Article Cite This: J. Agric. Food Chem. 2019, 67, 7926−7934
pubs.acs.org/JAFC
Characterization of Key Aroma-Active Compounds in Black Garlic by Sensory-Directed Flavor Analysis Ping Yang,† Huanlu Song,† Lijin Wang,*,† and Hao Jing‡ †
Downloaded via GUILFORD COLG on July 17, 2019 at 08:48:37 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Research Center for Food Additive Engineering Technology, Laboratory of Molecular Sensory Science, Beijing Technology and Business University (BTBU), Beijing 100048, P. R. China ‡ College of Food Science and Nutritional Engineering, China Agricultural University (CAU), Beijing 100083, P. R. China ABSTRACT: Black garlic is a new garlic product produced through fermentation of fresh garlic and is very popular in Asia countries due to its health benefits. Its key aroma-active compounds were characterized by gas chromatography−olfactometry− mass spectrometry (GC-O-MS), gas chromatography−time-of-flight mass spectrometry (GC-TOFMS), and sensory evaluation. In total 52 aroma compounds were identified, and 15 of them with high flavor dilution (FD) factors based on aroma extract dilution analysis (AEDA) were selected and quantitated. Finally, 9 key aroma-active compounds, including acetic acid (sour), allyl methyl trisulfide (cooked garlic), Furaneol (caramel), diallyldisulfide (garlic), diallyltrisulfide (sulfur), (E,Z)-2,6-nonadien1-ol (cucumber), 3-methylbutanoic acid (sweat), 5-heptyldihydro-2(3H)-furanone (apricot), and diallyl sulfide (garlic), were determined through aroma recombination and omission experiment. In addition to the sulfur-containing compounds, heterocyclic compounds were the major aroma contributors in black garlic. Sensory evaluation revealed that the flavor profile of black garlic mainly consisted of sulfur, sour, sweet, fresh, sauce, gasoline, and roasted odors. KEYWORDS: black garlic, aroma-active compounds, aroma extraction dilution analysis, odor activity value, gas chromatography−olfactometry−mass spectrometry
■
genic effects on black garlic extract.10 Hence, black garlic, as a functional food, is beneficial for human health. Researchers have studied processing technology, nutrient content, functions, and physicochemical properties of black garlic.10 It is generally known that flavor is important for food quality and consumer perception. However, there are only a few studies on black garlic flavor analysis. Luo et al.5 detected 25 kinds of volatile compounds in black garlic, including thiophene, aldehyde, ester, acid, alcohol, furan, and ketone (47.42%, 9.73%, 9.67%, 8.24%, 2.94%, 2.85%, and 0.72%, respectively), among which 3-methyl-2-carboxyamide thiophene (36.510%), 2-propylthiophene (7.221%), 5-methylthiophene-2-carboxylic acid methyl ester (4.828%), and furfural (4.641%) were the main volatiles. Zhang et al.11 detected 50 kinds of volatile compounds in black garlic using simultaneous distillation extraction (SDE) method and revealed 3-vinyl-1,2dithiacyclohex-5-ene, diallyl disulfide, 2-ethyltetrahydrothiophene, 2-vinyl-1,3-dithiane, and N,N-dimethyl thiourea as the main compounds according to the relative percentage of peak area. Molina-Calle et al.12 identified 51 volatile compounds by headspace gas chromatography mass spectrometry (HS-GCMS) analysis. These compounds were classified into Salk(en)yl-L-cysteine derivatives, flavor compounds, and others. It was determined that the evolution of flavor compounds, especially sulfur volatiles, presented a remarkable difference in flavor between black garlic and fresh garlic. The heating
INTRODUCTION
