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Kaneko et al., 2014 (Isolasi Seny Umami Dari Soy Sauce)

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Kaneko et al., 2014 (Isolasi Seny Umami Dari Soy Sauce)
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  Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=tbbb20 Bioscience, Biotechnology, and Biochemistry ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20 Isolation and Identification of the UmamiEnhancing Compounds in Japanese Soy Sauce Shu KANEKO, Kenji KUMAZAWA & Osamu NISHIMURA To cite this article:  Shu KANEKO, Kenji KUMAZAWA & Osamu NISHIMURA (2011) Isolationand Identification of the Umami Enhancing Compounds in Japanese Soy Sauce, Bioscience,Biotechnology, and Biochemistry, 75:7, 1275-1282, DOI: 10.1271/bbb.110041 To link to this article: https://doi.org/10.1271/bbb.110041 Published online: 22 May 2014.Submit your article to this journal Article views: 410View related articles Citing articles: 20 View citing articles  Isolation and Identification of the Umami Enhancing Compoundsin Japanese Soy Sauce Shu K  ANEKO , y Kenji K  UMAZAWA , and Osamu N ISHIMURA  Analytical Laboratory, Functional Ingredient Department, Ogawa & Co., Ltd.,15-7 Chidori, Urayasu, Chiba 279-0032, Japan Received January 17, 2011; Accepted April 15, 2011; Online Publication, July 7, 2011[doi:10.1271/bbb.110041] To clarify the key compounds that account for theumami taste of soy sauce, a typical Japanese soy sauce,  Koikuchi Shoyu , was separated by preparative chroma-tography, and the umami enhancing fractions werescreened on the basis of an umami intensity of a 6.0m M monosodium  L -glutamate (MSG) solution. Liquid chro-matography-time of flight mass spectrometry (LC-TOFMS), 1D/2D nuclear magnetic resonance spectros-copy (NMR) studies of the umami enhancing fractionsled to the identification of   N  -(1-deoxy- D -fructos-1-yl)pyroglutamicacid(Fru-pGlu),  N  -(1-deoxy- D -fructos-1-yl)valine (Fru-Val),  N  -(1-deoxy- D -fructos-1-yl)methionine(Fru-Met), pyroglutamylglutamine (pGlu-Gln), andpyroglutamylglycine (pGlu-Gly). Although all the com-pounds identified were at sub-threshold concentrationsin the soy sauce, a taste reconstitution experimentrevealed that they contributed part of the umami tasteof the soy sauce.Key words:  soy sauce; amadori compound; pyroglutamylpeptide; umami; taste enhancer Traditionally-brewed soy sauce has been one of themost popular seasonings in Japan for hundreds of years.It is used not only as a sauce for food, but as a seasoningin cooking. Five typical varieties of soy sauce, catego-rized based on Japan Agricultural Standards, areprovincially brewed for traditional dishes. Because of the differences in the materials for brewing, an individ-ual soy sauce has a characteristic aroma and taste. Inrecent years, a typical Japanese soy sauce, called Koikuchi Syoyu , that accounts for about 85% of totalsoy sauce consumption in Japan is widely consumed allover the world. 1) Nearly equal amounts of soy beans boiled todenature the protein and wheat comminuted afterroasting are used as starting materials in brewing Koikuchi Syoyu , followed by a seed mold called  koji added to them. This incubated to produce enzymes overseveral days under high humidity conditions. 1) Afterincubation, NaCl solution is mixed in and this istraditionally fermented for 6 to 8 months. Aftermaturation, the brew is compressed to separate thefiltrate and residue. The soy sauce is produced byheating the filtrate to deactivate the enzymes and tomake a complex flavor. Koikuchi Shoyu , produced through heating and anenzymatic reaction, is a dark-red, clear liquid exhibitinga well-balanced strong salty taste as well as a richumami taste and the other tastants besides fresh, roast,and sweet aromas. 2–6) Since the sensory profile of soysauce is one of the key criteria in judging productquality, some investigations have been done to correlateflavor quality and components, but the data reported todate on the molecules imparting the umami taste of soysauce are very limited. It is known that the umami tasteof soy sauce is due to acidic amino acids, includingglutamic acid and aspartic acid from soy protein andwheat gluten enzymatic hydrolysate. 