The sensory and flavor characteristics of Shaoxing Huangjiu (Chinese rice wine) were significantly influenced by micro‐oxygen and electric field

Abstract In order to improve the high cost of equipment and difficult management caused by the natural aging of Chinese rice wine (Huangjiu), micro‐oxygen (MO) and electric field (PEF) technology are used to accelerate the aging of Huangjiu. The results showed that micro‐oxygen and electric field have a significant effect on the sensory characteristics and flavor characteristics of Huangjiu. Compared with the naturally aged Huangjiu, the flavor compounds of Huangjiu treated with micro‐oxygen and electric field increase significantly. Based on principal component analysis, Huangjiu processed at 0.35 mg L/day or 0.5 mg L/day combined electric field exhibited similar flavor to the natural aged Huangjiu, which was highly associated with long‐chain fatty acid ethyl esters (C13–C18). Moreover, partial least squares regression demonstrated that sensory attributes of cereal aroma and astringency were highlighted after aging time, while fruit aroma, continuation, and full body were dominant after micro‐oxygen and electric field treatment. Micro‐oxygen and electric field effectively enhanced the quality of Huangjiu, which could be applied in other alcoholic beverages.

steps such as steamed rice, yeast fermentation, fried wine, and storage (Ren et al., 2016;Shen et al., 2018). The taste of young Huangjiu is rough, and the aroma is insufficient. However, natural aging needs a long storage time, large space, and high equipment costs, which is a major problem to limit the Huangjiu market (Jiao, Xu, & Jin, 2017).
For this reason, people are exploring ways to speed up the aging of Huangjiu.
Micro-oxygen technology refers to a technology that meets the oxygen demand of various chemical and physical reactions in the wine body through different oxygen levels .
In recent years, it has been involved in sludge digestion, water quality improvement, and biological denitrification (Duc & Kumar, 2018;Zhang et al., 2019). However, little is known about the efficacy of micro-oxygen technology in the treatment of Huangjiu. Electric field treatment is an emerging nonthermal sterilization technology in the fields of food sterilization, preservation, and ingredient extraction (Caminiti et al., 2011;Yang et al., 2019b). Its processing cycle is short, heat production is small, and it can effectively protect the nutrients of food (Dalvi-Isfahan et al., 2016;Gabri et al., 2017). Besides, electric field treatment on wine flavor compounds has produced conclusive evidence (Teusdea et al., 2017), indicating that it can improve the sensory properties of fermented red wine.
However, the effect of electric field treatment on the quality of Huangjiu has rarely been studied, although Huangjiu has a broad sales prospect in the wine market. During Huangjiu brewing, the degradation of raw materials by cofermentation with lactic acid bacteria, fungi, and yeast results of the production with a large number of amino acids, proteins, oligosaccharides, vitamins, and mineral elements (Lv et al., 2016). Amino acids are nutrient components of Huangjiu and the precursors for aroentma compounds, which contribute to the formation of the flavor of Huangjiu (Miao et al., 2015;Shen et al., 2010). Several studies have reported that applying appropriate pretreatment, fermentation, or saccharification starters could greatly contribute to the aroma and flavor profiles of wines (Takahashi et al., 2017;Wu et al., 2015). However, the flavor compounds in Huangjiu need longer aging time to accumulate, so it is suggested to use micro-oxygen and electric field treatment instead of traditional natural aging to produce high quality Huangjiu. Researchers and winemakers need to understand the effect of aging on the flavor properties of Huangjiu in different accelerated aging treatments.
As amino acids and aroma were important in Huangjiu, this study aimed at analyses the differences of brewing characteristics, free amino acids, flavor, and sensory characteristics of Huangjiu under three treatment conditions of natural aging, micro-oxygen, and electric field. This study could provide advice for winemakers to choose appropriate accelerated aging treatments to produce high quality Huangjiu or other alcoholic beverages.

| Huangjiu sample material
Huangjiu with different aging time: Provided by Zhejiang Guyue Longshan Shaoxing Wine Co., LTD. The Huangjiu was fermented in the cellar at constant temperature and aged in pottery jars. The same original wine age is 1, 3, 5, and 8 years Huangjiu, and the wine sample is put in a clean plastic bottle, stored in a 4°C constant temperature refrigerator, ready to use.

