Lichenysin, a Cyclooctapeptide Occurring in Chinese Liquor

The solvents were water containing 0.1% formic acid as solvent A and methanol as ... (4-EG) (Figure 1) were respectively dissolved in the model soluti...
0 downloads 0 Views 634KB Size
Download PDF
Article pubs.acs.org/JAFC

Lichenysin, a Cyclooctapeptide Occurring in Chinese Liquor Jiannanchun Reduced the Headspace Concentration of Phenolic OffFlavors via Hydrogen-Bond Interactions Rong Zhang, Qun Wu, and Yan Xu* State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China ABSTRACT: Nonvolatile compounds play important roles in the quality of alcoholic beverages. In our previous work, a type of cyclooctapeptide lichenysin was newly identified in Chinese strong-aroma type liquor. In this work, it was found that lichenysin could selectively affect aroma volatility in strong-aroma type (Jiannanchun) liquor. Interaction of lichenysin and volatile phenolic compounds (off-odors in strong-aroma type liquor) was characterized using headspace solid-phase microextraction coupled with gas chromatography−mass spectrometry (HS-SPME-GC-MS). HS-SPME results indicated that lichenysin very efficiently suppressed the volatility of phenolic compounds by 36−48% (P < 0.05). Thermodynamic analysis showed that the binding process was mainly mediated by hydrogen bonding. Furthermore, the mixture of lichenysin and 4-ethylguaiacol revealed intermolecular cross peaks between the aH (Val) of lichenysin and the 1H of 4-ethylguaiacol, by using nuclear Overhauser effect spectroscopy. This study will help to further understand the interaction mechanisms between flavor and nonvolatile matrix components in Chinese liquors. KEYWORDS: lichenysin, phenolic off-flavors, aroma volatility, hydrogen-bond interaction

■

INTRODUCTION Chinese liquor is a traditionally indigenous and famous distilled beverage in China.1 The various aroma profiles of Chinese liquors result from their complexity in manufacturing practices. During the production, Chinese liquor is typically distilled from fermented sorghum. The sorghum used for liquor fermentation is cooked and then mixed with a fermentation starter (Daqu powder). Daqu made from wheat is rich in various microorganisms including bacteria, yeast, and fungi. They are typically carried out at 28−32 °C under anaerobic conditions in the solid-state fermentation. After fermentation, the liquor is distilled out with steam from fermented sorghum by the means of traditional solid distillation. Then, it is aged in a china jar (sealed tightly) for more than 3 years at 15−25 °C.1 The quality of Chinese liquor depends on the volatile and nonvolatile compounds. In Chinese strong-aroma type liquors, a sum of 132 aroma compounds was detected using gas chromatography olfactometry (GC-O) coupled with mass spectrometry (MS).2,3 Besides the volatile compounds, nonvolatile compounds also play important roles in the liquor quality. In past research, we isolated and identified a cyclooctapeptide lichenysin in many different Chinese liquors as a nonvolatile compound, using preparative high-performance liquid chromatography (HPLC) coupled with a quadrupoletime-of-flight (Q-TOF) mass spectrometer. Lichenysin is produced by Bacillus licheniformis in the fermented sorghum and is most likely to be distilled into the liquor with steam by means of traditional solid distillation. The concentration of this nonvolatile compound is the highest to reach 112 μg/L in the Dongjiu liquor (unpublished data). However, little information has been reported about the nonvolatile compounds affecting © 2014 American Chemical Society

aroma perception in Chinese liquors, especially their interaction mechanism with aroma compounds. The nature of interactions between odor and nonvolatile matrix components may depend on the physicochemical properties of the aroma compound and the binding mechanisms. The most frequent method to measure the interactions that occur between aroma compounds and other food or beverage constituents is headspace analysis, the headspace solid-phase microextraction (HS-SPME) method.4−6 Aronson and Ebeler7 have shown that gallic acid (in 1% ethanol solution) significantly decreased the volatility of 2methylpyrazine by HS-SPME. To further explore the nature of the interactions between aroma compounds and nonvolatile wine compounds, NMR spectroscopy has been proven as one of the most powerful techniques. It was found that the interactions between the galloyl ring of gallic acid and the aromatic ring of methylpyrazine were stabilized by hydrogen bonds, using NMR techniques.8 β-Cyclodextrin (β-CD) is usually used for the microencapsulation of aroma compounds in food processes. The binding nature of β-CD with benzyl alcohol and 2-methylbutyric acid was studied by using 1H and 13 C NMR experiments.9 Up to now, there is no report about the interaction mechanism between nonvolatile compounds and aroma compounds in Chinese liquor. In this study, we characterized that the cyclooctapeptide lichenysin reduced the headspace concentration of phenolic offReceived: Revised: Accepted: Published: 8302

