Effects of metals released in strong-flavor baijiu on the evolution of aroma compounds during storage.

Abstract Storage is essential in improving the quality of strong‐flavor baijiu (SFB). Here, we investigated the release behaviors of metals from containers into SFB and their effects on the evolution of aroma compounds during storage. Twenty‐six metals were identified in SFB samples. The concentrations of Na, K, Ca, Mg, Al, and Fe obviously increased after storing in pottery jar, whereas those of Fe and Cu greatly increased after storing in stainless‐steel vessel. The volatility of most esters, alcohols, ketone, furan, and aldehyde decreased, whereas that of most acids increased after adding the metal ions into fresh SFB. The fluorescence intensity of SFB decreased with increased aging time in pottery jar, whereas the fluorescence intensity of acids was quenched with adding Fe3+ and Cu2+. All these results suggested that some metals released from containers had binding affinities with acids, thereby reducing SFB organoleptic stimulation by forming metal‐aroma compound complexes during storage.


| INTRODUC TI ON
Baijiu is an ancient Chinese liquor that belongs the six major distilled spirits in the world. Baijiu has a distinctive flavor different from other major distilled spirits, such as brandy, whiskey, rum, gin, and vodka (Jin, Zhu, & Xu, 2017;Liu & Sun, 2018). The flavors of baijiu are generally classified into twelve types, including three major and nine minor flavor types (Zheng & Han, 2016). Strong-flavor baijiu (SFB) is one of the major flavor types and accounts for approximately 70% of the total baijiu production in China. Production of SFB is unique and complex, involving solid-state fermentation, distillation, storage/ aging, and blending. Sorghum or a mixture of corn, rice, glutinous rice, and wheat are mostly used as raw materials for SFB production, and daqu is used as the saccharifying and fermentative agent.
Storage is an essential procedure in SFB production. Fresh distilled baijiu with unpleasant, rough and pungent flavor needs to be stored in a pottery jar or a stainless-steel vessel for months or years to improve SFB quality (Zheng & Han, 2016). After storage, fresh distilled baijiu is converted into aged/stored SFB, and its flavor becomes mellow and harmonious, and even appears chen-aroma.
During storage, some metals are released into SFB from the containers (Jiang, Xie, Wan, Chen, & Zheng, 2019). These metals can of alcoholic beverage. Cu, Fe, Mn, Al, and Zn influence the release and formation of some aroma compounds, such as hydrogen sulfide, methanethiol, and dimethyl sulfide in wine (Franco-Luesma & Ferreira, 2014;Viviers, Smith, Wilkes, & Smith, 2013). The presence of Cu at a low level leads to a better taste and aroma of brandy (Bonic et al., 2013). Therefore, understanding the effects of metals on the release and formation of aroma compounds of baijiu are important for modulating its flavor and sensory properties.
This work aimed to investigate the effects of metals released from pottery jar or stainless-steel vessel on the evolution of aroma compounds of SFB during storage. Release behaviors of metals into SFB were first investigated. Major metals Na, K, Ca, Mg, Al, and Fe in SFB samples were determined via inductively coupled plasma-optical emission spectrometry (ICP-OES), and trace metals Ag, As, Ba, Be, Cd, Cr, Cu, Li, Mn, Ni, Pb, Sr, Ti, Zn, La, Pr, Nd, Sm, Eu, and Gd were determined via inductively coupled plasma-mass spectrometry (ICP-MS). Then, changes of aroma compounds in SFB after adding various metal ions were analyzed via headspace solid-phase microextraction combined with gas chromatographymass spectrometry (HS-SPME-GC-MS), and the metal-binding abilities of aroma compounds were evaluated via fluorescence spectroscopy to further understand the effects of metals on aroma compounds of SFB.