| Micro-oxygen treatment
The oxygen content into Huangjiu is controlled by the flowmeter and then used the syringe to inject oxygen into Huangjiu, with a timer to control the interval cycle of predetermined oxygen content. The micro-oxygen has combined the advantages of both aerobic (Mass concentration of dissolved oxygen >1.0 mg/L) and anaerobic (Mass concentration of dissolved oxygen <0.3 mg/L and does not contain nitrate oxygen), usually refers to the environmental dissolved oxygen mass concentration in the reactor is 0.3 ~ 1.0 mg/L (Duc & Kumar, 2018), while the daily dissolved oxygen concentration of Huangjiu in the pottery jars is 0.35 mg/L. The method of adding oxygen from bottom to top is adopted to make oxygen fully contact with liquid and achieve better oxidation effect. In this experiment, the amounts of oxygens added and the action time were used as variables to observe the effects on various compounds in Huangjiu.
Accelerated aging treatments for 60 days were performed on microoxygen 0.35 mg L/day (MO36) and for 60 days on micro-oxygen 0.5 mg L/day (MO56). The temperature of micro-oxygen treatment should be kept at 15 ~ 18°C, because the temperature will affect the solubility of oxygen in Huangjiu. Second, there is a certain height requirement for the oxygenated container, so as to ensure that the tiny bubbles of oxygen can be fully dissolved in Huangjiu, so the container should be at least 2 m high.

| The electric field treatment
The electric field treatment of Huangjiu uses a static pulse treatment chamber. The pulse discharge waveform adopts exponential attenuation wave, and the electrode is stainless steel parallel electrode.
Considering the effect of electric field strength on the features of Huangjiu, the wine samples were processed under the optimal parameters of electric field strength of 2 kv/cm, pulse number of 36, and frequency of 0.5 Hz. The samples of traditional natural aged for 1 year (N1), natural aged for 3 years (N3), natural aged for 5 years (N5), natural aged for 8 years (N8), 2 kv/cm (PF2), 0.35 mg L/day 2 kv/cm (PM32), and 0.5 mg L/day 2 kv/cm (PM52) were tested after different treatments.

| Enological properties of Huangjiu
The total sugar content was determined by dinitrosalicylic acid method. Total acidity (in terms of lactic acid) and alcohol content were measured by Chinese standard method (GB/T 13662-2018).
The analysis carried out by high-performance liquid chromatography (Waters e2695-Empower System Waters Corporation, Milford, MA) equipped with exclusion column (Carbomix H-NP, 300 mm × 7.8 mm, Sepax Technologies, Inc., Newark, DE) at 55°C with a mobile phase of sulfuric acid (2.5 mM) at a flow rate of 0.6 ml/min. In order to determine the total nitrogen, 5 ml of wine sample and catalyst was digested at 420°C for 150 min and analyzed by an automatic Kjeldahl apparatus (Kjeltec 8400; FOSS., Hilleroed, Denmark). Turbidity was measured by nephelometric analysis using a WGZ-1 turbidity meter (WGZ-1; Shanghai Xinrui Instrument Co., Ltd, Shanghai, China) and calibrated with a standard solution. The turbidity index of Huangjiu samples is expressed in units of turbidity (NTU).
The red, orange, and yellow produced by the saccharifying agent have the maximum absorption at 510, 465, and 410 nm, respectively.
Appropriate dilutions of wine samples were measured for absorbance at 510, 465, and 410 nm with a spectrophotometer (V-1800; Shanghai Meipuda Instrument Co., Ltd, Shanghai, China). The color value was defined as absorbance unit at maximum absorption wavelength multiplied by a dilution factor per milliliter of wine sample (U/ml).

| Analysis of free amino acids
According to the method described in Xia et al (Xia et al., 2016), the previously filtered Huangjiu sample was precipitated at 4°C for 2 hr with an equivalent amount of 10% sulfosalicylic acid to remove the large peptides and then centrifuged at 12,000 g for 20 min. The 20 μl supernatant was injected into t/L8900 automatic amino acid analyzer (Hitachi, Tokyo, Japan). Determination of free amino acids in the Huangjiu sample was calculated by calibrating with standard amino acids.

| Volatile flavor compounds
The 5 ml Huangjiu sample was diluted and placed in a 20 ml headspace glass vials. Then, add 2 g sodium chloride and 10 μl internal standard of 4-methyl-2-pentanol (250 μg/ml in absolute ethanol) (Yang et al., 2017). The fiber (50 μm DVB/CAR/PDMS) in a solidphase microextraction (SPME) device (Supelco, Bellefonte, PA) was inserted into the vials and then extracted at 50°C for 30 min. The volatile compounds were then desorbed at 250°C into the GC inlet with an auto-sampler for 7 min. A GC-MS system (SCION SQ-456; Bruker Daltonics Inc., Billerica, MA) equipped with a DB-WAX column (60 m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA) and programmed from 40°C (holding for 3 min) to 210°C at 6 °C/min and then 210°C to 230°C at 8 °C/min (holding for 15 min).
The Kovats indices (KI) of unknown compounds were determined by sample injection with a homologous series of alkanes (C7-C30).
The extracted volatile compounds were compared with the mass spectral libraries (NIST 1.6 and Wiley 6.0), and the mass spectra and KIs of the tentatively identified compounds were compared with those of authentic standards for positive identification. The content of volatile compounds was determined by internal standard method.
After preliminary identification of mass spectra and KIs in the literature, semi-quantitative data of volatile compounds are calculated according to the following formula (Mo et al., 2012): C is the relative concentration of the analyte; C is is the final concentration of the internal standard; A c is the peak area of the analyte; A is is the internal standard of the peak area.