6) As for the othercontributors, acidic peptides exhibiting umami havebeen isolated from the soy sauce, 7,8) but the contributionof these peptides to the umami taste of soy sauce wasnegligible due to low concentrations. 8,9) Other reportshave suggested that low-molecular-weight compoundsin soy sauce contribute to the salty and the umamitaste, 10) and that phenylalanine enhanced the umamitaste of soy sauce because of its enhancing effect on thatof monosodium  L -glutamate (MSG), 11) but the keydriver of the umami taste of soy sauce on a molecularlevel is still unclear.The objectives of the present investigation weretherefore to clarify the umami taste contributors in Koikuchi Syoyu  using instrumental analyses and sensoryanalyses in order to isolate and identify the keycompounds, and to evaluate their taste impact on thebasis of model experiments. Materials and Methods Chemicals.  Amino acids, organic acids, sugars, minerals, caffeine,and quercetin-3-rutinoside hydrate were purchased from Wako PureChemical Industries (Osaka, Japan), Kanto Chemical (Tokyo, Japan),and Tokyo Chemical Industry (Tokyo). Pyroglutamylglutamine (pGlu-Gln) and pyroglutamylglycine (pGlu-Gly) were purchased fromBachem (Bubendorf, Switzerland) (Fig. 1).  N  -(1-deoxy- D -fructos-1-yl)pyroglutamic acid (Fru-pGlu),  N  -(1-deoxy- D -fructos-1-yl)valine(Fru-Val), and  N  -(1-deoxy- D -fructos-1-yl)methionine (Fru-Met) weresynthesized following the literature. 12) All solvents were of HPLCgrade (Kanto Chemical, Tokyo). Deuterium oxide was purchased from y To whom correspondence should be addressed. Tel: +81-47-305-1408; Fax: +81-47-305-1423; E-mail: kaneko.shu@ogawa.net  Abbreviations : COSY, correlation spectroscopy; DoT, dose-over-threshold; Fru-Met,  N  -(1-deoxy- D -fructos-1-yl)methionine; Fru-pGlu,  N  -(1-deoxy- D -fructos-1-yl)pyroglutamic acid; Fru-Val,  N  -(1-deoxy- D -fructos-1-yl)valine; GPC, gel permeation chromatography; HILIC, hydrophilicinteraction chromatography; HMBC, hetero-nuclear multiple quantum coherence; HMQC, hetero-nuclear multiple-bond connectivity; LC-TOFMS,liquid chromatography-time of flight mass spectrometry; MSG, monosodium  L -glutamate; NMR, nuclear magnetic resonance spectroscopy;pGlu-Gln, pyroglutamylglutamine; pGlu-Gly, pyroglutamylglycine; RP-HPLC, reverse phase-high performance liquid chromatography  Biosci. Biotechnol. Biochem. ,  75  (7), 1275–1282, 2011  Euriso-Top (Gif-sur-Yvette, France).  Koikuchi Syoyu  was purchasedfrom Marushinhonke (Yuasa, Japan). Preparative separation of soy sauce on reverse phase-high performance liquid chromatography (RP-HPLC).  Two mL of thesoy sauce was separated by RP-HPLC using an ODS RP18 column(10 m  m ,  250    20 mm i.d., Fuji Silysia Chemical, Kasugai, Japan).Monitoring the effluent at 214nm, chromatography was performedwith a 0.1% formic acid solution (methanol:water=3:97) at a flow rateof 12.0mL/min for 5min, then increasing the methanol content to 15%over 15min, and finally increasing the methanol content to 100% overa further 10min. In total, 8 fractions, A to H, were collectedindividually 100 times, and then the eluates of the correspondingfractions were combined and freeze-dried. The yields of the individualfractions are summarized in Table 1. Preparative separation of fraction B on gel permeation chroma-tography (GPC).  An aliquot (1.0g) of fraction B was dissolved indistilled water (20mL) containing 0.1% formic acid, and afterfiltration, 2mL of the solution was separated by GPC using a steelcolumn ( 500    22 mm i.d.) filled with an acidic (0.1% formic acid)water slurry of a Sephadex G-10 (55–165 m  m , GE Healthcare UK,Little Chalfont, UK). Monitoring the effluent with a refractive indexdetector, chromatography was performed with a 0.1% formic acidsolution at a flow rate of 0.5mL/min. In total, 4 fractions, B-I to B-IV,were collected individually 10 times, and then the eluates of thecorresponding fractions were combined and freeze-dried. The yields of the individual fractions are summarized in Table 2. Preparative separation of fraction B-II on hydrophilic interactionchromatography (HILIC).  