Yeast extracts weakened warmed-over flavor in surimi gels made from silver carp due to the masking effect by high concentrations of pyrazines and esters.

Warmed-over flavor (WOF) is an off-flavor in surimi gels. Yeast extract (YE) could improve the aroma properties of food. However, the effect of YE on the WOF in surimi gels and its mechanism was still unclear. In this study, aroma profiles, the composition of aroma compounds and aroma precursors, concentrations of WOF compounds, and thiobarbituric acid reactive substances (TBARS) of surimi gels with different amounts of YE were investigated by molecular sensory science and chromatographic techniques. Moreover, the effect of pyrazines and esters introduced by YE on WOF was also tested by sensory analysis. The addition of no less than 1% YE to surimi gels significantly weakened WOF. However, YE did not decrease the concentrations of WOF compounds and did not change the fatty acid composition and TBARS in surimi gels. Conversely, the addition of YE significantly increased the contents of free amino acids, N-containing compounds, and esters in surimi gels. The contents of total free amino acids, 2,6-dimethylpyrazine, and ethyl acetate in surimi gels with 2.5% YE were 1.5, 21, and 2.1 times higher than those in the control, respectively. Additionally, the sensory results of the spiked aroma models containing WOF compounds, 2,6-dimethylpyrazine, and esters showed that more than 9.4 µg/kg of 2,6-dimethylpyrazine with a baked-potato note and more than 6.1 µg/kg of ethyl acetate and 11.2 µg/kg of butyl acetate with a fruity note could significantly mask WOF. In conclusion, WOF in surimi gels could be masked by YE due to the high concentrations of pyrazines and esters. Practical Application: Yeast extracts could decrease the warmed-over flavor (WOF) due to the high concentrations of pyrazines (baked-potato note) and esters (fruity note). This finding extends the application of yeast extracts in the food industry. On the other hand, this study presents a reasonable solution for the reduction of WOF in surimi products.

Development of the gas chromatography/mass spectrometry-based aroma designer capable of modifying volatile chemical compositions in complex odors.

Many volatile organic compounds (VOCs) are used to produce various commercial products with aromas mimicking natural products. The VOCs responsible for aromas have been identified from many natural products. The current major strategy is to analyze chemical compositions and aroma qualities of individual VOCs using gas chromatography/mass spectrometry (GC/MS) and GC-olfactometry. However, such analyses cannot determine whether candidate VOCs contribute to the characteristic aroma in mixtures of many VOCs. In this study, we developed a GC/MS-based VOC collection/omission system that can modify the VOC compositions of samples easily and rapidly. The system is composed of GC/MS with a switching unit that can change gas flow routes between MS and a VOC collection device. We first applied this system to prepare gas samples for omission tests, and the aroma qualities of VOC mixtures with and without some VOCs were evaluated by panelists. If aroma qualities were different between the 2 samples, the omitted VOCs were likely key odorants. By collecting VOCs in a gas bag attached to the collection device and transferring some VOCs to MS, specific VOCs could be omitted easily from the VOC mixture. The system could prepare omission samples without chemical identification, preparation of each VOC, and laborious techniques for mixing VOCs, thus overcoming the limitations of previous methods of sample preparation. Finally, the system was used to prepare artificial aromas by replacing VOC compositions between different samples for screening of key odorants. In conclusion, the system developed here can improve aroma research by identifying key odorants from natural products.

Characterization of key aroma compounds in roasted chicken using SPME, SAFE, GC-O, GC-MS, AEDA, OAV, recombination-omission tests, and sensory evaluation.

Aroma compounds in the roasted breasts, thighs and skins of chicken were isolated by solvent-assisted flavor evaporation (SAFE), quantitated by gas chromatography-olfactometry-mass (GC-O-MS), analyzed by aroma extract dilution analysis (AEDA), and determined by recombination-omission tests and sensory evaluation. Forty-seven aroma compounds in total, including aldehydes, ketones, furans, pyrazines, and furanones, were selected by AEDA. Twenty-five compounds were selected as pivotal odorants (Odor Activity Value, OAV ≥ 1). Twenty aroma compounds significantly were identified by recombination and omission experiments. Anethole (fennel odor) was the highest OAV (＞ 1843). Hexanal (grassy) and (E, E)-2,4-decadienal (meaty) were the most abundant aldehydes identified in roasted chicken. 1-octen-3-ol (mushroom), methanethiol (cabbage) and dimethyl trisulfide (areca, sulfur) were considered the key compounds of the breast and thighs of roasted chicken. Notably, furanone and pyrazines, 4-hydroxy-5-methyl-3(2H)-furanone (caramel, sweet and burning odor), 3-ethyl-2,5-dimethylpyrazine (nutty, toasty) and 2,3-dimethyl-5-ethylpyrazine (nutty, toasty) had the most significant effect on roasted chicken odor, especially in the skin.

