Effects of simultaneous and sequential cofermentation of Wickerhamomyces anomalus and Saccharomyces cerevisiae on physicochemical and flavor properties of rice wine

Abstract Microorganism species and inoculation fermentation methods have great influence on physicochemical and flavor properties of rice wine. Thus, this work investigated microbial interactions and physicochemical and aroma changes of rice wine through different inoculation strategies of Wickerhamomyces anomalus (W. anomalus) and Saccharomyces cerevisiae (S. cerevisiae). The results underlined that inoculation strategies and non‐Saccharomyces yeasts all affected the volatile acidity, total acidity, and alcohol content of rice wine. The sequential cofermentation consumed relatively more sugar and resulted in the higher ethanol content, causing reduced thiols and increased alcohols, esters, phenylethyls, and terpenes, which was more conducive to improve rice wine flavor than simultaneous cofermentation. Moreover, simultaneous cofermentation increased fatty aroma of rice wine, while sequential cofermentation increased mellow and cereal‐like flavor. These results confirmed that sequential cofermentation of S. cerevisiae and W. anomalus was a choice for the future production of rice wine with good flavor and quality.

widely applied in the food and beverage industry. However, few reports were involved in the changes of main functional microorganisms and flavor substances for saccharified rice solution during fermentation.
Fungi molds and yeasts are used as main starter in rice wine, which are responsible for starch degradation and alcohol fermentation, respectively (Sanoppa, Huang, & Wu, 2019;Yang et al., 2017). Meanwhile, wine industrial fermentation tends to use S. cerevisiae to ensure the smooth progress of wine fermentation, but that reduces the flavor diversity of wine to some extent (Krogerus, Magalhães, Vidgren, & Gibson, 2017). Recently, research has found that the microbial and brewing characteristics of non-Saccharomyces impact on wine flavor positively Kutyna, Varela, Henschke, Chambers, & Stanley, 2010;Varela, Sengler, Solomon, & Curtin, 2016). It can synthesize many kinds of enzymes and transform the precursor materials into ester, acid, higher alcohol, and other flavor substances, while causing weak alcohol resistance, low fermentation power, and high yield of acetic acid . It has been reported that mixed mold cultures can influence flavor compounds in the fermentation process of rice wine production Yang et al., 2019).
Presently, mixed fermentation of different yeast strains was used in rice wine brewing. Among them, simultaneous cofermentation means that non-Saccharomyces yeasts and S. cerevisiae are inoculated at the same time, while sequential fermentation means that S cerevisiae is inoculated 1-3 days later after non-Saccharomyces yeasts are inoculated (Shi, Wang, Chen, & Zhang, 2019). Acidity and astringency were the lowest in mixed co-inoculations, mouthfeel and bitterness were the lowest in S. cerevisiae wines, and tasters were preferred to mixed co-inoculated wines (Minnaar, du Plessis, Jolly, van der Rijst, & du Toit, 2019). The contents of alcohols were significantly decreased by cofermentation of S. cerevisiae with Torulaspora delbrueckii, but the contents of esters were increased (Liu, Laaksonen, & Yang, 2019). Furthermore, the sequential fermentation of Hanseniaspora uvarum and S. cerevisiae improved the contents of medium-chain fatty acid ethyl ester compared with their simultaneous cofermentation (Hu, Jin, Mei, Li, & Tao, 2018). Previous studies have reported that S. cerevisiae and non-Saccharomyces did not coexist passively. Instead, they showed interesting interactions that may affect quality of wine (Lencioni et al., 2016). Due to its specific winemaking properties, it may have an additive effect on the flavor and aroma of rice wine. For example, Yang et al. (Yang et al., 2017) studied the volatile compounds of Chinese rice wine fermented by S. cerevisiae FC 15 and S. cerevisiae BR 30, finding that mixed fermentation rice wine has been highly scored in the overall sense, which indicated that the flavor characteristic of Chinese rice wine can be adjusted by the combination of yeast fermentation. Previous studies pointed that W. anomalus was the main strain producing ethyl acetate, which made a special contribution to the Baijiu flavor and quality (Fan et al., 2019). Our previous research also found that W. anomalus fermentation produced a large amount of esters and alcohols, which had a strong fruit flavor (Chen, Ren, Li, & Ma, 2020). However, the effects of mixed fermentation of W. anomalus and S. cerevisiae on aroma and chemical components of rice wine have not been reported.
Thus, this study focused on evaluating the effects of simultaneous and sequential cofermentation of W. anomalus with S. cerevisiae on aroma, microbial interactions, and physicochemical changes of rice wine through different inoculation strategies.
Principal component analysis (PCA) was used to evaluate the influence of inoculum type and inoculation method on volatile compound profile of rice wine. Our study was expected to provide a new starter culture and inoculation method for the rice wine production.