April 30, 2014 July 13, 2014 July 26, 2014 July 26, 2014 dx.doi.org/10.1021/jf502053g | J. Agric. Food Chem. 2014, 62, 8302−8307

Journal of Agricultural and Food Chemistry

Article

flavors in strong-aroma type liquor and the model solution, using HS-SPME-GC-MS and sensory evaluation. Furthermore, we used thermodynamic analysis and nuclear Overhauser effect spectroscopy (NOESY) to elucidate the nature of binding between lichenysin and phenolic compounds.

■

cm, Supelco Inc., Bellefonte, PA, USA) was used for analyte extraction. A total of 8 mL of sample was spiked with 10 μL of internal standard (hexyl formate for nonpolar compounds, 760 μg/L in ethanol, 2octanol for polar compounds, 810 μg/L in ethanol). The sample was transferred to a screw-capped, straight-sided headspace vial with a 15 mL volume. The vial was tightly capped with a Teflon-faced silicone septum. The samples were equilibrated at 30 °C for 5 min and extracted for 45 min at the same temperature6,7 under stirring (500 rpm, on for 20 s, off for 0 s). After extraction, the fiber was inserted into the injection port of the gas chromatograph (250 °C) for 5 min to desorb the analytes. All analyses were made in triplicate, using a modified HS-SPME method.6,7 GC-MS Analysis and GC-MS Data Analysis. For the GC-MS analysis, an Agilent 6890N gas chromatograph coupled to an Agilent 5975 mass selective detector (MSD) was used. The capillary column used was a DB-Wax column (30 m length × 0.25 mm i.d., 0.25 μm film thickness; Phenomenex, Torrance, CA, USA). The injector temperature was 250 °C, and the splitless mode was used. The conditions were as follows: starting temperature 50 °C (holding 2 min), then raised to 230 °C at the rate of 6 °C/min, and held at 230 °C for 30 min. The column carrier gas was helium with a purity of 99.9995% at a constant flow rate of 2 mL/min. The electron impact energy was 70 eV, and the ion source temperature was set at 230 °C. Full-scan acquisition was used in the ranges of masses (30−350 amu). Identification of aroma compounds in Chinese liquor has been carried out on a DB-Wax column and a DB-5 column (30 m × 0.25 mm i.d., 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA). It was based on the following criteria: comparing determined mass spectra and retention indices (RIs) with those of reference compounds. RIs were determined using a series of standard linear alkanes C5−C23 in accordance with a modified Kovats method.11 Peak area integration of unique masses was conducted using MSD Chemstation (G1701-90057, Agilent, Santa Clara, CA, USA). We compared the peak areas of aroma compounds in the control group (the liquor sample) with that of experiment group (the liquor sample added with lichenysin) by the means of HS-SPME-GC-MS. The net changes in peak areas were obtained by dividing the peak area of the control group subtracted from that of experiment group by the control. To eliminate the effect of other factors, we kept the control group and the experiment group at the same condition. From the net changes in peak areas, we studied about the effect of lichenysin on the volatility of different aroma compounds in the Chinese liquor. The use of peak area data to express aroma release was sufficient for the purpose of studying about aroma volatility in this work. Sensory Evaluation of Model Solutions. The model solutions were presented to 10 panelists (China Distilled Spirit Appraisal Commissioner) at room temperature of 25 °C. According to Aronson’s method,7 the judges were trained in three test sessions and then carried up the experiment. They learned how to use the intensity scales by practicing with a number of samples. They familiarized themselves with the aroma and the low (zero) and high (the following concentrations) standards for each flavor compound. The model solution is 46% ethanol/water solution by volume, in which the phenol concentration is 1.28 mg/L, the 4-MP concentration is 3.20 mg/L, the 4-EP concentration is 0.38 mg/L, the guaiacol concentration is 0.38 mg/L, and the 4-EG concentration is 1.54 mg/L. Each flavor compound was evaluated respectively for two weeks. Samples of 30 mL were randomly presented in coded clear white wine glasses covered by tinfoil plates. The samples consisted of five controls and five phenolic/lichenysin solutions. The five phenolic compounds were respectively dissolved in the model solutions. Standards were used to anchor the ends of the scale for the judges. The aroma intensity of the samples was marked on a 10 cm line scale in terms of deviation. The line scale was labeled “less than reference” and “more than reference” at the two ends. Judges were asked not to swirl the samples so as not to disturb the equilibrium. Lastly, they were allowed to resniff samples as long as they waited approximately 1 min before smelling the same sample a second time. All evaluations were conducted in a well-ventilated room. Each phenol and lichenysin combination was replicated during the triangle tests.