| Materials
SFB samples were the same level base baijiu and provided by Luzhoulaojiao Co., Ltd. (Luzhou, China). Samples were stored in pottery jar or type 304 stainless-steel vessel for 0, 1, 2, 3, 5, and 10 years. The volume of the pottery jar is 1 m 3 , and its surface area is approximately 5.8 m 2 in comparison to 1 m 3 of SFB. The volume of the stainless-steel vessel is 10 m 3 , and its surface area is approximately 2.1 m 2 in comparison to 1 m 3 of SFB. During storage, the pottery jar was sealed and put in a cave at 20°C. The stainless-steel vessel was sealed and put in the shade at a room temperature which varied with the seasons. Standard solutions of Na, K, Ca, Mg, Al, Fe, Ag, As, Ba, Be, Cd, Cr, Cu, Li, Mn, Ni, Pb, Sr, Ti, Zn, La, Pr, Nd, Sm, Eu, and

| Analysis of metals in the stored SFB by ICP-OES and ICP-MS
SFB samples (25 ml) were heated at 80°C until their volume was reduced to 2-3 ml. Then, the concentrated samples were digested at 120°C with 5 ml nitric acid and 1 ml hydrogen peroxide. After cooling to room temperature, these digested solutions were diluted to 25 ml with Milli-Q water. The concentrations of major metals, such as Na, K, Ca, Mg, Al, and Fe, were determined by ICP-OES (Optima 8000; PerkinElmer), and the concentrations of trace metals, such as Ag, As, Ba, Be, Cd, Cr, Cu, Li, Mn, Ni, Pb, Sr, Ti, Zn, La, Pr, Nd, Sm, Eu, and Gd, were determined by ICP-MS (ELAN 9000/DRC-e; PerkinElmer).

| HS-SPME extraction and GC-MS analysis of aroma compounds in SFB
Aqueous solutions of NaCl, KCl, CaCl 2 , MgCl 2 , AlCl 3 , FeCl 3 , and CuCl 2 , in which the metal ion has a concentration of 1,000 mg/L, were prepared individually. Different volumes of the above-mentioned solutions were added into fresh SFB samples to obtain 2 and 10 mg/L of Na + , K + , and Ca 2+ , 0.5 and 2 mg/L Mg 2+ , Al 3+ , Fe 3+ , and Cu 2+ . SFB samples with addition of metal were regarded as experimental groups, and SFB sample without addition of metal was the control group. All samples were stored at 25°C for a week.
Changes in aroma compounds of fresh SFB after adding various metal ions were analyzed through HS-SPME-GC-MS using the reported method with some modifications (Huang et al., 2019;. SFB sample (1 ml) and methyl hexanoate (10 μL, 1,310 μg/L in 70% ethanol/water solution v/v) as internal standard were added into a 20-mL screw-capped headspace vial. The vial was placed in thermostatic water bath, equilibrated at 40°C for 10 min and extracted for 45 min at the same temperature by being exposed to a 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane fiber (2 cm; Supelco, Inc.). Subsequently, the fiber was transferred into an injector to desorb at 250°C for 5 min.
Injector was maintained at 250°C. Oven temperature was started at 40°C for 5 min, raised to 100°C at rate of 4°C/min, then ramped to 230°C at a rate of 6°C/min and held for 15 min. For MS analysis, electron ionization was used at 70 eV. Temperatures of the interface and the ion source were set at 250 and 230°C, respectively. The mass range scanned was from 30 to 450 amu at a scan rate of 0.3 s.
Volatile aroma compounds were identified by comparing their mass spectra (MS) with the NIST14 mass spectra database and comparing their retention indices (RIs) with those of reference compounds. Net changes were used to evaluate aroma volatility and were calculated according to the equation: Net change (%) = (mean peak area of control-mean peak area of experimental group)/ mean peak area of control × 100% .

| Statistical analysis
Each sample was analyzed in triplicate for each determination. The results in tables are presented as mean ± standard deviation. Oneway analysis of variance (ANOVA) and Duncan's multiple range tests were carried out with SPSS 25.0 software to evaluate significant differences of assay (p < .05).