Garlic (Allium sativum L.) has been widely cultivated and used as spice and condiment as well as traditional medicine for centuries.1 However, some people are reluctant to use raw garlic because of the strong pungent odor and the gastrointestinal problems caused by excessive consumption.1 Black garlic is a processed food made by aging fresh garlic under high temperature (65−90 °C) and high humidity (60%−80%) for 60−90 days.1 The whole garlic clove turns black after fermentation, and so it is called black garlic.2 It acquires a sweet and sour taste and a chewy, jellylike texture during the process, which is more acceptable by consumers.3 Black garlic has gained popularity in recent years in Asian countries like Japan, Singapore, and China due to high nutrient content.4 Compared to fresh garlic, the nutrients such as microelements, amino acids, reduced sugars, and vitamins are significantly higher in black garlic whereas water and fat contents are lower.2,5 Moreover, the antioxidant activity of black garlic is higher than that of fresh garlic.6 More than 10fold increase in superoxide dismutase-like activity and scavenging activity against hydrogen peroxide has been reported in black garlic extract compared with garlic extract in vitro.7 Liu et al.8 found that black garlic improves heart function in patients with coronary heart disease by improving circulating antioxidant levels. The improvement in antioxidant activity of black garlic was correlated with the increase in polyphenols as well as 5-hydroxymethyl furfuraldehyde (5HMF), which is an important intermediate of Maillard reaction during the aging process.9 Besides, there were also some antiallergic, antidiabetic, anti-inflammatory, and anticarcino© 2019 American Chemical Society
Received: Revised: Accepted: Published: 7926
May 25, 2019 June 20, 2019 June 28, 2019 June 28, 2019 DOI: 10.1021/acs.jafc.9b03269 J. Agric. Food Chem. 2019, 67, 7926−7934
Article
Journal of Agricultural and Food Chemistry
related to potato chips; sulfur-like, a kind of garlic flavor. Each attribute was evaluated on a 0−3 point scale with 0.5 steps. A necessary rest (1 min) between samples was allowed for sensory recovery. Coffee beans were prepared for olfactory recovery. Aroma assessors were unaware of the nature of sample being tested, and each sample was evaluated three times by each panelist. The final score was represented as the average of three replicate scores. Fresh garlic sample was also evaluated in triplicate by panelists to compare with black garlic. Solvent-Assisted Flavor Evaporation. Black garlic (50 g) was weighed, crushed, and mixed with ethyl ether (150 mL) in a Teflon bottle at 180 rpm and 4 °C for 5 h. Additionally, 2-methy-3heptanone as the internal standard (0.816 μg/μL, 50 μL) was added before the extraction procedure. The extract was distilled and purified using solvent-assisted flavor evaporation (SAFE) installation according to the method described by Engel et al.17 Then anhydrous sodium sulfate was added to remove excess water. After removing water, the extract was concentrated to ∼10 mL by passing through a Vigreux distillation column (50 cm × 1 cm internal diameter; Ban Xia Science and Technology Development Co., Ltd., Beijing, China). Approximately 500 μL of the concentrate was obtained under gentle flow of nitrogen. Approximately 1 μL of the sample was injected using a 10 μL syringe into the GC column for analysis. Each sample was analyzed 3 times. Solid-Phase Microextraction. Black garlic (2 g) was weighed, crushed, and placed in a headspace vial (40 mL) with 1 μL of 2methy-3-heptanone (0.816 μg/μL) and was incubated in a thermostatic water bath at 55 °C for 20 min. A solid-phase microextraction sampler with a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (50/30 μm, Supelco, Bellefonte, PA, U.S.A.) was