MATERIALS AND METHODS

Chemicals. DMSO-d6 and tetramethylsilane (TMS) were purchased from Sigma Technology (St. Louis, MO, USA). The chemical standards of all the aroma compounds and internal standards (hexyl formate and 2-octanol) were purchased from Acros Organics (Fair Lawn, NJ, USA). Formic acid was purchased from Acros Organics. Methanol and absolute alcohol were purchased from Tedia (Fairfield, OH, USA). The reference standards were prepared in an ethanol solvent. All standard solutions were flushed with nitrogen to avoid oxidation of the phenols and kept in the freezer at −20 °C in the dark. We got the C15−lichenysin from the Chinese liquor sample Dongjiu liquor, from the Dongjiu distilleries. The compound was isolated from the fractions by means of preparative HPLC (autopurity system, Waters, Milford, MA, USA) material, by using an Xbridge Prep C18 (250 mm × 19 mm, 10 μm) reversed-phase Xbridge steel column. The solvents were water containing 0.1% formic acid as solvent A and methanol as solvent B. The program was conducted by means of gradient elution, and the detection was selected at wavelength of 210 nm. The structure of C15−lichenysin was confirmed by the Waters Synapt Q-TOF system. It is built from a heptapeptide and a β-hydroxy fatty acid with a chain of 15 carbon atoms. The purity of C15− lichenysin that we got from the liquor can reach 99%. The Chinese liquor sample Jiannanchun liquor was purchased from the market. Jiannanchun is one of the most famous strong-aroma type liquors, which typically have strong fruity, pineapple-like, and banana-like aromas.1 This liquor is the typical representatives of strong-aroma type. Sample Preparation for SPME Extraction and SPME Conditions. An aliquot of C15−lichenysin was dissolved in Chinese liquor (strong-aroma type liquor) and model solution. Phenol, 4methylphenol (4-MP), 4-ethylphenol (4-EP), guaiacol, and 4-ethylguaiacol (4-EG) (Figure 1) were respectively dissolved in the model

Figure 1. Chemical structures of C15-Lichenysin and five phenolic compounds. solution (46% ethanol/water solution, v/v). The phenol concentration was 1.28 mg/L, the 4-MP concentration was 3.20 mg/L, the 4-EP concentration was 0.38 mg/L, the guaiacol concentration was 0.38 mg/L, and the 4-EG concentration was 1.54 mg/L. The concentration of phenolic compound in the model solution reached close to the strong aroma type Jiannanchun liquor determined by HS-SPME-GCMS.10 The C15−lichenysin is the highest to reach 75% of the total in Chinese liquor. Therefore, the effect of C15−lichenysin on aroma compounds release was evaluated by HS-SPME-GC-MS. For the SPME, an automatic headspace sampling system (Multi Purpose Sample MPS 2 with a SPME adapter, from GERSTEL Inc., Baltimore, MD, USA) with a 50/30 μm DVB/CAR/PDMS fiber (2 8303

dx.doi.org/10.1021/jf502053g | J. Agric. Food Chem. 2014, 62, 8302−8307

Journal of Agricultural and Food Chemistry

Article

Table 1. Effect of C15−Lichenysin on Mean HS-SPME-GC-MS Peak Areas for Aroma Volatility in Chinese Liquor (n = 3) RI compd

DB-Wax

DB-5

odor descriptorsa

ethyl acetate ethyl butanoate ethyl pentanoate ethyl hexanoate ethyl heptanoate ethyl 2-hydroxypropanoate ethyl octanoate ethyl decanoate 2-methylpropanol 3-methylbutanol acetic acid butanoic acid hexanoic acid furfural benzaldehyde ethyl 2-phenylacetate ethyl 3-phenylpropanoate phenol 4-methylphenol 4-ethylphenol guaiacol 4-ethylguaiacol

