low scores (Fig. 2A). The intensities of the five basictastes in tempeh hydrolysate were significantly different to the in-tensities of standard taste agents (p < 0.05) and generally the in-tensities of the five basic tastes found in the tempeh hydrolysate werelower than those of the five standards.

In order to identify the key component that contributed to theumami taste, the hydrolysate was used for further sensory-guidedfractionation to identify the sources of the taste. Since the umami tastemay be caused by small peptides, ultrafiltration was used to get pep-tides with low molecular weight and high umami taste intensity. In thisstudy, the umami taste intensity of the small peptide mixture washigher than that of the water soluble extracts of tempeh (p < 0.05)(Fig. 2B). This work was in agreement with the results of Su et al.(2012), who found that as the protein hydrolysate was fractionated togive peptides with lower molecular weight, the umami taste roughlyincreased with decrease of peptide molecular weight.

In this work, ultrafiltration was used to separate molecules ac-cording to their molecular weights under centrifugal force (Jang & Lee,

2005). During this process, the free amino acids, salts, organic acids,and peptides smalled than the 3 kDa cutoff pass through the filtermembrane, but the free amino acids and salts may have synergisticeffects on the peptides, which could interfere with sensory evaluation ofumami taste (Wang, Maga, & Bechtel, 1996). Therefore, it was alsonecessary to conduct sensory evaluation for purified peptides. SyntheticGK-15 (purity > 95%) was used for taste profile evaluation and theresults are shown in Fig. 2C. This peptide elicited all of the basic tastes.Umami taste had the highest intensity, followed by salty, bitter, sweet,and sour. The hydrophilic peptides were commonly associated withdesirable flavours such as sweet, meaty, and brothy, while the hydro-phobic peptides were usually associated with more undesirable bitter(and often sour) taste (Spanier & Edwards, 1987).

GK-15 has some hydrophilic amino acids such as glutamic acid,aspartic acid, and threonine, and those amino acids were assessed assweet taste eliciting amino acids, and are often considered to be themain contributors of sweet and umami taste (Lioe, Takara, & Yasuda,2006). By contrast, the hydrophobic amino acids of GK-15, such asvaline, alanine, and isoleucine, could form a hydrophobic surface pre-dicted to face the receptor (cf. Fig. S3), which could reduce thesweetness (Xue, Szczepankiewicz, Thulin, Linse, & Carey, 2009), andthe existence of valine close to the C terminal may explain why bittertaste also was elicited by this peptide. The hydrophobic amino acidsmight play an important role to suppress the umami taste (Salles,Septier, Roudot-Algaron, Guillot, & Etievant, 1995). When the peptidewas compared with the small peptide mixture, the umami taste in-tensity of GK-15 was not significantly different from that of the small

Fig. 1. A. LC-MS chromatogram of the< 3 kDa fraction The inset figure shows the precursor ion of m/z 789.64; B. ESI-LC-MS/MS spectrum of precusor ion of m/z789.64.

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peptide mixture (p > 0.05). Hence, this peptide may be one of severalkey peptides that contribute to the overall umami taste of the fraction.

3.3. Docking results for the interaction between the umami peptide andreceptor T1R1/T1R3

The computational work was designed to predict the possiblebinding poses between the umami peptide and each umami taste re-ceptor conformation. We commenced with prediction of the umamipeptide’s secondary structure. In total, 25 conformations of GK-15 weregenerated using the ps2-v2 web server and their electrostatic solvationenergies are given in Table S3, among which the most negative ispredicted to correspond to the most stable conformation in the proteinenvironment. The lowest 10 conformations were considered for furtheranalysis (Fig. S4). The heterodimers of the receptor protein for each

form were separated using PYMOL before docking. Previous researchshowed that umami substances bind into each monomer. Therefore, wehad four receptor conformations to use in this work: T1R1 in closedconformation, T1R1 in open conformation, T1R3 in closed conforma-tion, and T1R3 in open conformation.

Previous work reported the docking of L-Glu to T1R1/T1R3 andfound a strong interaction (López Cascales et al., 2010). Therefore, inthis work the docking procedures were first confirmed by re-docking L-Glu to T1R, after which we analyzed the interactions and calculated thebinding free energies. The results showed that the binding pocketamino acid residues of each protein receptor conformation (Table S1)have strong interactions with L-Glu.

Considering the four receptor conformations and ten GK-15 con-formations, a total of 40 complexes were submitted to the ZDOCK webserver. Peptide GK-15 was expected to have interactions with amino

Fig. 2. A. Taste profile of water soluble extract from tempeh; B. The difference in taste attributes between water soluble extract (standard) and<3 kDa cutofffraction; C. The difference in taste attributes between the< 3 kDa fraction (standard) and the peptide GK-15.

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acid residues in T1R similar to those of T1R–L-Glu. The complex of theT1R receptor and GK-15 that had the best interaction resulted in theZDOCK scores ≤ 730 (Pierce, Wiehe, Hwang, Kim, Vreven, & Weng,2014). By contrast, the high ZDOCK scores represent that GK-15 in-teracted on the surface of the protein or diffused away from the bindingpocket. From among all the 40 complexes, there were four peptideconformations that had favourable interaction with T1R3 in its closedconformation and two peptide conformations that had favourable in-teraction with T1R3 in its open conformation (Table 2).

We used the best docked poses of GK-15 in the T1R binding pocketsfor force field minimization and binding free energy calculations usingCHARMm, the results of which are shown in Table S4. Binding of GK-15to T1R3 in the open conformation was found to be more favorable thanto T1R3 in the closed conformation or to either of T1R1’s

conformations, based on the binding free energy calculations, for whichthe lower energy (more negative) is the more favorable interaction(Table S4). In T1R1, all of GK-15’s conformations bound to the surfaceof protein T1R1 both in the open and closed conformation (Fig. 3),predicting that T1R1 is not the favorable receptor for this peptide.