| Sensory evaluation
Sensory evaluation of Huangjiu was conducted by a training panel of 10 judges (five males and five females 20-50 years old). The judges were selected and trained according to ISO 8586-1 (ISO, 1993). A total of 12 descriptors (appearance: yellowness, redness, and turbidity; aroma: alcohol, fruit, and cereal; taste: sweet, sour, and bitter; mouthfeel: astringency, continuation, and full body) were generated, to characterize the sensory properties of Huangjiu (GB/T 13662-2018). During the training, guide the judges to learn the Huangjiu scoring standards ( There was a uniform source of lightening, absence of noise, and distracting stimuli in the training and wine-tasting test. Put different Huangjiu samples (15 ml) into transparent glass, cover with petri dishes, and label them randomly. Provide drinking water to panelists to reduce taste disturbance.

| Statistical analysis
Perform triplicate chemical analysis for each Huangjiu sample, and the result was expressed as mean value ± standard deviation (SD).
One-way ANOVA, followed by Duncan's test, was performed to analyze the significant differences between data. The mean difference was considered significant at p <.05. PCA using a correlation matrix with no rotation was performed to research the relationship between different processing method wine samples and volatile compounds. PLSR studied to the relationship among wine samples, were used to deal with the GC-MS and chemical analysis data.

| Effects on enological properties of Huangjiu
Characteristics of naturally aged, micro-oxygen and electric field treated Huangjiu (total sugar, total acidity, amino acid nitrogen, and ethanol) in Table 2. After natural aging, the acidity of Huangjiu increased and the alcohol content decreased. Due to the inevitable loss of volatile alcohols and aldehydes caused by aging, the total acid content of naturally aged Huangjiu was significantly lower than that of the slightly oxygenated Huangjiu. Ethanol is closely associated with the oxidation and esterification of Huangjiu (Wang et al., 2014). Compared with the natural aged Huangjiu, the decline trend of ethanol in Huangjiu was relatively flat after electric field treatment, indicating that electric field treatment promoted the pro- The wine is light, slightly bitter, and ripe (25 ~ 34 scores) The wine is not mellow and has a bitter, spicy taste (

TA B L E 2 Different treatment of Huangjiu brewing characteristics
F I G U R E 1 Effects of different accelerated aging treatments on total free amino acid content. (a) Content of free amino acids in Huangjiu (mg/L). (b) Total content of free amino acids with taste characteristic in different treatments wines. (c) Difference in amino acid content between natural aging and accelerated aging treatment of Huangjiu lowest ethanol content, indicating that part of the ethanol will volatilize during the aging process. The esterification of Huangjiu in the aging process is also one of the reasons for the decrease of ethanol content . The proportion of alcohol, acid, and ester can be changed by controlling the electric field intensity, so as to accelerate the aging process. After the electric field treatment, the change trend of amino acid nitrogen was relatively stable, and the total sugar content decreased, while the total acid was just the opposite. MO56 exhibited the highest absorbance at 420 nm (0.685), followed by MO36 (A420 = 0.617), indicating that micro-oxygen treatment could markedly improve the speed of Maillard reaction in Huangjiu. Since electric field treatment can improve the absorbance of Huangjiu (Rajha et al., 2016), the sugars and amino acids in Huangjiu accelerate Maillard reaction under the action of electric field, which promotes the reduction of total sugar content. The aldehydes in Huangjiu are oxidized to acids which increase the total acidity (Tian et al., 2016). Nevertheless, the electric field treatment maintained the total acid content of Huangjiu.