887 1031 1144 1233 1316 1348 1410 1624 1112 1210 1473 1641 1857 1467 1501 1775 1878 2007 2090 2185 1862 2031

585 800 900 1010 1095 815 1195 1390 610 783 N.D. 795 N.D. 830 960 1247 1353 970 N.D. 1181 1009 1281

pineapple pineapple apple fruity, floral fruity fruity fruity fruity, grape wine, solvent rancid, nail polish acidic, vinegar rancid, cheesy sweaty, cheesy sweet, almond fruity, berry rosy, honey fruity, floral phenol, medicinal animal, phenol smoky spicy, animal clove, spicy

identificationb MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,

RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI

controlc 2 085 585 7 392 103 942 935 3 581 850 2 360 905 164 962 234 2 411 046 2 176 588 3 325 027 382 271 2 391 796 273 365 368 519 153 321 413 593 5 256 621 148 759 1785 1594 3457 62 831 4774

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

129 699a 376 057a 29 573a 156 172a 55 039a 25 280 185a 115 910a 42 168a 143 751a 133 554a 39 720a 10 054a 17 128a 6164a 1787a 346 629a 498a 276a 261a 846a 24 086a 541a

lichenysin, 160 μg/L

net change

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.62% 2.29% 0.90% 1.21% 1.89% 4.53% 2.80% 3.36% 3.40% 0.69% 1.17% 6.67% 3.71% 14.68% 4.77% 11.42% 5.33% 76.86% 58.85% 47.98% 63.35% 55.14%

2 051 879 7 222 882 951 414 3 538 357 2 405 441 157 489 181 2 478 513 2 249 724 3 438 090 384 928 2 419 804 291 606 382 181 175 827 433 321 4 656 515 156 688 413 656 1798 23 025 2142

61 274a 241 102a 17 454a 156 168a 61 676a 27 761 731a 178 558a 175 299a 107 324a 163 154a 35 247a 7584a 24 427a 12 850a 22 105a 850 976a 6358a 272b 201b 844b 6931b 664b

a

Odor descriptors of the aroma compounds were from refs 2 and 3. bIdentification based on comparing determined MS and RI with that of reference compounds. cDifferent letters within a row denote significantly different means at p ≤ 0.05. Three samples include one control and two experiment groups. Judges periodically took some time to rest during the experiment to minimize fatigue. The responses for the test were decoded manually in centimeters and entered into an Excel spreadsheet. NMR Procedures. NMR measurements were performed on a Bruker Avance 500 MHz spectrometer at 25 °C. Samples were prepared in DMSO-d6; tetramethylsilane (TMS) were added to the DMSO-d6 at a level of 0.05%. To investigate the concentration dependence of chemical shift changes, a 2.5 mM phenolic compound solution (0.5 mL) was mixed with various amounts of lichenysin covering molar ratios of 1:4, 1:2, 3:4, 1:1, 2:1, and 2.5:1 in DMSO-d6 and observed by the single-pulse method. The chemical shift changes of protons were measured relative to the reference chemical shift. The experiments were carried out at 25, 45, and 60 °C, from which equilibrium association constants (Ka) and thermodynamic parameters (ΔH and ΔS) were obtained using a nonlinear estimation program, ORIGIN (Microcal Software Co. Inc., Northampton, MA, USA). Then, a 2.5 mM lichenysin solution (0.5 mL) was mixed with various amounts of 4-EG to cover molar ratios of 1:0, 4:1, 4:2, 1:1, and 1:2 in DMSO-d6. The NOESY spectrum was observed by the mean of NOESY measurement. The NOESY spectra were acquired with a spectral width of 7 kHz in 2K data points using 16 scans for each of the 512 t1 increments. The spin lock power level and mixing time were set to 29 dB and 60 ms, respectively. Data processing was performed by using Topspin NMR software (version 2.1; Bruker, Rheinstetten, Germany). Statistical Analysis Software. Each sample was analyzed in triplicate for each analysis. Results presented in the tables were shown with standard deviations and the arithmetic means. All data were statistically assessed using SPSS version 19.0 for Windows statistical package (SPSS Inc.; Chicago, IL, USA). The significance of differences between mean values obtained was determined using a one-way ANOVA (P < 0.05).