| Strains and media
S. cerevisiae (SITCL254) and non-Saccharomyces yeasts (SITCY125) with high fermentability and fragrance production had been isolated from Kijo of Ningbo in Zhejiang and Chongming in Shanghai, respectively. Identification was corroborated by sequencing the D1/ D2 variable domains of the 26S rRNA, and their colony morphology is shown in Figure 1.
Glutinous rice was purchased from Chongming. All chemicals and reagents were purchased at Tansoole. 2-Octanol standards of chromatographic grade were purchased from Sigma-Aldrich.

| Saccharification of rice
The glutinous rice was completely grinded to powder, passed through a 60-mesh sieve, and stored at −20°C until used. Fifty grams of glutinous rice flour was mixed with distilled water at a certain ratio (1:8 w/v), followed by soaking in a 90°C water bath for 15 min for starch gelatinization. Then, the rice was cooled down to room temperature, and amylase was added (1000 U/g, 0.16%, rice) to water bath at 80°C for 45 min, cooling to room temperature; glucoamylase F I G U R E 1 Colony morphology of selected strains SITCL254 and SITCY125 on WLN medium was added (5000 U/g, 4.8%, rice), bathed at 60°C for 6 hr, and autoclaved at 121°C for 30 min.

| Fermentation conditions
Four types of rice wine were prepared as follows: (a) inoculated at 5.05 × 10 6 CFU/mL SITCL254, (b) inoculated at 5.05 × 10 6 CFU/ mL SITCY125, (c) co-inoculated at 5 × 10 4 CFU/mL SITCL254, and 5 × 10 6 CFU/mL SITCY125, and (d) inoculated at 5 × 10 6 CFU/mL SITCY125, followed by sequential inoculation of 5 × 10 4 CFU/mL SITCL254. Fermentations were done in triplicate at 28°C under static conditions. Sampling was carried out every 12 hr to analyze microbial colony count, reducing sugar content, and pH until the end of fermentation. Starter cultures of all yeast strains were grown YPDA liquid medium at 28°C for 24 hr and 120 r/min and were used to start the rice wine fermentation.

| Measurement of physiochemical properties
Rice wine samples were clarified and centrifuged at 8,000 rpm for 8 min and then stored at −4°C. The 3,5-dinitrosalicylic acid (DNS) colorimetric method was used to determine the reducing sugar in the rice wine. WLN medium was used to distinguish the SITCY125 from SITCL254 according to the different color and size of their colonies on plates. Changes in pH were monitored using a pH meter (Mettler Toledo). Alcohol, total acidity, and volatile acidity were determined through methods recommended by Agricultural Industry Standard of the People's Republic of China (NY/T 1885-2017). Total acidity was expressed as lactic acid (g/L), and volatile acidity was expressed as acetic acid (g/L).

| Analysis of the volatile compounds by HS-SPME/GC-MS
Volatile compounds were identified and quantified as described by Yu et al. (Yu, Xie, Xie, Ai, & Tian, 2019), with slight modifications.
The volatile compounds were extracted by headspace solid-phase microextraction with 50/30 μm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) and analyzed using gas chromatography-mass spectrometry (GC-MS). Agilent 7,890 gas chromatograph with a HP-INNOWax column (30 m × 0.25 mm × 0.25 μm, Agilent) coupled to an Agilent 7,890 mass spectrometer was used. 4 ml rice wine samples, 1.5 g NaCl, and internal standard (2-octanol, 1,760 μg/L) were held in the 20 ml headspace bottle, which was stirred by a magnetic bar in the 50°C water bath for 15 min. After that, the fiber was exposed to the sample headspace for 30 min and immediately followed by desorption of the fiber in the gas chromatography injector at 250°C for 5 min. The GC was operated at the following conditions: initial temperature of 40°C increased to 100°C at 3°C/min and then to 230°C at 10°C /min, a temperature at which it was maintained for 8 min. The injector and detector temperature were all set at 250°C.
The flow rate of the carrier gas (helium, 99.999%) was 1 ml/min. The mass spectrometer was operated in electron impact ionization mode at 70 eV, and ion source temperature was 230°C. Compounds were identified by comparing their retention time and MS spectra with their standard compounds, and other compounds were identified by comparing the MS fragmentation patterns which were obtained from database NIST11.