An aliquot (81mg) of fraction B-II wastaken up in a 90% acetonitrile aqueous solution (acetonitrile:water=9:1) containing 0.1% formic acid (16.3mL), and membrane filtered,and then 0.5mL of the sample was separated by HPLC using anAtlantis HILIC Silica column (5 m  m ,  250    4 : 6 mm i.d., Nihon Waters,Tokyo). Monitoring the effluent at 214nm, chromatography wasperformed with 95% acetonitrile containing aqueous formic acid (0.1%in water) at a flow rate of 1.0mL/min, then decreasing the acetonitrilecontent to 91.5% over 16.7min, and then rapidly decreasing it to 70%over a further 1.3min. This condition was maintained for 15min. Intotal, 20 peaks, B-II-1 to B-II-20, were collected individually 32 times,and then the eluates of the corresponding fractions were combined,evaporated, and freeze-dried.  Isolation and identification of Fru-pGlu and pGlu-Gly in fraction B-II-6.  An aliquot (6.0mg) of fraction B-II-6 was taken up in a solution(acetonitrile:water=98:2) containing 0.1% formic acid (0.6mL) andthen 50 m  L of the sample was separated by HPLC using an AtlantisHILIC Silica column (5 m  m ,  250    4 : 6 mm i.d., Nihon Waters, Tokyo).Monitoring the effluent at 214nm, chromatography was performedwith 98% acetonitrile containing aqueous formic acid (0.1%) at a flowrate of 1.0mL/min over 20min, then decreasing the acetonitrilecontent to 86% over 40min. Fru-pGlu and pGlu-Gly were obtainedfrom 46min to 48.4min, and 48.4min to 52min respectively. In total,the respective fractions were collected individually 10 times, and thenthe eluates of the corresponding fractions were combined, evaporated,and freeze-dried.Fru-pGlu:  13 C{ 1 H} NMR data and the ratio of the cyclic form atequilibrium in a  D 2 O solution agreed with the data reported in theliterature. 12)  1 H NMR (400MHz,  D 2 O; COSY):    2.13 ( m , 1H,H-C(3 0 )), 2.43–2.62 ( m , 3H, H-C(3 0 ), H-C(4 0 )), 3.18–4.11 (7H,H-C(1), H-C(3), H-C(4), H-C(5), H-C(6)), 4.56 ( dd  , 0.24H, 3  J   ¼  8 : 70 Hz,  3  J   ¼  2 : 70 Hz, H-C(2 0 ) in   -furanose form), 4.61 ( d  ,0.12H,  3  J   ¼  8 : 25 Hz, H-C(2 0 ) in   -furanose form), 4.67 ( dd  , 0.64H, 3  J   ¼  9 : 15 Hz,  3  J   ¼  2 : 75 Hz, H-C(2 0 ) in   -pyranose form).pGlu-Gly:  1 H NMR (400MHz,  D 2 O; COSY):    2.12 ( m , 1H,H-C(3)), 2.42 ( m , 2H, H-C(4)), 2.57 ( m , 1H, H-C(3)), 4.02 ( s , 2H,H-C(2 0 )), 4.37 ( dd  , 2H,  3  J   ¼  4 : 91 Hz,  3  J   ¼  8 : 93 Hz, H-C(2));  13 C{ 1 H}NMR (100MHz,  D 2 O; HMQC, HMBC):    25.5 [C(3)], 29.6 [C(4)],41.4 [C(2 0 )], 57.3 [C(2)], 173.5 [C(1 0 )], 176.0 [C(1)], 182.8 [C(5)].  Identification of pGlu-Gln in fraction B-II-13.  pGlu-Gln:  1 H NMR(400MHz,  D 2 O; COSY):    2.01–2.17 ( m , 3H, H-C(3 0 ), H-C(3)), 2.25( m , 1H, H-C(3 0 )), 2.39–2.48 ( m , 4H, H-C(4), H-C(4 0 )), 2.58 ( m , 1H,H-C(3)), 4.40 ( dd  , 1H,  3  J   ¼  5 : 03 Hz,  3  J   ¼  9 : 08 Hz, H-C(2)), 4.44 ( dd  ,1H,  3  J   ¼  5 : 11 Hz,  3  J   ¼  9 : 18 Hz, H-C(2 0 ));  13 C{ 1 H} NMR (100MHz,  D 2 O; HMQC, HMBC):    25.5 [C(3)], 26.6 [C(3 0 )], 29.6 [C(4)], 31.6[C(4 0 )], 52.7 [C(2 0 )], 57.2 [C(2)], 175.2, 175.5 [C(1 0 ), C(1)], 178.4[C(5 0 )], 182.7 [C(5)].  Isolation and identification of Fru-Val and Fru-Met in fraction B-II-19.  An aliquot (12.0mg) of fraction B-II-19 was taken up in distilled Fru-Val Fru-MetpGlu-GlypGlu-GlnFru-pGlu 534625’2’11’ 3’4’345625’2’11’3’4’345625’2’11’3’4’ OHHOOHHOHHOHNOHOOC 123451’2’ NHHNOOCOOHOHHOOHHOHHOHNHCOOHHOHOOHHOHHOHNHCOOHSHNH 343’4’5’  NH 2 O 1251’2’ NHHNOOCOOH 4’2’ Fig. 1.  Chemical Structures of Pyroglutamylpeptides and Amadori Compounds (  -pyranose form). Table 1.  Yields of ODS FractionsFraction Yield fromthe soy sauce (%)A 30.5B 1.9C 1.8D 0.7E 0.9F 0.2G 1.9H 0.03 Table 2.  