The study examined the effect of lichenysin on the volatility of aroma compounds in Chinese liquor (strong-aroma type liquor). In this study, a sum of 22 volatile compounds in Chinese liquor was identified and quantitated by using the HSSPME method (Table 1). These following compounds were eight esters, two alcohols, three acids, two aldehydes, two aromatic compounds, and five phenolic compounds. Addition of lichenysin (at concentration of 160 μg/L) to the liquor sample resulted in significant but variable suppressing effects on headspace concentration (HS-GC peak area) depending on the nature of flavor compound examined (Table 1). For esters, the headspace concentrations were influenced little by lichenysin, where the volatility of esters decreased by 1−5% in the liquor sample. For alcohols and aldehydes, the volatility of these compounds was affected the least by 1−15%. For volatile acids, the headspace concentrations were influenced by 1−7%. The most pronounced effects were interactions between lichenysin and phenolic compounds (phenol, 4-MP, 4-EP, guaiacol, and 4-EG). It might be because the phenolic hydroxyl group of these compounds formed hydrogen bonds with the carboxyl group of the lichenysin. The headspace concentration of phenol, 4-MP, 4EP, guaiacol, and 4-EG significantly decreased by 77%, 59%, 48%, 63%, and 55%, respectively, in the liquor sample. The volatilities of esters, alcohols, aldehydes, and volatile acids were affected little. Thus, lichenysin selectively affected aroma volatility in Chinese liquor. Interaction between C15−Lichenysin and Phenolic Compounds in Model Solutions by HS-SPME-GC-MS and Sensory Evaluation. Volatile phenolic compounds in Chinese liquor are considered as off-odors,10 in which 4-MP and 4-EG, respectively, give horsy and medical smells. We chose phenol, 4-MP, 4-EP, guaiacol, and 4-EG as aroma compounds. Interactions between lichenysin and phenolic

■

RESULTS AND DISCUSSION Interaction between C15−Lichenysin and Aroma Compounds in Chinese Liquor by HS-SPME-GC-MS. 8304

dx.doi.org/10.1021/jf502053g | J. Agric. Food Chem. 2014, 62, 8302−8307

Journal of Agricultural and Food Chemistry

Article

ation with lichenysin in DMSO-d6. Exchangeable protons such as those in carboxylic acids or alcohols do not give separate resonances in D2O or ethanol-d6. Therefore, the solvent (DMSO-d6) was chosen in which exchange is so slow that coupling between OH and adjacent protons can be observed. A titration process was then used to determine the concentration equilibrium constant, Ka, based on the chemical shift changes of phenolic protons. The 1H proton of each phenolic compound, which was most significantly affected by lichenysin complexation (Figure 3), was selected to obtain the association

compounds were studied in model solution by using the HSSPME-GC-MS method. In Figure 2, lichenysin significantly

Figure 2. Effect of C15−lichenysin concentration on mean HS-GC peak area for phenolic compounds in model solution (46%, ethanol/ water solution, n = 3). Figure 3. Nonlinear curve fitting of chemical shift data for mixtures of 4-EG (2.5 mM) and lichenysin in DMSO-d6 at 25 °C.

decreased the headspace concentration (HS-GC peak area) of the whole phenolic compounds relative to control group (without the addition of lichenysin), and the effect was the most pronounced when the concentration of lichenysin was 160 μg/L. The headspace concentrations of phenol, 4-MP, and 4-EP decreased by 39%, 45%, and 48%, respectively, (p < 0.05). The volatility of guaiacol and 4-EG in model solution decreased by 30% and 45% (p < 0.05). Later from that concentration, the effect of lichenysin on the volatility of phenolic compounds tended to reach an equilibrium state. When the concentration of lichenysin was 16 μg/L, the headspace concentrations of phenolic compounds decreased by 17−22%. Ten trained sensory panelists were asked to test the aroma intensity scores of the model solution and also the one with lichenysin addition. Lichenysin (160 μg/L in model solution) significantly suppressed aroma perception of phenolic compounds (p < 0.05, Table 2). Thus, lichenysin could efficiently suppress the volatility of phenolic off-flavors in Chinese liquor. Thermodynamic Parameters and Acting Forces between Lichenysin and Phenolic Compounds. To further study the nature of the phenolic/lichenysin interaction, the changes in the chemical shifts of protons in the phenolic compounds were evaluated by using 1H NMR, upon complex-