| Statistical analysis
Microbial cell enumeration and physicochemical tests were conducted in triplicate. The results were presented as means ± standard deviation. Significant differences among means were tested

| Microbial concentration and physiochemical properties of rice wine after different fermentations
According to a previous pure fermentation experiment, W. anomalus needs to reach 10 6 ~ 10 7 cfu/mL to start fermentation in order to prevent the vigorous propagation of S. cerevisiae, and two inoculation concentrations of 5.0 × 10 6 cfu/mL and 5.0 × 10 4 cfu/mL were selected, respectively, in this study. Figure 2 shows the pure culture fermentation, and S. cerevisiae grows faster than W. anomalus. Although the initial inoculation amount of S. cerevisiae was not high, the cell concentration reached 10 8 cfu/ mL in 24 hr. In the mixed fermentation, the number of W. anomalus decreased rapidly after reaching its maximum of 8.51 × 10 7 cfu/mL. It may be related to the competitive effect of nutrients in the mixed fermentation, the formation concentration of toxic substances (such as ethanol), the population induction of cells, and other factors. In contrast, S. cerevisiae maintained a relatively stable rate at a higher order of magnitude (10 7 -10 8 cfu/mL) until the end of fermentation after reaching its maximum quantity. The results showed that there was obvious competition between the two kinds of yeast. This is consistent with the previous results. S. cerevisiae can use the nitrogen source in the substrate faster and more effectively (Liu, Arneborg N, & Toldam-andersen, 2017), which shows higher fermentation capacity than non-Saccharomyces yeasts in mixed fermentation (Ruiz et al., 2019). Furthermore, in the simultaneous cofermentation, S. cerevisiae still kept a high colony number at the end of fermentation.
It indicated that S. cerevisiae was the dominant yeast, which was similar to the conclusion of Luan .
The maximum biomass of W. anomalus and S. cerevisiae in sequential cofermentation was significantly higher as compared with those in simultaneous cofermentation. It showed that sequential cofermentation could reduce the inhibition of S. cerevisiae on W. anomalus from Figure 2d, which were also observed by Shi (Shi et al., 2019). This may be due to the synergistic effect between S. cerevisiae and W. anomalus in the sequential cofermentation process, and the relationship between them needs further study.
Sugar is the important substrate of alcohol fermentation.
Sequential cofermentations had the fastest sugar consumption than simultaneous cofermentation (Lu, Chua, Huang, Lee, & Liu, 2017;Wei et al., 2020). Compared with the S. cerevisiae, the access of non-Saccharomyces yeasts to a certain extent delayed the fermentation process, which is a reflection of the relatively weak fermentation capacity (Domizio et al., 2011). The pH value of fermentation broth shows the same change trend under different strains and their mixed fermentation modes. When the pH value drops to a certain extent, it will rise slowly. Yeasts use sugar in the fermentation broth for growth and reproduction and produce a large number of acid substances, so the pH value in the fermentation broth decreases. In the later stage of F I G U R E 2 Growth kinetics, reducing sugar, and pH of W. anomalus and S. Cerevisiae growth during rice wine fermentation with pure culture of W. anomalus (a), pure culture of S. Cerevisiae (b), simultaneous cofermentation of W. anomalus/S. Cerevisiae (c), and sequential fermentation, yeast is in the stage of vigorous alcohol fermentation, and some acids react with alcohols produced in the fermentation process and increase pH value (Kai, Guo, Yin, & Yong, 2018).