Characterization of key aroma compounds in aged Qingxiangxing baijiu by comparative aroma extract dilution analysis, quantitative measurements, aroma recombination, and omission studies.

Aging aroma is an intriguing but an understudied phenomenon in baijiu. The aromatic characteristics of Qingxiangxing (QXX) baijiu stored for 45 years (aged) were investigated using the sensomics approach and were compared with those of young baijiu (0 year-old). Aroma extract dilution analysis revealed 59 odorants, with many long-retained components exhibiting greater flavor dilution values in the aged than the young sample. The analysis of variance of the quantitative data showed significant differences between the young and aged baijiu for 26 compounds. Based on the variations in the content of these compounds, QXX baijiu aged for 0, 5, 15, 20, and 45 years was analyzed and twelve compounds were significantly correlated with aging duration. Finally, eight positively correlated compounds were omitted from the recombinant model, and sotolon, methional, vanillin, dimethyl trisulfide, benzaldehyde, and 3-hydroxy-2-butanone were confirmed to primarily contribute toward the aging aroma of QXX baijiu.

Comparison of potent odorants in raw and cooked mildly salted large yellow croaker using odor-active value calculation and omission test: Understanding the role of cooking method.

Cooking improves food aroma, but few studies have explored how cooking affects food aromas. Here, aroma changes in mildly salted large yellow croaker (Larimichthys crocea, MSLYC) after steaming, baking, frying, and deep frying was investigated. The raw fish was dominated by fishy notes but after cooking, the aroma became dominated by fatty notes. Nine volatiles, including hexanal, nonanal, (E, Z)-2, 6-nonadienal, (E, E)-2, 4-decadienal, 1-octen-3-ol, linalool, ethyl hexanoate, acetic acid and anethole, were identified as key odor-active compounds using GC-MS, OAV, and omission tests analyses. Changes in the concentrations of key odor-active compounds were mainly due to evaporation, oxidation of linolenic acids, and thermal catalyzed reactions. Interestingly, anethole was the key odor-active compound, providing new insight into the underlying reactions of cooked fish aroma.

Characterization of Key Odor-Active Compounds in Sun-Dried Black Tea by Sensory and Instrumental-Directed Flavor Analysis.

The aroma profile of sun-dried black tea (SBT) was identified by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O). A total of 37 scents were captured by using the GC-O technique, and 35 scents with odor intensities ranging from 1.09 ± 1.93 to 9.91 ± 0.29 were identified. Twenty-one compounds were further identified as key odor-active compounds with odor activity values (OAVs) greater than or equal to one. These key odor-active compounds were restructured with their detected concentrations, and the aroma profile of the selected SBT sample was successfully imitated to a certain extent. An omission test was performed by designing 25 models and confirmed that (E)-ß-damascenone, ß-ionone, dihydro-ß-ionone, linalool, and geraniol were the key odor-active compounds for the aroma profile of SBT. Meanwhile, phenylethyl alcohol, (E)-2-decenal, hexanal, and methyl salicylate were also important to the aroma profile of SBT. This study can provide theoretical support for the improvement of the aroma quality of sun-dried black tea.

Flavoromic determination of lactones in cheddar cheese by GC-MS-olfactometry, aroma extract dilution analysis, aroma recombination and omission analysis.

