Introduction

There have been numerous studies on Wickerhamomyces anomalus, also called Pichia anomala and Hansenula anomala, in recent years due to its unique physiological characteristics and metabolic features (Morales et al., 2020; Schneider et al., 2012; Ye et al., 2014). They include the following aspects: (1) tolerance to various extreme environmental conditions such as high/low pH, high osmotic pressure, and anaerobic conditions; (2) capability of synthesizing many glycosidases that can hydrolyze flavor precursor compounds containing glucosidic bonds in raw materials, which results in the release of flavor compounds; (3) generation of numerous volatile ester compounds with floral and fruity aromas. Research results show that β-D-glucosidase, β-D-xylosidase, and α-D-rhamnosidase can be secreted by W. anomalus. When wine is fermented with W. anomalus, various amounts of ester compounds (such as ethyl acetate, isoamyl acetate, and 2-phenethyl acetate) with floral and fruity aromas can be added to improve the sensory characteristic scores of wines (Padilla et al., 2018). In apple ciders fermented using W. anomalus, the varieties and amounts of various flavor compounds (including acetate esters, ethyl esters, higher alcohols, aldehydes, and ketones) increase and result in a higher sensory evaluation score (Ye et al., 2014); In addition, the application of W. anomalus also can increase the ethyl acetate in Prunus salicina Lindl. cv. “Kongxinli” fruit wine when it is produced using mixed inoculation with W. anomalus and Saccharomyces cerevisiae (Liu et al., 2020a). Thus, W. anomalus delivers the capability of regulating the aromatic and sensory characteristics of wines, and has promising application prospects in wine production.

Longan (Dimocarpus longan Lour.) belongs to the Sapindaceae family (Han et al., 2020), whose fruits contain abundant nutrients and show medicinal value, including relieving uneasiness and enhancing the functions of the heart and spleen (Park et al., 2016). Longan fruit is considered to be medicine and food and has a favorable development value. As for fermentation of longan wine, Yuan et al. (2020) carried out research on fermentation with aroma-producing yeast and S. cerevisiae. They performed co-inoculation using active dry wine yeasts and aroma-producing yeast in the ratio of 1:1 to analyze the influence of different fermentation parameters (sugar content, acidity, fermentation temperature, and fermentation time) on basic physicochemical indexes and sensory characteristics of longan wine.

Sanoppa et al. (2019) probed the effects of inoculation of Torulaspora delbrueckii on the nonvolatile and volatile compounds in longan wines. They found that longan wine fermented with T. delbrueckii by co-inoculation produced the highest number of total volatile aromatic compounds. In addition, co-inoculation generated high levels of the main volatile compounds. However, the different influences of mixed fermentation with W. anomalus and S. cerevisiae on various qualities (such as basic physicochemical parameters, aromatic characteristics, and sensory characteristics) of longan wines have not yet been clarified.

In the present study, based on sequential inoculation and co-inoculation, longan wine was fermented by mixing W. anomalus with S. cerevisiae to analyze the influence of both inoculation methods on the quality of wine from different aspects. These included basic physicochemical indexes, electronic sensory characteristics, and aromatic substances. This provides a theoretical basis for the application of both yeasts in the production of longan wine and a reference for the production of other fruit wines.

Materials and Methods

Reagents

Glucose, sucrose, yeast extract, peptone, sulfur dioxide (SO₂), and pectinase were reagent pure grade and obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Sodium hydroxide (NaOH), ethanol (95%), sodium chloride (NaCl), and cyclohexanone were analytically pure grade and purchased from Luoyang Chemical Co., Ltd. (Luoyang, China).

Yeast strains and culture conditions

Commercial Saccharomyces cerevisiae X16 obtained from the Laffort Company (France) was used as a reference strain. The W. anomalus C11 strain was isolated from the fruit of Rosa roxburghii Tratt. All yeast cells were cultured on yeast extract peptone dextrose medium (1% yeast extract, 2% peptone, 2% glucose, and 2% agar) containing 100 mg/L of chloromycetin at 28°C for 72 h, and then were maintained at 4°C for later use.