L-Glu was selected as a control in this work because, first, it isconsidered a standard for umami taste and, second, the binding modebetween L-Glu and T1R1/T1R3 has been previously studied. Thedocking of GK-15 into T1R can be compared to that of L-Glu. In thiswork, the ligand interactions analysis showed that GK-15 had somesimilar interactions to those of L-Glu in the binding pocket of T1R3 inthe open and closed conformation (Fig. 4; Table S5).

The lysine (K) residue in GK-15 has similar interactions to those of L-Glu in the binding pocket of T1R3, and its glycine residue also had onesimilar interaction to that of L-Glu in the binding pocket of T1R3 in theclosed conformation. In T1R3 in the open conformation, valine, lysine,glutamic acid, and aspartic acid each had one similar interaction tothose of L-Glu in the binding pocket of T1R3.

According to the molecular docking results, we can consider pos-sible reasons why L-Glu has more favourable umami taste than GK-15: L-Glu can bind into the binding pocket of all T1R protein receptor con-formations and it has strong interactions with more amino acid residuesin the binding pockets. By contrast, peptide GK-15 only could bind intothe binding pocket of protein receptor T1R3′s open and closed con-formations. Although the ΔG for complex T1R–peptide GK-15 is muchmore negative than that of T1R–L-Glu (Table S4), that is because theamino acid residues in GK-15 interact with other amino acid residuesoutside the binding pocket of T1R1 and T1R3 in their open and closedconformations (Fig. 4; Table S5). The hydrophobic amino acid residuesin peptide GK-15 could form a hydrophobic surface predicted to facethe receptor, which could reduce the savory taste (Fig. S3) (Xue et al.,2009). The docking interactions support that GK-15 is a significantsource of the umami taste in tempeh.

Table 2ZDOCK score for umami receptor–umami peptide interaction.

GK-15Rank

GK-15Conf

GK-15SolvationEnergya

(kcal/mol)

ZDOCK score

Form 1 Form 2

T1R1 closed T1R3 open T1R1 open T1R3 closed1 12 −904.65 994.23 863.30 891.86 932.802 4 −892.99 891.70 963.12 958.90 1040.343 24 −891.19 922.88 987.61 1078.58 702.744 22 −882.91 1025.93 968.34 919.93 667.405 18 −881.79 918.75 683.97 1239.47 565.896 23 −876.46 936.70 969.30 873.34 729.487 6 −875.62 907.52 1019.79 997.92 978.258 20 −867.52 910.25 589.22 1017.88 1024.859 3 −864.45 916.54 1117.34 950.45 1082.2210 7 −857.67 881.60 996.78 898.59 1085.71

a From PBEQ solver CHARMm GUI.

Fig. 3. Docking complexes (obtained from ZDOCK web server) of protein receptor T1R and peptide GK-15.

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4. Conclusion

In this study, a novel umami peptide GK-15 was identified fromtempeh using LC-MS/MS analysis and database-assisted identification.

The water-soluble extract of tempeh was collected using a 3 kDa mo-lecular weight cutoff ultrafiltration membrane. LC-MS/MS identified 4peptides from the ultrafiltration fraction, including GK-15, the sequenceof which was confirmed by comparison to a synthetic peptide with the

Fig. 4. Receptor-ligand interactions for L-Glu (left) and peptide GK-15 (right) with the binding pocket of T1R: A and B. T1R1 closed; C and D. T1R1 open; E and F.T1R3 closed; G and H. T1R3 open.

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same sequence. According to the sensory evaluation, the small peptidefraction showed that umami was the most intensive taste among thefive basic tastes and GK-15 also showed a similar trend, which sug-gested that the umami taste of the small peptide (< 3 kDa) fraction maybe contributed by GK-15. Molecular docking using ZDOCK showed thatGK-15 could bind to the binding pocket of T1R3 in either its open orclosed conformation. Hence this peptide may be the major source of theumami taste of tempeh. As far as we know, GK-15 is the first reportedumami peptide identified in tempeh.

CRediT authorship contribution statement

Muhamad Nur Ghoyatul Amin: Conceptualization, Methodology,Validation, Investigation, Data curation, Writing - original draft,Writing - review & editing, Visualization. Joni Kusnadi:Conceptualization, Methodology, Writing - review & editing, Fundingacquisition. Jue-Liang Hsu: Conceptualization, Methodology,Resources, Writing - review & editing, Supervision, Project adminis-tration, Funding acquisition. Robert J. Doerksen: Methodology,Writing - review & editing, Supervision, Project administration. Tzou-Chi Huang: Conceptualization, Methodology, Resources, Writing - re-view & editing, Supervision, Project administration, Funding acquisi-tion.

Declaration of Competing Interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgements

This work was financially supported by Taiwan MOST (Ministry ofScience and Technology) grants (MOST 104-2113-M-020-001 for Dr.Jue-Liang Hsu and NSC 102-2811-B-020-005 for Dr. Robert J.Doerksen). We also appreciate the instrument support by ResearchCenter for Active Natural Products Development in NPUST. Thanks toProfessor Eric Walters (Rosalind Franklin University of Medicine andScience) for contributing the protein models of T1R1/T1R3, ProfessorJenn-Kang Hwang and Shih-Chung Yen (National Chiao-TungUniversity) for releasing a knowledge based method for peptide mod-eling, and Professor Bo-Kang Liou (Central Taiwan University ofScience and Technology) for giving an opportunity to use an electronictongue machine to conduct the sensory evaluation.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2020.127411.

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