| Analysis of physicochemical characteristics
Physicochemical characteristics of rice wine samples in different fermentations are shown in Table 1. The mass concentration of reducing sugar in rice wine was lower than 4 g/L, which indicated that rice wine had been fermented completely. The ethanol volume fraction of S. cerevisiae pure-fermented rice wine was the highest, compared with mixed fermented wine. The results showed that S. cerevisiae had the strongest ability of reducing sugar transformation. The total acid mass concentration was between 4.32 and 5.02 g/ L. There were some differences among rice wines, and the difference in volatile acid content was the most significant. The concentration of volatile acids in S. cerevisiae-fermented rice wine reached 0.36 g/L, while the mass concentration of volatile acids in simultaneous cofermentation and sequential cofermentation rice wine was only 0.22 and 0.25 g/L. Cofermentations had lower volatile acidity than S. cerevisiae fermentation, which was in accordance with results reported by Liu (Liu, Laaksonen, Kortesniemi, Kalpio, & Yang, 2018). The acidity of simultaneous cofermentation was lower than that of sequential cofermentation. These results indicated that fermentation methods, inoculation methods, and non-Saccharomyces yeasts may affect the volatile acidity, total acidity, and alcohol content of rice wine.

| Volatile compounds of rice wine samples in different fermentations
Aroma is one of the most important indicators to measure the quality of rice wine. In this study, ninety-one aroma compounds were identified in different fermentation wine samples as shown in Table 2. The odor activity value (OAV) is a commonly index used to evaluate the contribution of volatile components of rice wine to the actual aroma. It is widely used in the screening and identification of key odor active compounds in food and can be calculated by the ratio of the concentration to the olfactory threshold of the substance (Wang, Capone, Wilkinson, & Jeffery, 2016). It is generally believed that an OAV greater than 1 indicates that it contributes to the odor, and a larger odor activity value indicates a greater individual contribution of the compound. Compared with S. cerevisiae and W. anomalus fermentation (376.72 and 766.49 mg/L, respectively), higher content of varietal aroma compounds was detected from cofermentation wine samples. Compared with the corresponding simultaneous cofermentation (870.07 mg/L), varietal aroma compounds (1568.17 mg/L) in sequential cofermentation were higher.
These results indicated that the varietal aroma content was affected by the use of non-Saccharomyces, fermentation method, and inoculation strategies. This result was in agreement with a previous study .
Alcohol is one of the most important component types in the rice wine. Higher alcohols are mainly produced by transamination of amino acids as substrates and reduction of alcohol dehydrogenase. Compared with pure fermentation of S. cerevisiae (127.19 mg/L), the content of higher alcohols in cofermentations was significantly increased, which was in agreement with a previous report which pointed that more ethanol was produced in mixed culture fermentation with S. cerevisiae and W. anomalus fermentation and accumulation of primary metabolites could influence microbial interaction, end-product flavor, and Baijiu quality (Zha, Sun, Wu, Yin, & Wang, 2018). Moreover, the content of higher alcohols was also higher in the simultaneous cofermentation (294.85 mg/L) than that in sequential cofermentation (414.87 mg/L). It has been reported that when the concentration of higher alcohols exceeds 400 mg/L, they have a negative effect on wine flavor, and the concentration of 300-400 mg/L is acceptable, whereas the optimal level (below 300 mg/L) imparts a pleasant character . These results indicated that the simultaneous cofermentation of non-Saccharomyces yeasts and S. cerevisiae was more conductive to producing appropriate content of higher non-Saccharomyces yeast alcohols in the wine.
Interestingly, 3-methyl-1-butanol which was reported to have the nail polish odor  was not detected in sequential cofermentations and relatively abundant especially in the rice wine from the pure fermentation of S. cerevisiae and simultaneous cofermentation. The reason may be that W. anomalus can provide nutrients for S. cerevisiae in the later stage of fermentation, or W. anomalus has some enzyme activities, which can provide nutrients for S. cerevisiae. Additionally, C 6 alcohols usually have the characteristics of "plant" and "turf," which have a negative impact on the aroma of wine . Compared with S. cerevisiae, C 6 alcohol (3-methyl-pentanol) was not detected in sequential cofermentation. Sequential cofermentation method can effectively decrease C 6 alcohol formation. The high alcohol in