| Effect of total free Amino acids on Huangjiu
The effect of different accelerated aging treatments on the total free amino acid content is shown in Figure 1. Eighteen common free amino acids were quantitatively analyzed in wine samples.  treated at 0.5 mg L/day 2 kv/cm was better. Some accelerated aging treatments reduced the content of Thr, Glu, Ala, and Met, while the sweetness was increased by the combination of micro-oxygen and electric field. While other treatments reduced the content of bitter amino acids such as Val, Ile, His, and Leu (Figure 1c), Met, Ile, Gly, Asp, Phe, Tyr, Arg and so on are more sensitive to accelerated aging treatments. Since free amino acids are precursors of flavor compounds (Krivoruchko & Nielsen, 2015), we speculated that these 8 free amino acids were highly correlated to the complex synthesis of flavor compounds in Huangjiu and great significance to the overall aroma of Huangjiu.

| Effects on volatile flavor compounds of Huangjiu
The Almost all of those compounds have been detected in Huangjiu, and the concentration distributions of flavor compounds were consistent with previous research results. (Jung et al., 2014;Tian et al., 2016). It can be seen from Table 3 that the total content of volatile compounds in the Huangjiu treated with micro-oxygen (0.5 mg L/day) and electric field was 1,122,972.96 mg/L, which was higher than that in the naturally aged Huangjiu. In addition to the instability of the 1-year-old Huangjiu system, the longer the natural aging time was, the more volatile flavor substances were found in Huangjiu, followed by N8 (1,135,951.17 mg/L) and Huangjiu contained a large number of volatile flavor compounds, the most abundant were alcohols, esters, and aldehydes, which constitute the structural components of the aroma of Huangjiu (Rang et al., 2016;Ren et al., 2019). Oxidation, reduction, esterification, and hydrolysis reactions occurred in wine, influencing the contents and numbers of alcohols, aldehydes and esters (Tian et al., 2016).
After aging, most alcohols in Huangjiu were on the rise, and the content of N5 (735,226.63 mg/L) fluctuated in 5 years (Table 3).
The total content of those three alcohols in different Huangjiu is F I G U R E 3 Loadings plot for the 33 volatile flavor compounds (a) and different treatments of Huangjiu scores plot (b) shown in Figure 2. The three alcohols fluctuated during the natural aging process, and the other two alcohols, except 2-Phenylethanol, showed a decreasing trend. The content of the three alcohols was increased after the treatment of micro-oxygen and electric field, among which the trend of 2-phenylethanol increased obviously.
2-Phenylethanol, as the most abundant compound in alcohols, was greatly affected (Osada et al., 2015). It is mainly produced through the Ehrlich pathway, which transforms L-phenylalanine into 2-Phenylethanol by aminotransferase, decarboxylase, and dehydrogenase (Ivanov et al., 2013). Compared with Huangjiu treated with electric field only, the content of phenylalanine in PM52 and PM32 was relatively low (Figure 1c), which could indirectly explain the results. Huangjiu at the electric field intensity of 2 kv/cm, the microoxygen is over 0.5 mg L/day, could activate enzymes and contribute to the synthesis of 2-phenylethanol ( Figure 2a).
The results showed that the total ester content of Huangjiu treated with micro-oxygen or electric field was equivalent to that of natural aging. The total ester loss of natural aging was faster in the initial storage, with a reduction of 21.55% (Table 3) (Table 3). In the Huangjiu treated with micro-oxygen and electric field, with the increment of oxygen content from 0.35 mg L/day to 0.5 mg L/day, the content of SCFAEE in the 2 kv/cm electric field treatment decreased by 17.07%, while in the Huangjiu treated with micro-oxygen treatment, the content of SCFAEE decreased by 35.63% (Figure 2b, Table 3). The MCFAEE content of PM53 and PM32 is higher than that of N5, while the MCFAEE content of PF2 is higher than that of N3 (Figure 2c, Table 3). LCFAEE content of N8 decreased by 25.51% compared with N1, and the LCFAEE in PF2 and MO56 was close to that in N3 (Figure 2d, Table 3). It is worth noting that the high content of LCFAEE has a great influence on the results.
The contents of aromatic compounds among accelerated agingtreated Huangjiu were significantly different, especially PM52.
2-Phenylethanol, which has a floral scent, greatly affects the total contents of aromatic compounds. PM52 contained the highest content of 2-phenylethanol (364,816.82 mg/L), which was about 12.07% higher than N1. At 0.5 mg L/day 2 kv/cm, and microoxygen and electric field were most beneficial to the growth of the total content of aromatic compounds.
The results showed that FAEE in Huangjiu treated with microoxygen and electric field could reach the level of natural aged Huangjiu, especially in MCFAEE and LCFAEE. Accelerated aging treatments achieve natural aging levels in the production of 2-methyl-1-propanol, 3-methyl-1-butanol, 2-phenylethanol, and aromatic compounds.