used to extract the volatile compounds in headspace for 40 min at 55 °C. The sampler was inserted into the GC injector for thermal desorption at 250 °C for 5 min. Three replicates were maintained for each analysis. Gas Chromatography−Olfactometry−Mass Spectrometry Analysis. A gas chromatography−mass spectrometry (GC-MS) instrument (7890A-7000B, Agilent Technologies, Inc., Santa Clara, CA, U.S.A.) equipped with an olfactory detection port (Sniffer 9000, Brechbühler, Schlieren, Switzerland) was used to analyze and identify the aroma compounds. The polar DB-WAX capillary column (30 mm × 0.32 mm, 0.25 μm film thickness; J & W Scientific, Folsom, CA, U.S.A.) was used, and the column temperature was programmed to increase from 40 °C (after a 3 min hold) to 200 °C at a rate of 5 °C/ min and then to 230 °C at a rate of 10 °C/min with a 5 min final hold. Ultrahigh-purity helium (99.999%, Beijing AP BAIF Gases Industry Co., Ltd., Beijing, China) was used as the carrier gas and set at a flow rate of 1.2 mL/min. Electron-impact mass spectra were generated at 70 eV ionization energy with an m/z scan range from 50 to 350. MS source temperature was 230 °C. GC × GC-TOFMS System. Agilent 7890B GC combined with EI0610 TOF (He Xin Analytical Instrument Co., Ltd., Guangzhou, China) was used to further identify the aroma compounds of black garlic. We used a nonpolar HP-5 ms UI (60 m × 0.25 mm, 0.25 μm film thickness; Agilent Technologies) primary column and a midpolar DB-17 ms (1.85 m × 0.18 mm, 0.18 μm film thickness; Agilent Technologies) secondary column. The column temperature was programmed to increase from 40 °C (after a 3 min hold) to 280 °C at a rate of 5 °C/min with a 5 min final hold. Both columns were housed inside the same GC oven. Ultrahigh-purity helium was set at a constant flow rate of 2 mL/min. The split ratio was 20:1. Electronimpact mass spectra were generated at 70 eV ionization energy with an m/z scan range from 40 to 500. TOFMS source temperature was 220 °C. A solid-state modulator SSM1800 (J&X Technologies, China) was used for the heating and cooling stage between the two columns. The cold zone temperature was set at −50 °C. The temperatures of entry hot zone and exit hot zone were 30 and 120 °C offset relative to oven temperatures, respectively, with a cap temperature of 320 °C for both hot zones. The modulation period was 4 s.18
process decreased the concentration of the sulfur volatiles, while Maillard reaction increased the concentration of sweet ́ and roasted volatiles. Martinez-Casas et al.3 found that there was a significant difference between the volatile profiles of black garlic and fresh garlic and emphasized that the relative concentration of derivatives of S-alk(en)-yl-L-cysteine was decreased in black garlic compared with fresh garlic. Zhang et al.13 indicated that raising the temperature to 90 °C could speed up the aging time; however, it produced bitter and sour taste. It is still not clear which aroma-active compounds contribute to the final aroma profile of black garlic. Therefore, the characterization of aroma compounds of black garlic is indispensable. This research aimed to characterize the key aroma-active compounds in black garlic by modern separation and extraction technology and sensory-directed flavor analysis, such as gas chromatography−olfactometry−mass spectrometry (GC-O-MS), aroma extract dilution analysis (AEDA), odor activity value (OAV), and aroma recombination and omission experiment.14 This study will help further study on the formation pathways of black garlic flavor during processing and makes it possible to control aroma generation by adjusting the production conditions.