To systematically identify and quantify the γ- and δ-lactones in Cheddar cheeses, 20 samples from three sources (Ireland, the UK and the USA) were analysed by gas chromatography-mass spectrometry (GC-MS), gas chromatography-olfactometry (GC-O), aroma extract dilution analysis, aroma recombination analysis, and aroma addition/omission analysis. Nine lactones were detected in these samples, and one of these lactones, γ-undecalactone, was identified in Cheddar cheese for the first time. The quantitative results showed that the concentration of lactones in these cheeses usually increased as the length of their maturity period increased. γ-Octalactone, γ-undecalactone, γ-dodecalactone, δ-octalactone, δ-decalactone and δ-dodecalactone were identified as aroma-active substances based on their odour activity values and aroma extract dilution analysis, with flavour dilution factors ranging from 2 to 128. Aroma recombination and omission experiments based on a newly developed deodorised Cheddar matrix further validated the important contributions of these lactones to the overall aroma of Cheddar cheeses. The addition of each lactone to aroma recombination models reduced the aroma intensity of sour and rancid properties to various extents and improved the acceptability of the overall flavour.

Identification, quantitation and sensorial contribution of lactones in brandies between China and France.

Lactones are important flavor compounds in lots of foodstuffs. They also play an important role in brandy, but have not been studied at large. In this study, solid-phase extraction (SPE) and stir bar sorptive extraction (SBSE) combined with comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry (GC × GC-TOFMS) were applied to identify and quantify lactones in brandies between China and France. Totally 17 lactones were identified, four of which were detected only in SBSE. Among them, γ-valerolactone, γ-heptalactone, δ-octalactone, γ-undecanolactone and δ-dodecalactone were detected in brandy for the first time. The results of partial least squares-discriminant analysis (PLS-DA) revealed that lactones distinguished regional characteristics among different brandies. The omission test showed that four lactones (OAV > 1) had direct impact on the aroma of brandy, and other seven lactones at sub-threshold (0.1 < OAV < 1) provided peach and apricot aroma characteristics through synergistic effects.

Characterization of key odor-active compounds in sweet Petit Manseng (Vitis vinifera L.) wine by gas chromatography-olfactometry, aroma reconstitution, and omission tests.

Petit Manseng (Vitis vinifera L.) has become a popular variety in China for the production of semisweet and sweet wines. However, few studies focused on investigating the molecular odor code of its key odorants. In this study, the key odor-active compounds of Chinese sweet Petit Manseng wine were identified by gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). Fifty-five odorous zones were sniffed and identified by application of aroma extraction dilution analysis on a distillate prepared by liquid-liquid extraction and solvent-assisted flavor evaporation. Among them, isoamyl alcohol, ethyl octanoate, isovaleric acid, (E)-ß-damascenone, and phenylethanol particularly displayed with highest flavor dilution factors above 1024. The quantification of volatiles by headspace solid-phase microextraction coupled with GC-MS and GC coupled with triple quadrupole MS/MS and a calculation of odor activity values (OAVs) indicated 23 volatiles with OAVs above 1. Ethyl hexanoate showed the highest OAV with 208.8, followed by (E)-ß-damascenone (189.0), 3-mercaptohexanol (60.3), isoamyl acetate (45.4), and furaneol (40.1). The aroma of the sweet wine was successfully reconstituted by combining 42 aroma compounds in a model wine solution. 3-Mercaptohexanol, (E)-ß-damascenone, furaneol, γ-octalactone + γ-decalactone + γ-hexalactone, and ethyl cinnamate had important influence on the aroma of sweet Petit Manseng wine assessed by omission tests. Moreover, the discrimination of wines from three regions was successfully achieved by partial least squares discriminant analysis based on quantitative results of key odorants. 3-Mercaptohexanol was considered as the most responsible for the region discrimination and had highest concentrations in Petit Manseng wines from Jiaodong Peninsula. PRACTICAL APPLICATION: Understanding of the knowledge in key odorants of Petit Manseng wines could be useful to improve wine quality through viticultural and enological practices.

Identification of characteristic aroma components of butter from Chinese butter hotpot seasoning.

The potent aroma compounds in butter from four kinds of Chinese butter hotpot seasoning were first investigated by analyzing the isolates from solid-phase microextraction. A total of 49 aroma compounds were identified, and 23 of them were highly correlated with the aroma profiles of the butter by partial least squares regression analysis. Aroma extract dilution analysis and odor activity value calculations were applied to further reveal the dominant odorants. Fifty-three odorants with flavor dilution factors between 1 and 1024 were identified and OAVs of 17 odorants were greater than 1. Finally, an aroma recombination experiment was prepared by mixing the aroma-active compounds (OAVs > 1), and the aroma profile of the recombination showed good agreement with that of the original sample. Omission tests showed that 2-furfurylthiol, 2-acetylthiazole, anethole, (E)-2-decenal, and 1,8-cineole were the key odorants for the overall aroma of butter.