| Effects on flavor characteristic of Huangjiu
The main component analysis (

| Sensory evaluation
To profile the sensory profiles of Huangjiu, descriptive sensory analysis was conducted. The average strength levels of natural aging, micro-oxygen treatment, and electric field treatment are shown in Figure 4. Natural aging exhibited a high level of yellowness, while PM52 showed high intensity of redness, which was in agreement with the color analysis results (Figure 1). Micro-oxygen and electric field-treated wines exhibited higher levels in redness than natural aging, suggesting appropriate electric field treatment reduced the loss of red pigment. All 9 wines exhibited similar clarity in turbidity, which were all scored lower than 3. Turbidity index of the nine samples ranged from 0.7 to 0.9 NTU, which exhibited a good clarification. Different processing had little effect of Huangjiu. MO36 had the highest alcohol-aroma index and PM52 had the lowest alcohol-aroma index, which was consistent with the determination of alcohol content (Table 3). Esters with pleasant aroma were responsible for the perception of fruit aroma. Except for PM52 which scored higher than 4 in fruit aroma, other treatments rice wine were assessed with low intensity in fruit aroma. In terms of cereal aroma, MO36 score was the highest among the treatment Huangjiu, followed by PM52 and PM32, and the score of Huangjiu treated with micro-oxygen was significantly higher than that of naturally aged Huangjiu. Except that the scores of PM32 and PM52 were higher than 4, all other indexes of sweet taste were improved. The unpleasant attributes in N1 (relative to the industry), where astringency intensity increased slightly with aging time and bitterness intensity decreased. PM52 was intense in fruit aroma, mouthfeel of continuation, and full body (pleasant attributes relative to the industry) and was the most highly assessed Huangjiu among the nine wines.
Among naturally aged Huangjiu, N8 scored highest for pleasant properties and lowest for unpleasant ones, meaning that the closer the effect was to N8, the better the flavor. However, in contrast to traditional aged wines, micro-oxygen with electric field-treated wines presented a more mellow taste which was preferred by the panelists.

| Relationship between sensory attributes and flavor compounds
Multivariate PLSR was performed to study the relationship between sensory attributes and volatile compounds. All volatile flavor F I G U R E 5 Load diagram of sensory indexes and 33 volatile components of Huangjiu under natural aging and accelerated aging treatment. (The codes were defined in Table 3, respectively) compounds and nine sensorial attributes (fruit aroma, cereal aroma, alcohol aroma, sweet, astringency, sour, bitter, full body, and continuation) were designed as dependent variables. PLSR modeling between the matrices of volatile compounds and sensory attributes provided a two-factor model explaining 50% of the variance in X (volatile compounds) and 51% of that in Y (sensory attributes).
The small ellipse indicated 50% of the explained variance, and the big ellipse is the unit-circle indicating 100% of the explained variance (Xiao et al., 2014). Aroma compounds between the two ellipses could be considered as correlated with sensory attributes, and those inside the inner ellipse were poorly connected with sensory attributes. Eleven Y variables (N1, N3, N5, N8, PF2, MO36, MO56, PM32, PM52, full body, continuation, fruit aroma, astringency, cereal aroma, and sweet) and thirty-three X variables were located be- Therefore, sensory characteristics of Huangjiu could be enhanced by improving the contents of aromatic compounds, MCFAEE, and LCFAEE. After aging treatment, MCFAEE and LCFAEE in PM52 can reach the natural aged 5 years, which is 11.01% higher than the natural aged 5 years.

| CON CLUS IONS
The effects of traditional natural aging, micro-oxygen and electric field-treatments on oenological properties, free amino acid, flavor characteristics, and sensory profiles were investigated. After aging, the brewing characteristics of Huangjiu changed significantly, but the amino content could reach the level of natural aged Huangjiu after being treated with micro-oxygen and electric field. The Huangjiu treated with 0.35 mg L/day or 0.5 mg L/day combined with electric field 2 kv/cm showed similar effects, and the flavor characteristics of naturally aged Huangjiu were compared with those of naturally aged Huangjiu, and it was found that the flavor characteristics of naturally aged Huangjiu were closely related to CFAEE.
The sensory profiles of Huangjiu could be enhanced by improving the contents of aromatic compounds, MCFAEE, and LCFAEE. In this experiment, the aging treatment of 0.5 mg L/day and 2 kv/cm could maximize the sensory profiles. Because natural aging has a significant effect on free amino acids, flavor characteristics, and sensory characteristics, our results suggest that micro-oxygen, electric field treatments can be used instead. Among them, the flavor characteristics of Huangjiu treated with micro-oxygen are similar to those of naturally aged Huangjiu, while the combination of electric field treatment can improve the flavor intensity of fermented Huangjiu to a greater extent.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data of this study are openly available.