Yields of GPC FractionsFractionYield (%)From the soy sauce From fraction BB-I 0.1 3.2B-II 1.4 72.9B-III 0.3 16.5B-IV 0.1 5.31276 S. K  ANEKO  et al.  water containing 0.1% formic acid (1.2mL), and then 50 m  L of thesample was separated by HPLC using a Develosil C30-UG column(5 m  m ,  250    4 : 6 mm i.d., Nomura Chemical, Aichi, Japan). Monitor-ing the effluent at 214nm, chromatography was performed with 100%distilled water containing aqueous formic acid (0.1%) at a flow rate of 1.0mL/min over 15min. Fru-Val and Fru-Met were obtained from6.8min to 7.6min, and 11.0min to 11.8min respectively. In total, therespective fractions were collected individually 20 times, and then theeluates of the corresponding fractions were combined, evaporated, andfreeze-dried.Fru-Val:  13 C{ 1 H} NMR data and the ratio of the cyclic form atequilibrium in a  D 2 O solution agreed with the data reported in theliterature. 12)  1 H NMR (400MHz,  D 2 O; COSY):    0.96 (3H, H-C(4 0 )),1.04 (3H, H-C(5 0 )), 2.20 (1H, H-C(3 0 )), 3.23–3.36 (2H, H-C(1)), 3.47–4.18 (6H, H-C(3), H-C(4), H-C(5), H-C(6), H-C(2 0 )).Fru-Met:  13 C{ 1 H} NMR data and the ratio of the cyclic form atequilibrium in a  D 2 O solution agreed with the data reported in theliterature. 12)  1 H NMR (400MHz,  D 2 O; COSY):    2.12 (3H, H-C(5 0 )),2.17–2.22 (2H, H-C(4 0 )), 2.61–2.65 (2H, H-C(3 0 )), 3.27–3.33 (1H,H-C(1)), 3.63–4.20 (6H, H-C(3), H-C(4), H-C(5), H-C(6), H-C(2 0 )). Quantitative analysis.  Amino acids were quantified using anL-8500A amino acids analyzer (Hitachi High Technologies, Tokyo)by ninhydrin detection. 13) Organic acids were quantified by HPLCusing a Shim-pack SCR-102H column (7 m  m ,  300    8 : 0 mm i.d.   2 ,Shimadzu, Kyoto, Japan) and detected with a conductivity detector. 14) Sodium, potassium, magnesium, and calcium were quantified by HPLCusing a Shim-pack IC-C3 column (7 m  m ,  100    4 : 6 mm i.d., Shimadzu)detected with a conductivity detector. 15) Chloride was determined bypotentiometry. 16) Sugars were quantified by HPLC using an Asahipak NH2P-50 4E column (5 m  m ,  4 : 6    250 mm i.d., Showa Denko, Tokyo)detected with a refractive index detector. Chromatography wasperformed under isocratic conditions of 80% acetonitrile at a flowrate of 1.0mL/min for 40min. Pyroglutamylpeptides and amadoricompounds were quantified by LC-TOFMS using an ACQUITY UPLCBEH C18 column (1.7 m  m ,  100    2 : 1 mm i.d., Nihon Waters, Tokyo).All quantitative data represent the mean values of triplicate measure-ments.  Liquid chromatography-time of flight mass spectrometry (LC-TOFMS).  LC-TOFMS measurements were done with an ACQUITYUltraPerformance LC equipped with an ACQUITY UPLC BEH C18column (1.7 m  m ,  100    2 : 1 mm i.d., Nihon Waters) and a MicromassLCT Premier system (Nihon Waters, Tokyo) in ESI þ mode.Chromatography was done with a 0.1% formic acid solution(methanol:water=3:97) at a flow rate of 0.5mL/min for 2min, thenincreasing the methanol content to 100% over 3min. After that,conditions were maintained for 5min. The TOFMS system wasinitially calibrated with sodium formate, and analysis was carried outunder the following conditions: electrospray positive mode; MScapillary voltage, 3,000V; sample cone, 50V; source temperature,100  C; cone gas flow, 50l/h; desolvation gas temperature, 350  C;desolvation gas flow, 800l/h; internal reference, leucine enkephalinprotonated peak at  m =  z  556.2771; and scan,  m =  z  100–1000 at0.5s/scan.  Nuclear magnetic resonance spectroscopy (NMR).  1 H,  13 C, COSY,HMQC, and HMBC spectroscopic experiments were done using aBruker AVANCE-400 spectrometer (Bruker Biospin, Yokohama,Japan).  D 2 O was used as the solvent, and chemical shifts weremeasured using methanol as internal standard. Training of the sensory panel.  