| Quantification of metals in SFB
Twenty-six metals in fresh and stored SFB samples were analyzed via ICP-OES and ICP-MS, and the results are listed in Table 1. Na, K, Ca, Mg, Al, and Fe are the major metals with high content in baijiu (Xie, Jiang, & Wu, 2017). Hence, their concentrations were determined by ICP-OES, which is usually used to analyze samples with high total dissolved solids content. Besides, the minor metals Ag, As, Ba, Be, Cd, Cr, Cu, Li, Mn, Ni, Pb, Sr, Ti, Zn, La, Pr, Nd, Sm, Eu, and Gd were analyzed by ICP-MS, which is more sensitive, but limited to samples with low total dissolved solids content (Martin, Watling, & Lee, 2012;Šelih, Šala, & Drgan, 2014). Fresh SFB had little amounts of metals because most of metals remained in Zaopei after distillation (Li et al., 2016;Zou et al., 2018). However, some metals were dissolved out from containers during storage. SFB sample stored in pottery jar for 10 years The concentrations of most metals in SFB increased during storage ( Figure S1). When SFB was stored in pottery jar, the concentrations of Na, K, Ca, Mg, Al, and Fe increased considerably, and concentrations of Ba, Be, Cu, Li, Mn, Ni, Sr, Ti, and Zn increased slightly. Pottery jar is made from fired clay and covered with glaze over the surface. Its main components are SiO 2 and Al 2 O 3 and contain small amounts of metallic oxides, such as CaO, Fe 2 O 3 , CuO, NiO, and TiO 2 (Schleicher, Miller, Watkins-Kenney, Carnes-McNaughton, & Wilde-Ramsing, 2008). These metallic oxides can be slowly dissolved into baijiu during storage (Yang, Zhao, & Liu, 2001); therefore, most metals in SFB increased during storage in pottery jar. When SFB was stored in stainless-steel vessel, concentrations of Fe and Cu in SFB increased greatly and concentrations of Cr, Mn, Ni, Sr, Ti, and Zn increased slightly. The Cu in SFB should be the impurity, which was introduced from welding process in production of the stainless-steel vessel (Magnabosco, Ferro, Bonollo, & Arnberg, 2006).   Note: Values are mean ± standard deviation of triplicates. P0, P1, P3, P5, and P10 are SFB samples stored in pottery jar for 0, 1, 3, 5, and 10 years, respectively. S0, S1, S3, S5, and S10 are SFB samples stored in stainless-steel vessel for 0, 1, 3, 5, and 10 years, respectively. Values in the same row with different superscript letters are significantly different (p < .05). 2.14 Note: Odor descriptors of aroma compounds were from the reports by , and . Identification was based on comparing determined MS and RIs with those of reference compounds.

RI
The concentration of each metal ion is 2 mg/L in SFB sample.
the color of spirits to yellow or even brown (Pohl, 2007). Hence, metal concentrations in SFB should be limited according to the international statutory limits in alcoholic beverages recommended by the WHO.

| Effect of main metals on aroma compounds in SFB
Na, K, Ca, Mg, Al, Fe, and Cu, which had relatively high concentrations in stored SFB, were chosen for further study of their effects on volatile aroma compounds. HS-SPME method is widely used for identification and quantification of volatile components in baijiu (Fan & Qian, 2005;. In this study, a total of 30 volatile aroma compounds, including 17 esters, 6 alcohols, 4 acids, 1 ketone, 1 aldehyde, and 1 furan, were identified in SFB and quantified through the HS-SPME method (    (Figure 1b). This result implied that Fe 3+ had a stronger ability to interact with carboxyl of acids in SFB than other metal ions (Na + , K + , Ca 2+ , Mg 2+ , Al 3+ , and Cu 2+ ). Esters and acids were the main aroma compounds in baijiu. Total acid content increased and total ester content decreased during the aging period (Qiao, Zhang, & Wang, 2013). Therefore, the above results indicated that the quality of aged SFB was associated with Na, K, Ca, Mg, Al, Fe, and Cu, and these metals had an aging-advancing function.