Enzymatic hydrolysis and auto-isomerization during ß-glucosidase treatment improve the aroma of instant white tea infusion.

The aroma changes in instant white tea resulting from ß-glucosidase treatment was investigated by quantitative descriptive analysis (QDA), gas chromatography-mass spectrometry (GC-MS), odour activity value analysis (OAV), aroma reconstruction and omission tests. The grassy, floral and sweet notes increased significantly (P < 0.05), and the roasted note decreased significantly (P < 0.05) upon ß-glucosidase treatment. Quantitative analysis showed that the concentrations of benzaldehyde, benzeneacetaldehyde, (Z)-3-hexen-1-ol, linalool, phenylethyl alcohol, cis-linalool oxide, trans-linalool oxide, hexanol, hotrienol and (E)-2-hexen-1-ol increased significantly (P < 0.05) after treatment; however, (Z)-3-hexen-1-ol isomerized to (E)-2-hexen-1-ol. OAV analysis, aroma reconstruction and the omission test showed that the grassy, floral and sweet notes increased as the (Z)-3-hexen-1-ol, cis/trans-linalool oxide and benzeneacetaldehyde increased, whereas the roasted note declined under the same conditions. The enzymatic hydrolysis of glycosidic precursors and the auto-isomerization of volatile compounds provide new information for understanding how ß-glucosidase treatment improves the aroma of tea products.

Elucidating the Odor-Active Aroma Compounds in Alcohol-Free Beer and Their Contribution to the Worty Flavor.

Alcohol-free beers (AFBs) brewed by cold-contact fermentation exhibit a flavor reminiscent of wort which affects consumer acceptability. The aims of this study were to identify the odor-active compounds in AFB and elucidate the contribution of these to the overall aroma and worty character of the beer. Using a sensomics approach, 27 odor-active aroma compounds were identified and quantitated using gas chromatography-mass spectrometry. The most odor-active compound was methional (boiled potato-like aroma), followed by 3-methylbutanal (cocoa-like), (E)-ß-damascenone (apple, jam-like), 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (curry, spicy-like), and phenylacetaldehyde (floral, honey-like). The important contribution of these flavor compounds to the worty and honey aroma of AFB was determined by sensory assessment of the recombinate in a beer-like matrix with omission tests. The role of 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone in AFB aroma was reported for the first time. The outcomes from this study are of relevance for the brewing industry to design strategies for the reduction of the wortiness of AFB.

Characterization of the Aroma of an Instant White Tea Dried by Freeze Drying.

The aroma of an instant white tea (IWT) was extracted through simultaneous distillation-extraction (SDE) and analyzed by sensory evaluation, gas chromatography-mass spectrometry-olfactometry (GC-MS-O), aroma reconstruction, omission test and synergistic interaction analysis. Sensory evaluation showed the IWT was dominated with floral and sweet notes. The SDE extract had the aroma similar to the IWT. The main volatile components in the SDE extract were benzyl alcohol, linalool, hotrienol, geraniol, α-terpineol, coumarin, camphene, benzeneacetaldehyde, 2-hexanone, cis-jasmin lactone and phenylethyl alcohol. GC-MS-O and aroma reconstruction experiments showed 16 aroma-active compounds. Linalool, trans-ß-damascenone and camphene were the major contributors to floral, sweet and green notes based on flavor dilution analysis and omission test. Linalool and trans-ß-damascenone had synergistic effect to promote floral and sweet notes. Camphene and trans-ß-damascenone had synergistic effect to reduce green and sweet notes. The study helps to understand the aroma of IWT and antagonism interactions among aroma-active volatiles.

Insights into the aroma profiles and characteristic aroma of 'Honeycrisp' apple (Malus × domestica).