■
MATERIALS AND METHODS
Samples. Fresh garlic was purchased from Jinxiang county, Shandong province. Black garlic was prepared from fresh garlic according to a previously described method.15 Briefly, fresh garlic cloves were heated from 30 to 90 °C using an incremental temperature scheme with a relative humidity of 75%. The color of black garlic and the contents of alliin, S-allylcysteine, and phenolic substance were monitored during the thermal process (data not shown). Chemicals. Chemicals (purity >99%) used in the study are as follows: ethyl ether, n-hexane, and anhydrous sodium sulfate were purchased from Lab Gou e-mall (Beijing, China). Ethyl ether was freshly distilled prior to use. Other chemicals including 2-methyl-3heptanone, n-alkanes (C7−C30), diallyl trisulfide, 3-(methylthio)propionaldehyde, 3-methylbutanoic acid, Furaneol, ethanol, nonanal, furfural, acetoin, 1-octen-3-one, dimethyl disulfide, dimethyl trisulfide, 2,6-dimethylpyrazine, butyrolactone, 2-furanylmethanol, ethyl acetate, and acetic acid methyl ester were purchased from Sigma-Aldrich (St. Louis, U.S.A.). Dihydro-4-hydroxy-2(3H)-furanone and 1-hydroxy-2butanone were bought from Macklin Biochemical Co., Ltd. (Shanghai, China). The chemicals 2(5H)-furanone, 1-(2-furanyl)ethanone, (E,Z)-2,6-nonadien-1-ol, dimethyl sulfoxide, and 5heptyldihydro-2(3H)-furanone were purchased from TCI (Shanghai, China). Acetic acid and propanoic acid were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Diallyl sulfide and diallyl disulfide were purchased from Chromadex, Inc. (Los Angeles, U.S.A.). Allyl methyl trisulfide was bought from Li Da Flavors and Fragrances Co., Ltd. (Beijing, China). Nitrogen gas (99.9992% purity) was purchased from Beijing AP BAIF Gases Industry Co., Ltd. (Beijing, China). Liquid nitrogen was bought from XianHeyu Trading Co., Ltd. (Beijing, China). Aroma Profile Evaluation. Aroma profile was evaluated by 12 trained panelists who were recruited from the Laboratory of Molecular Sensory Science, Beijing Technology and Business University (Beijing, China). Daily training sessions were conducted for a week to ensure that the panelists could accurately recognize the aroma of black garlic. Flavor attributes were described following a discussion among the panelists and included sulfur-like, sour, sweet, fresh, sauce-like, gasoline-like, and roasted odors. The specific descriptions and training session standard of each attribute are as follows:16 sour, the smell similar to vinegar; sweet, the caramel-like flavor; fresh, the flavor cleared to cucumber; sauce-like, the same smell as soy sauce; gasoline-like, the petrol-like smell; roasted, a flavor 7927
DOI: 10.1021/acs.jafc.9b03269 J. Agric. Food Chem. 2019, 67, 7926−7934
Article
Journal of Agricultural and Food Chemistry Table 1. Fifty-Two Aroma Compounds Detected in Black Garlic RIb no.
compounds
DB-WAX
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
methylthiiranea ethyl acetate ethanol dimethyl disulfide allyl alcohola 2-methyl-1-butanola diallyl sulfide 3,4-dimethylthiophenea allyl methyl disulfidea acetoin acetic acid methylester unknownf 1-hydroxy-2-butanone dimethyl trisulfide 2-acetyl-1-pyrrolinea nonanal 1-octen-3-one allyl propyl disulfidea 3-(methylthio)propionaldehyde 2,6-dimethylpyrazine 2-methyl-1,3-dithianea diallyl disulfide benzofurana 3H-1,2-dithiolea unknownf propanoic acid acetic acid unknownf 1-(2-furanyl)-1-propanonea dimethyl sulfoxide furfural allyl methyl trisulfide 1-(2-furanyl)ethanone 2-methylpropanoic acida 2-propionylfurana 2(5H)-furanone (E,Z)-2,6-nonadien-1-ol butyrolactone diallyl trisulfide 2-furanylmethanol 3-methylbutanoic acid 3-vinyl-1,2-dithiacyclohex-4-enea 2-hydroxy-3-methylcyclopent-2-en-1-onea unknownf 2-vinyl-4H-1,3-dithiinea unknownf allyl propyl trisulfidea 4-methoxy-2,5-dimethyl-3(2H)-furanonea Furaneol 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-onea 5-heptyldihydro-2(3H)-furanone dihydro-4-hydroxy-2(3H)-furanone
844 908 984 1043 1087 1190 1208 1224 1252 1255 1270 1277 1341 1351 1354 1368 1400 1401 1421 1428 1439 1451 1470 1490 1491 1498 1507 1528 1540 1546 1552 1560 1605 1670 1682 1712 1732 1738 1756 1767 1783 1856 1940 1958 1967 2005 2016 2050 2159 2219 2428 2547
HP-5