TA B L E 2 (Continued)
sequential cofermentation had a similar response to rice as other three ways of fermentation, but their amounts were higher than those of them, especially with phenylethyl alcohol, 1-propanol, 2-ethyl-1-hexanol and glycerin (contributing to "smoothness," "sweetness," and "complexity" notes for wine) (Binati et al., 2020), and the total contents. The increase of 2-phenylethanol during mixed fermentation seems to be related to the synergistic effect of these two different strains. In pure culture, both of yeasts are producers of low-2-phenylethanol, as previously noted for other pairings of yeast species (GOBBI, Comitini, Domizio, Romani, & Lencioni, 2013). This indicated that sequential cofermentation had a relatively strong ability to synthesis higher alcohol.

Esters (including acetate esters and fatty acid ethyl esters)
were one of the main products of fermentation, and it is mainly produced by yeast metabolism and esterification reaction in wine, with flower and fruit fragrance (Cao, Wu, & Weng, 2020).
Compared with pure fermentation, the content of total esters in sequential cofermentation was higher. Cofermentations significantly enhanced the production of ethyl acetate, hexanoic ethyl ester, and acetic acid phenethyl ester. Interestingly, sequential cofermentation produces significantly higher amounts of hexanoic ethyl ester and acetic acid phenethyl ester than simultaneous co-inoculation. This may be due to the sequential cofermentation of non-Saccharomyces yeasts and S. cerevisiae contributed to the formation of esters, which was also reported by other researchers (Shi et al., 2019;Tristezza et al., 2016;. The mixed fermentation of W. anomalus and S. cerevisiae not only increased the yield of ethyl acetate, but also increased the content of other flavor substances such as β-phenethyl alcohol and phenethyl acetate, which provided an opportunity to change the aroma and flavor of liquor (Fan et al., 2019). In addition, it is reported that low concentrations of ethyl acetate (<150 mg/L) will bring fruity and pleasant aromas to wine (Xiao et al., 2015).
Since the concentrations of ethyl acetate ranged between 2.80 and 8.44 mg/L in our study, it was likely that the presence of this compound positively affected rice wine quality.
When the concentration of fatty acids is low, they are creamy and cheesy, while when the concentration is too high, they will produce sour and sour taste (Niu et al., 2019). The highest total amounts of fatty acids, decanoic acid, and octanoic acid were produced in pure fermentation of W. anomalus, that W. anomalus strains produced lower levels of fatty acid (decanoic acid) than S. cerevisiae.
It was interesting to notice that the octanoic acid in sequential cofermentation was 61.89% lower than that in pure fermentation of W. anomalus. This meant that the rice wine aroma is more harmonious and balanced. Likewise, in mixed fermentation, the content of hexanoic acid and octanoic acid is higher than that of pure fermentation. These results indicated the sequential cofermentation of non-Saccharomyces yeasts and S. cerevisiae would contribute to the formation of fatty acid in the wine. This conclusion is consistent with the previous results of Ma and Wang (Ma, Yan, Wang, Zhang, & Tao, 2017;Wang, Tao, Wu, An, & Yue, 2017).
Terpenes have strong physiological activity to the human body.
Generally, it exists in the form of glycosides, which also contributes to the aroma of wine. The mixed fermentation of S. cerevisiae and W. anomalus was beneficial to the formation of citronellol (strong smell of rose) (Pratibha et al., 2018), which was not detected in pure fermentation. The content of terpenoids produced by sequential cofermentation was the highest, reaching 7.33 mg/L. Compared with pure fermentation, the content of linalool (lavender), geraniol (rose), and 6-methyl-1-heptene slightly increased after mixed fermentation.
Therefore, sequential cofermentation can improve the aroma complexity of rice wine.