'Honeycrisp' is a popular apple cultivar because of its superior appearance and flavor. We investigated its aroma profiles and characteristic aroma. Whereas the aroma profiles of developing fruits were dominated by aldehydes, postharvest fruit accumulated abundant esters, alcohols, and sesquiterpenoids. Most of these components showed maxima at the fruit's climacteric peak. There were more types and higher contents of sesquiterpenoids, aldehydes, and esters in the fruit skin than in the pulp, while alcohol contents and types were comparable between the pulp and skin. Aroma extract dilution and gas chromatography-olfactometry analyses revealed that hexyl 2-methylbutyrate, α-farnesene, 1,3-octanediol, hexanal, (E)-2-hexenal, hexanol, butanol, and 2-methyl-butanol are the most potent odor compounds in 'Honeycrisp' apple. Aroma reconstruction and omission tests combined with sensory analyses suggested that hexyl 2-methylbutyrate is responsible for the strong fruity note of 'Honeycrisp' apple, and that other alcohols, aldehydes, and α-farnesene are essential for its background aroma notes.

Characterization of the Key Aroma Compounds in Traditional Hunan Smoke-Cured Pork Leg (Larou, THSL) by Aroma Extract Dilution Analysis (AEDA), Odor Activity Value (OAV), and Sensory Evaluation Experiments.

The key aroma compounds in smoke-cured pork leg were characterized by gas chromatography-olfactometry coupled with aroma extract dilution analysis (GC-O/AEDA), odor activity value (OAV), recombination modeling, and omission tests. Ranking analysis showed that pork leg smoke-cured for 18 days had the best sensory qualities, with strong meaty, smoky, roasty, woody, and greasy attributes. Thirty-nine aroma-active regions with flavor dilution (FD) factors ranging from 9 to 6561 were detected. Overall, 3-ethylphenol had the highest FD factor of 6561, followed by 2,6-dimethoxyphenol, 3,4-dimethylphenol, 4-ethylguaiacol, 4-methylguaiacol, 3-methylphenol, and 2-acetyl-1-pyrroline, with FD ≥243. Among 39 aroma compounds, 27 compounds with OAVs ≥1 and were potent odorants. A similarity of 90.73% between the recombination model and traditional Hunan Smoke-cured Pork Leg (THSL) sample was obtained. Omission tests further confirmed that (E)-2-nonenal, 2-methoxy-4-vinylphenol, guaiacol, 3-ethylphenol, 2,6-dimethylphenol, 2-acetyl-1-pyrroline, and methional were key odorants in smoke-cured pork leg. Additionally, 2-acetyl-1-pyrroline (38.88 µg/kg), which contributes to a roasty aroma, was characterized here as a key odorant of smoke-cured pork leg for the first time.

Characterization of the key aroma compounds in Semnostachya menglaensis Tsui by gas chromatography-olfactometry, odor activity values, aroma recombination, and omission analysis.

The aroma profile of Semnostachya menglaensis Tsui (S. menglaensis) was identified by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). A total of 29 odor-active compounds, which have aroma intensities ranging from 0.2 ± 0.5 to 10.0 ± 0.0 were detected by GC-O, whereas 14 compounds were detected as important odorants by quantitative analysis and odor activity values (OAVs). An aroma recombination model prepared by mixing 12 odor-active compounds with OAVs ≥ 1 in an "aroma-free" S. menglaensis matrix showed good similarity to the aroma of original S. menglaensis. Omission tests showed that in the overall aroma of S. menglaensis, 2-propionyl-3,4,5,6-tetrahydropyridine, 2-propionyl-1-pyrroline, 1-octen-3-ol, and linalool were the key odorants, and 2-acetyl-3,4,5,6-tetrahydropyridine, 2-propionylpyridine, and α-ionone were also important.

Aroma enhancement of instant green tea infusion using ß-glucosidase and ß-xylosidase.