Preparation of fermented longan wine

Fresh longan fruit produced in Thailand was purchased from a local fruit market in Guiyang, Guizhou Province, China. The longan fruit was selected, peeled, pitted, and squeezed to obtain its must. Sulfur dioxide (50 mg/L), pectinase (200 mg/L), and dimethyl dicarbonate (600 mg/L) were added to the must, which was maintained at room temperature for 12 h. After completing the treatment, the sugar was adjusted to 24° Brix. Then, the mixture was divided into four groups and poured into 2.5-L sterile Erlenmeyer flasks with rubber plugs and respirators, with three parallel repetitions for each. In terms of the first group (S. cerevisiae), only S. cerevisiae X16 was inoculated, at a concentration of 10⁷ colony-forming units (cfu)/mL; in the second group (W. anomalus), only W. anomalus was inoculated, at a concentration of 10⁸ cfu/mL. As for the third group (co-inoculation), W. anomalus and S. cerevisiae were co-inoculated, separately showing concentrations of 10⁸ and 10⁷ cfu/mL. For the fourth group (sequential inoculation group), W. anomalus with a concentration of 10⁸ cfu/mL was inoculated at the beginning of fermentation. On the 4th day of fermentation, S. cerevisiae with a concentration of 10⁷ cfu/mL was inoculated. The samples in different groups were statically maintained and fermented at 22°C in an incubator with a thermostat until the fermentation was complete.

Detection of basic physiochemical parameters for longan wine

The determination of the alcohol content, total acidity, and volatile acid of longan wine was performed according to the method proposed by Xia et al. (2018). The total sugar content in longan wine was determined by applying the optimized anthrone method (Liu et al., 2020a). The pH value was measured by employing a pH meter (Model PT-10, Sartorius AG, Germany).

Detection of electronic sensory characteristics for longan wine

The electronic sensory indexes for longan wine were determined using an electronic tongue system (SA402B, Insent, Japan). Measurements were obtained according to the instrument instructions and the method proposed by Liu et al. (2020b). Each wine sample was measured in three parallel repetitions, and the data were collected four times in each parallel repetition.

Detection of the aromatic composition of longan wine

The aromatic composition of longan wine was determined by combining headspace solid phase microextraction (TQ8040, Agilent, USA) with gas chromatography-mass spectrometry (GC-MS) (Tandee et al., 2020). The qualitative analysis was conducted by searching the National Institute of Standards and Technology (NIST) standard library for matches with the GC-MS data. By using cyclohexanones as internal standards, the aromatic compositions were quantitatively analyzed by applying the internal standard method.

Data Analysis

Principal component analysis (PCA) and significance analysis were conducted on the data with the aid of SPSS 25 software. Excel 2010 software was used for data processing and plotting. Adobe Photoshop CS was applied to edit the figures. The data are expressed in the form of the mean ± standard deviation. P < 0.05 indicates that the difference shows statistical significance.

Results and Analysis

Influence of mixed fermentation of W. anomalus and S. cerevisiae on the basic physiochemical parameters of longan wine

The basic physiochemical parameters of fermented longan wine in the different groups are shown in Table 1. The longan wines fermented by inoculation with mixed S. cerevisiae with W. anomalus exhibited lower alcohol content than that fermented by inoculation with S. cerevisiae alone (control group). No significant difference was found in the total acidities, volatile acid, or pH values of the four groups of longan wine.

Influence of mixed fermentation of W. anomalus and S. cerevisiae on the electronic sensory characteristics of longan wine

Figure 1 shows the electronic sensory characteristics of fermented longan wines in different groups analyzed using the electronic tongue detection system. The groups of longan wine produced by co-inoculation and sequential inoculation delivered lower acidity and richness than the group produced by inoculation with W. anomalus or S. cerevisiae alone.