Twelve subjects with no history of known taste disorders were trained to evaluate the tastes of aqueoussolutions (2mL each) of the following standard taste compounds indistilled water (pH 6.0) using a triangle test, as described in theliterature: 17) sucrose (50mmol/L) for sweet, lactic acid (20mmol/L)for sour, NaCl (20mmol/L) for salty, caffeine (1mmol/L) forbitter, MSG (8mmol/L) for umami, and quercetin-3-rutinoside (0.01mmol/L) for astringency. The assessors had participated at regularintervals for at least 2 years in sensory experiments and were thusfamiliar with the techniques applied. Sensory analyses were performedin a sensory panel room at 24–26  C in three different sessions. Evaluation of umami taste intensity.  ODS fractions diluted to one-fifth concentration in the soy sauce with distilled water (pH = 4.5) andthe five-fold diluted soy sauce with distilled water (pH = 4.5) werepresented to the sensory panel, who were asked to score umami tastequality on a scale from 0 (not detectable) to 5 (strong). To achieve this,samples (2mL) were applied to the tongue with a plastic pipette,briefly swirled around the mouth, and then expectorated. In order toevaluate the potential of the HPLC fractions in enhancing the umamitaste of MSG, the umami taste intensity of a 6.0m M  MSG solutioncontaining the fraction diluted to one-fifth concentration in the soysauce with distilled water was compared to a 6.0m M  MSG solutionas control.  Determination of recognition-threshold concentrations.  The tastethresholds of pyroglutamylpeptides and amadori compounds weredetermined by triangle test with two samples of distilled water ascontrol. To prevent fatigue, tasting began at a concentration level twosteps below the threshold concentration, which had been determined ina preliminary taste experiment. Whenever the panelist selectedincorrectly, the next trial took place at the next higher concentrationstep. When the panelist selected correctly, the same concentration waspresented again as proof for the correctness of the data. The geometricmean of the last and the penultimate concentration was calculated andtaken to be the individual recognition threshold. The recognitionthreshold value of the compound was calculated by averaging therecognition thresholds of the individual panelist. Taste reconstitution experiment.  In order to reconstitute the umamitaste of the soy sauce, the compounds summarized in Table 3 weredissolved in one-fifth concentrations of the natural source in distilledwater, which was adjusted to pH 4.5. The taste intensity of an aliquot(2mL) of the reconstituted solution was evaluated by a five-pointscoring method, and was compared to the taste of the authentic soysauce. Results and Discussion Screening for the umami taste contributors in the soysauce In order to get initial insight into the key drivers of thetypical umami sensation imparted by the soy sauce, thesauce was separated into eight fractions by preparativeRP-HPLC using an ODS column monitoring the effluentat 214nm (Fig. 2A). After freeze-drying, an aliquot of each individual fraction was dissolved in distilled waterat one-fifth concentration in the soy sauce, and theumami intensity was judged by the trained sensory panelon a scale from 0 to 5. As shown in Fig. 2B, fraction Aexhibited the strongest umami taste (1.9) among theODS fractions, and the other fractions did not exhibitany umami taste. However, the soy sauce and therecombinants of all the ODS fractions (rODS) exhibitedmuch more strongly (3.3 and 2.9). These results suggestthat there were umami enhancing compounds in ODSfractions B-H that did not exhibit any umami taste ontheir own.To clarify the umami-enhancing effect describedabove, each ODS fraction was dissolved in an aqueoussolution of a 6.0m M  MSG and screened on the basis of the umami intensity of a 6.0m M  MSG solution. Asshown in Fig. 2C, fraction A strongly enhanced theumami taste of MSG ( 1 : 1  !  4 : 0 ), followed by fractionB ( 1 : 1  !  2 : 5 ), but the other fractions did not enhancethe activity of the umami taste of MSG. Because fractionA contained almost all glutamic acid, aspartic acid, Na,and K, which are thought to be the dominant contrib-utors to the umami taste of soy sauce, these compoundsappeared to contribute to enhancement of the umami Umami-Enhancing Compounds in Japanese Soy Sauce 1277
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