ß-Glucosidase and ß-xylosidase were investigated for their ability to improve the aroma of instant green tea. The aroma and corresponding contributors were analyzed by sensory evaluation, gas chromatography-mass spectrometry, and odor activity value. Their specific contributions to aroma attributes were further examined by aroma reconstruction and omission experiments. The ß-glucosidase treatment significantly enhanced floral and grassy notes, on account of the increases of geraniol, nonanal, and cis-3-hexen-1-ol, and weakened the caramel note, attributable to the increases of nonanal, cis-3-hexen-1-ol, geraniol, methyl salicylate, and decanal. The co-treatment with ß-glucosidase and ß-xylosidase further enhanced the grassy note, with further increase in nonanal and cis-3-hexen-1-ol, and further weakened the caramel note, with additional increase in nonanal, cis-3-hexen-1-ol, methyl salicylate, and decanal. The synergistic action of ß-glucosidase and ß-xylosidase provides new clues to the production of instant green tea infusions with high aroma quality.

Characterization of Potent Odorants Causing a Pickle-like Off-Odor in Moutai-Aroma Type Baijiu by Comparative Aroma Extract Dilution Analysis, Quantitative Measurements, Aroma Addition, and Omission Studies.

A potent unpleasant aroma presenting a pickle-like off-odor in Moutai-aroma type Baijiu was studied by comparative aroma extract dilution analysis (AEDA). Two Moutai-aroma type Baijiu samples, one having no off-odor (A) and the other one presenting pickle-like off-odor (B), were selected for chemical analysis and sensory evaluation. The aroma compounds were isolated by headspace solid-phase microextraction and liquid-liquid extraction. AEDA, quantitative analysis, and odor activity value calculation were performed in both the A and B samples. The main differences between the two samples were obtained for 12 compounds, presenting significantly higher concentrations in the off-odor sample. A total of 30 Moutai-aroma type Baijiu samples having different intensities of pickle-like sensory defect were analyzed to confirm the differences. An aroma addition test indicated that the 12 compounds with higher concentrations contributed to the pickle-like off-odor when spiked into sample A. Finally, a triangle test involving omission of the aroma compounds from the spiked A sample proved that 2-methyl-3-furanthiol, methional, methyl 2-methyl-3-furyl disulfide, dimethyl trisulfide, 2-furfurylthiol, methanethiol, dimethyl disulfide, and bis(2-methyl-3-furyl)disulfide with higher concentrations were generally responsible for the pickle-like off-odor in Moutai-aroma type Baijiu.

Identification of the Non-Volatile Taste-Active Components in Crab Sauce.

Crab sauce is a traditional umami seasoning in the coastal cities in South East China. The putative non-volatile taste-active components in crab sauce were measured, and their impacts on the taste were evaluated on the basis of the taste activity value (TAV), omission test, addition test and equivalent umami concentration (EUC). The EUC used to evaluate the synergistic effect of the flavor nucleotides and umami amino acids was 19.3 g monosodium glutamate (MSG)/100 mL, which illuminated that crab sauce had a very intense umami taste. The key non-volatile taste-active components in crab sauce demonstrated by the omission test and addition test were glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), alanine (Ala), lysine (Lys), histidine (His), 5'-inosine monophosphate (IMP), 5'-guanosine monophosphate (GMP), NaCl, KCl, serine (Ser) and valine (Val), and most of these components also had a higher TAV. So, the TAV could be a high-efficiency tool to predict the taste-active components, and the TAV combined with the omission test and addition test could be a very useful method to determine the taste-active components in crab sauce.

Sensory-Guided Analysis of Key Taste-Active Compounds in Pufferfish (Takifugu obscurus).

To investigate key taste-active components in Takifugu obscurus, 28 putative taste compounds in cooked muscle of T. obscurus were quantitatively analyzed and the pivotal components were identified by taste reconstitution, omission, and addition tests. Moreover, the role of flavor peptides in the overall taste profile of T. obscurus was evaluated. Sensory evaluation revealed that glutamic acid, serine, proline, arginine, lysine, adenosine 5'-monophosphate, inosine 5'-monophosphate (IMP), succinic acid, sodium, potassium, phosphates, and chlorides were the core taste-active contributors to T. obscurus. Besides glutamic acid, IMP, succinic acid, and potassium, the characteristic T. obscurus-like umami and kokumi profiles were induced by adding flavor peptides, among which Pro-Val-Ala-Arg-Met-Cys-Arg and Tyr-Gly-Gly-Thr-Pro-Pro-Phe-Val were identified as key substances on the basis of the addition test and dose-response analysis. The present data may help to reveal the secret of the delicious taste of T. obscurus and provide the basis for the development of deeper flavor analysis of pufferfish.