| Effects of main metals on fluorescence of SFB and acids solution
Fluorescence analysis was used to study the mechanism of interaction between metals (Na, K, Ca, Mg, Al, Fe, and Cu) and SFB.
Fluorescence EEM landscapes of fresh and stored SFB samples are shown in Figure 2, and their spectral characteristics are listed in  (Lavrik & Mulloev, 2010;Zhang, Sahu, Xu, Wang, & Hu, 2017;Zhang, Shi, Sun, Xiong, & Peng, 2011). Hence, the above-mentioned change in SFB fluorescence might be ascribed to the binding of some metals released from pottery jar to the components in SFB during storage.   Figure 3.
Fluorescence intensity of SFB was slightly changed by adding Na + , K + , Ca 2+ , Mg 2+ , and Al 3+ at a low concentration (Figure 3a-e) but gradually decreased with increased concentrations of Fe 3+ and Cu 2+ (Figure 3f,g). Plots of F 0 /F for SFB versus metal ions at different concentrations exhibited that the binding affinity of Fe 3+ was higher than that of Cu 2+ (Figure 3h). Fluorescence intensities of acid solutions were almost unchanged after adding different concentrations of Na + , K + , Ca 2+ , Mg 2+ , and Al 3+ into the acids solution but decreased with increased concentrations of Fe 3+ and Cu 2+ ( Figure S2). These fluorescence quenching data showed that Fe 3+ and Cu 2+ had higher binding affinity with carboxyl of acetic acid, butanoic acid, and hexanoic acid in 70% (v/v) ethanol/water solution than Na + , K + , Ca 2+ , Mg 2+ , and Al 3+ . Irto et al. (2018) reported that Fe 3+ , Al 3+ , Cu 2+ , and Ca 2+ could bind with a compound derived from nitrilotriacetic acid. Hussain, Rahim, and Farooqui (2012) showed that the binary complexes of mandelic acid ligand with Fe 3+ and Cu 2+ had higher stability than those with Cd 2+ , Zn 2+ , Ni 2+ , and Co 2+ . Their results indicated that these metals, especially Fe 3+ and Cu 2+ , could chelate or bind with different organic compounds, which is consistent with our fluorescence analyses.
This study would be helpful to understand why metals can reduce organoleptic stimulation of alcoholic beverages.

| CON CLUS ION
Some metals were dissolved out from pottery jar and stainlesssteel vessel into the SFB during storage. Their concentrations were lower than the recommended allowable daily intake. When SFB was stored in pottery jar, the concentrations of Na, K, Ca, Mg, Al, and Fe remarkably increased, and concentrations of Ba, Be, Cu, Li, Mn, Ni, Sr, Ti, and Zn slightly increased. When SFB was stored in stainless-steel vessel, the concentrations of Fe and Cu greatly increased, and concentrations of Cr, Mn, Ni, Sr, Ti, and Zn slightly increased. Metals released from pottery jar and stainlesssteel vessel into SFB were consistent with the main components of the two containers. It is worth noting that the Cu released in SFB should be the impurity, which was introduced from welding process in production of the stainless-steel vessel. Headspace concentrations of most esters, alcohols, ketone, furan, and aldehyde in fresh SFB decreased, whereas headspace concentrations of most acids increased after adding Na + , K + , Ca 2+ , Mg 2+ , Al 3+ , Fe 3+ , and Cu 2+ . These metal cations promoted the decrease in total ester content and the increase in total acid content, indicating that these metal cations had an aging-advancing function.
Furthermore, maximum fluorescence intensity of SFB decreased with increasing aging time. This result might be ascribed to the binding between metals released from the container and aroma components, especially acids in SFB during storage. This binding can promote the formation of metal-aroma compound complexes or colloidal particles. This finding is helpful to explain the release of metal cations and the formation of aroma compounds of baijiu during storage, which is significant for modulating the flavor and sensory properties of baijiu.

ACK N OWLED G M ENTS
This work was financially supported by the National Engineering Research Center of Solid-State Brewing (K2019-243) and the Technology Development Program of Sichuan University (19H0393).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.