| Cluster heat map of volatile aroma compounds in different fermentations
According to the content of flavoring substances in different fermentation method (Table 2) Moreover, the aroma compounds were divided into two classes.
Class I mainly included acetic esters, fatty acid ethyl esters, higher alcohols, and terpene compounds. Class II mainly contained some kinds of C 6 compounds, but some kinds of higher alcohols, fatty acid ethyl esters, and other esters were also included. The simultaneous and sequential cofermentations were rich in class I compounds, while pure fermentations were abundant in class II compounds. The results showed that the aroma compound compositions of simultaneous cofermentation were closer to those of sequential cofermentation, which indicated that different inoculation strategies of cofermentations produced wine with different aroma composition profiles. The high content of ethyl ester could make the wine present cheese flavor, fruit flavor, and fatty acids present cream and cheese flavor at low concentration, and sour and rotten flavor will be produced at high concentration (Jolly, Varela, & Pretorius, 2014;Varela et al., 2016). Phenylethanol is a shikimic acid derivative, with roselike, bitter, sweet, and peach aromas ; isoamyl alcohol (malt aroma) as a typical representative of grain aroma in wine and the main component of higher alcohols, with apple brandy aroma and pungent taste (Jolly et al., 2014). High content of C 6 compounds would make wine present pungent and sour taste, while terpene compounds would give wine flower and fruit aroma, and improve the complexity of wine aroma. These results show that sequential cofermentations can improve the quality and sensory of rice wine.

| Principal component analysis of rice wine aroma components in different fermentations
In order to better explain the differences between the biological species and their inoculation sequence on the volatile compounds of rice wine, 62 aroma components (OAV > 1) were selected for principal component analysis (Figure 4).

| The cumulative odor activity comparison of different volatile chemicals from rice wine samples by different fermentation treatments
The OAV of the volatile components of different chemical categories in the tested rice wine was accumulated, and the data between different fermentation treatments were normalized. The comparison results are shown in Figure 5. Compared with pure fermentation of S. cerevisiae, pure fermentation of W. anomalus has obvious advantages in the odor activity values of some alcohols and aldehydes, which is related to 1-butanol, 5-methylfuran-2-carbaldehyde, maltol, and other substances. Fatty taste at low concentration is beneficial to increase the complexity of aroma, while pure fermentation of S. cerevisiae isoamyl alcohol, 3-methylbutanal, and other substances has low odor activity. Mixed fermentation improves the odor activity of fermented aroma components in rice wine. Simultaneous cofermentation has obvious advantages in the odor activity of ketones and acids, which is related to decanoic acid and heptanoic acid. Furthermore, there is higher 1-hexanol, 1-octanol odor activity.
According to the aroma characteristics of various odor components in rice wine fermentation Yu et al., 2019), pure fermentation of S. cerevisiae rice wine sample has a better fatty and herbaceous aroma and pure fermentation of W. anomalus rice wine sample has richer fruit aroma and caramel aroma. Sequential vor. Due to the cumulative effect of different esters on the aroma perception, the fruit aroma of rice wine comes from the effect of mixed esters. But the intensity of fruit aroma perception is not directly proportional to the total ester content, and it is related to the special ratio, which explains the reason that although the sequential cofermentation yield of ester is not high, the fruit aroma in the fermentation broth is strong. Simultaneous cofermentation also has a strong fatty and herbal aroma, and sequential cofermentation has a strong mellow and cereal aroma. The overall aroma is more coordinated and flower astringency. Therefore, the selection of suitable yeast and fermentation methods for wine production could be a promising way to regulate the characteristics of rice wine.

| CON CLUS IONS
The growth of W. anomalus was suppressed by the presence of S.
cerevisiae produced. Rice wines produced with cofermentations of W. anomalus and S. cerevisiae had lower volatile acidity, more kinds of flavor compounds, and fermentative aroma contents. Moreover, the sequential cofermentation was more conducive to improve rice wine flavor and quality than the simultaneous cofermentation, due to its reduced thiols, increased such fermentative compounds as higher alcohols, esters, phenylethyls, and terpenes. And the sequential cofermentation had better effect on regulating the odor activity of the mellow and cereal flavor components and has a better coordination on the overall flavor of rice wine. In general, the selection of suitable yeast and fermentation methods for rice wine production was important to improve rice wine quality. The sequential cofermentation with S. cerevisiae and W. anomalus was an available method to produce rice wine with good flavor. The results of this study would provide a guidance for mixed fermentation of other non-Saccharomyces yeast in rice wine brewing.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation of China (31771942) and Shanghai Natural Science Foundation (17ZR1429600).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interests.