constant, Ka, assuming a 1:1 stoichiometry between phenolic compound (2.5 mM) and lichenysin: LP Ka = (1) L×P .In eq1, L, P, and LP represent the mole fractions of the free lichenysin, the free phenolic compound, and the bound form of the phenolic compounds, respectively. When fast exchange processes take place, the NMR spectrum for each proton shows a single signal, averaged in the proportions of the partial mole fractions of the complexed and free molecules. Therefore, the observed chemical shift (δobs) of a signal is a weighted average of the chemical shifts in two possible environmentsthe chemical shift for the free flavor (δP) and the chemical shift for the bound flavor (δLP): δobs = δP + (δ LP − δ P)

control 8.6 7.8 8.0 8.2 7.9

± ± ± ± ±

0.6a 0.4a 0.5a 0.7a 0.5a

16 μg/L 6.8 6.6 7.2 7.6 7.2

± ± ± ± ±

0.8a 0.6a 0.8a 0.4a 0.4a

160 μg/L 4.0 3.5 3.0 5.1 4.2

± ± ± ± ±

(2)

,where P0 represents the total mole fraction of the phenolic compound. Values of Ka, δP, and δLP leading to a best fit of the proton chemical shift changes were estimated by a nonlinear curve fitting program, and theoretical curves were derived from eq2. The curve fit of a 4-EG (2.5 mM) and lichenysin mixture is shown in Figure 3. Quantitative analysis of NMR spectra recorded at 25, 45, and 60 °C yielded association constants (Ka) for the interaction of the five phenolic compounds with lichenysin (Table 3). The interaction forces between phenolic compounds and lichenysin mainly include four types: hydrogen bonding, hydrophobic interaction, electrostatic interaction, and van der Waals force. The thermodynamic parameters free energy change (ΔG), enthalpy change (ΔH), and entropy change

Table 2. Mean Perceived Aroma Intensity Scoresa for Phenolic Compounds with Added Lichenysin phenol 4-methylphenol 4-ethylphenol guaiacol 4-ethylguaiacol

LP P0

0.4b 0.8b 0.4b 0.5b 0.6b

a

Different letters within a row denote significantly different means at p ≤ 0.05, n = 10. 8305

dx.doi.org/10.1021/jf502053g | J. Agric. Food Chem. 2014, 62, 8302−8307

Journal of Agricultural and Food Chemistry

Article

the binding phenomenon between lichenysin and these two phenolic compounds. Meanwhile, the negative ΔH values may account for the involvement of hydrogen bonding in this interaction, as 1H atoms on the phenolic compounds can easily form hydrogen bonds with amino acid residues of lichenysin. Thus, the main acting force between lichenysin and phenolic compounds was hydrogen-bond interaction. 1 H NMR and Two-Dimensional NOESY in the Complexes of Lichenysin and 4-EG. To better evaluate the binding site of the interactions and determine the spatial proximity of interacting protons, we chose 1H NMR and twodimensional (2D) NOESY for the complexes of lichenysin and 4-EG. When passing from the free to the complex state, the 1H NMR chemical shifts change for individual molecules in the complex. By using 1H NMR, the changes in the chemical shifts of protons in the lichenysin were evaluated, upon complexation with 4-EG in DMSO-d6. For complexes of lichenysin and 4-EG, the special proton of both compounds underwent chemical shift changes compared to those of their free state. When the 4EG molar concentration was onefold with respect to that of lichenysin, a gentle downfield shift (4.10 ppm) of aH (Val) of lichenysin is observed in Figure 4, as compared with that (4.07 ppm) of its free state. Lichenysin has a potential of hydrogen bonding to 4-EG, as it has some acyl groups. The lichenysin/4-EG interactions were further evaluated using 2D NOESY. The cross peaks indicate spatial proximity among the peaks ( 0 and ΔS > 0, the contributions to these changes mainly arise from hydrophobic interactions; for ΔH < 0 and ΔS < 0, van der Waals forces and hydrogen-bond formation play major roles; for ΔH ≈ 0 and ΔS > 0, electrostatic forces are suggested as more important. The calculated ΔH and ΔS are summarized in Table 3. For 4-EP, guaiacol, and 4-EG, the values of both ΔH and ΔS were negative, which suggested the main acting force would be hydrogen bonding and/or van der Waals force. The carbonyl group of the amino acid residues of lichenysin is a neutral acceptor group for hydrogen bonding. For phenol and 4-MP, ΔS values were found to be positive, whereas ΔH values were negative. The positive ΔS values are supposed to provide evidence for hydrophobic interactions in

Figure 4. Change values of the chemical shift of lichenysin (2.5 mM) in DMSO-d6 solution at 25 °C following the addition of 4-EG at the ratio of mole. 8306

dx.doi.org/10.1021/jf502053g | J. Agric. Food Chem. 2014, 62, 8302−8307

Journal of Agricultural and Food Chemistry

Article

aroma perception during wine consumption. Anal. Bioanal. Chem. 2011, 401, 1497−1512. (6) Robinson, A. L.; Ebeler, S. E.; Heymann, H.; Boss, P. K.; Solomon, P. S.; Trengove, R. D. Interactions between wine volatile compounds and grape and wine matrix components influence aroma compound headspace partitioning. J. Agric. Food Chem. 2009, 57, 10313−10322. (7) Aronson, J.; Ebeler, S. E. Effect of polyphenol compounds on the headspace volatility of flavors. Am. J. Enol. Vitic. 2004, 55, 13−21. (8) Jung, D.-M.; Ropp, J. S. D.; Ebeler, S. E. Study of interactions between food phenolics and aromatic flavors using one- and twodimensional 1H NMR spectroscopy. J. Agric. Food Chem. 2000, 48, 407−412. (9) Goubet, I.; Dahout, C.; Sémon, E.; Guichard, E.; Le Quéré, J. L.; Voilley, A. Competitive binding of aroma compounds by βcyclodextrin. J. Agric. Food Chem. 2001, 49, 5916−5922. (10) Zhang, C.; Xu, Y.; Fan, W. Removal of off-odors from baijiu (Chinese liquor) with different adsorbents. Sci. Technol. Food Ind. 2012, 60−65. (11) Cates, V. E.; Meloan, C. E. Separation of sulfones by gas chromatography. J. Chromatogr. 1963, 11, 472−478. (12) Zhang, J.; Zhuang, S.; Tong, C.; Liu, W. Probing the molecular interaction of triazole fungicides with human serum albumin by multispectroscopic techniques and molecular modeling. J. Agric. Food Chem. 2013, 61, 7203−7211.

Figure 5. NOESY spectrum (4−9 ppm) of 2.5 mM lichenysin and 5.0 mM 4-EG in DMSO- d6 at 25 °C.

Jiannanchun. Though limitations still existed, our investigation could contribute to a better understanding of Chinese liquor at the molecular level.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 510 85918197; fax: +86 510 85918201; e-mail: [email protected]. Funding

This work was supported by National High Technology Research and Development Program of China (2012AA021301, 2013AA102108), National Natural Science Foundation of China (31000806, 31371822, 31271921), Cooperation Project of Jiangsu Province among Industries, Universities and Institutes (BY2010116), 169 Plan of Chinese liquor, the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-06), and the Priority Academic Program Development of Jiangsu Higher Education Institution. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Mu, X. Q.; Xu, Y.; Fan, W. L.; Wang, H. Y.; Wu, Q.; Wang, D. Solid state fermented alcoholic beverages. In Solid State Fermentation for Foods and Beverages; Chen, J., Zhu, Y., Eds.; Taylor & Francis Group: Boca Raton, FL, 2014; pp 288−299. (2) Fan, W.; Qian, M. C. Characterization of aroma compounds of Chinese “Wuliangye” and “Jiannanchun” liquors by aroma extract dilution analysis. J. Agric. Food Chem. 2006, 54, 2695−2704. (3) Fan, W.; Qian, M. C. Headspace solid phase microextraction and gas chromatography−olfactometry dilution analysis of young and aged Chinese “Yanghe Daqu” liquors. J. Agric. Food Chem. 2005, 53, 7931− 7938. (4) Villamor, R. R.; Ross, C. F. Wine matrix compounds affect perception of wine aromas. Annu. Rev. Food Sci. Technol. 2013, 4, 1− 20. (5) Munoz-Gonzalez, C.; Rodriguez-Bencomo, J. J.; Moreno-Arribas, M. V.; Pozo-Bayon, M. A. Beyond the characterization of wine aroma compounds: Looking for analytical approaches in trying to understand 8307

dx.doi.org/10.1021/jf502053g | J. Agric. Food Chem. 2014, 62, 8302−8307