Influence of mixed fermentation by W. anomalus and S. cerevisiae on the aromatic characteristics of longan wine

Ester compounds

Ester compounds are secondary metabolites generated during alcohol metabolism, most of which exhibit floral and fruity aromatic characteristics, and they are important aromatic compounds for various fermented wines (Song et al., 2019). As shown in Table 2, a total of 19 ester compounds were detected from the four groups of fermented longan wine, in which 16, 14, 15, and 19 ester compounds were separately detected from the S. cerevisiae group, W. anomalus group, co-inoculation group, and sequential inoculation group, respectively. Ethyl 3-hydroxybutyrate was only detected from longan wine produced through mixed fermentation (co-inoculation and sequential inoculation), and ethyl 9-decenoate and isoamyl octanoate are specific to longan wine fermented through sequential inoculation. In addition, the amount of aromatic ester compounds in longan wine fermented by co-inoculation was significantly lower than that fermented by inoculation with only S. cerevisiae or W. anomalus. There was no difference between longan wines fermented through sequential inoculation with S. cerevisiae and W. anomalus in terms of the amounts of ester compounds.

Ethyl acetate provides aromas of pineapple and sweet fruits, and it is one of the most predominant ester aromatic compounds in fermented wine, with a threshold of approximately 150 mg/L (Yan et al., 2020). Ethyl acetate at approximately 80 mg/L can favorably impart a fruity aroma to grape wine and increase the complexity of wines. The highest concentration ethyl acetate was measured in the longan wine fermented by sequential inoculation (66.87 ± 8.59 mg/L), while the ethyl acetate of the other three groups was approximately 30 mg/L (Table 2).

Alcohol compounds

Alcohol compounds are secondary metabolites generated through the metabolism of yeast cells during fermentation, and they contribute important regulatory effects on the aromatic type of fermented wines (Jolly et al., 2014). As shown in Table 3, a total of nine alcohol compounds were detected from the four groups of fermented longan wine, in which there were high amounts of isobutanol and phenethyl alcohol. Compared with the W. anomalus group, the longan wines fermented by mixed fermentation (including co-inoculation and sequential inoculation) delivered a significantly greater amount of alcohol compounds. The concentration of alcohol compounds in the co-inoculation group was the highest (95.48 ± 5.01 mg/L).

Higher alcohols are commonly known as fusel oil, which is a general term for monovalent alcohols with at least three carbon atoms. A moderate amount of higher alcohols can endow wines with a unique aroma and flavor. If the amount of higher alcohols is too low, a light-bodied wine will result, which leads to insufficient aroma. However, a high amount of higher alcohols is likely to lead to headache and drunkenness after consumption (Gonzalez and Morales, 2017). Research shows that there are 80–540 mg/L of higher alcohols in grape wine (Yan et al., 2020), and that the lowest amount (69.06 ± 5.03 mg/L) of higher alcohols was obtained in longan wine fermented by inoculation with W. anomalus alone. By contrast, the concentrations of higher alcohols in the other three groups were all higher than 80 mg/L, and the concentration of higher alcohols in longan wine produced by co-inoculation was the highest, at 94.04 ± 4.96 mg/L (Table 3).

Linalool, phenethyl alcohol, and citronellol are higher alcohols with floral and fruity aromas that were present in the four groups of longan wines. The three higher alcohols reached the highest concentration (45.92 ± 3.43 mg/L) in the co-inoculation group, while the lowest (39.29 ± 1.85 mg/L) was measured in the W. anomalus group (Table 3).

Volatile acidity

Volatile acidity is also considered as an important aspect of aromatic compositions in fruit wines. As shown in Table 4, only two volatile acid compounds were found in longan wine, that is, acetic acid and caprylic acid. Acetic acid was detected in the four groups of fermented fruit wine, but caprylic acid was not found in the co-inoculation or the sequential inoculation groups. Compared with the S. cerevisiae group, the volatile acidity in longan wine increased based on mixed inoculation with W. anomalus, in which the co-inoculation group delivered the highest concentration (79.25 ± 4.41 mg/L) of volatile acidity.

Aldehyde and ketone compounds

The varieties and amounts of aldehyde and ketone compounds also contribute to the aromatic characteristics of fruit wines. As shown in Table 5, a total of three aldehyde and ketone compounds (i.e., acetaldehyde, acetone, and 5-hexen-2-one) were detected from the four groups of fermented longan wine. To be specific, the three aldehyde and ketone compounds were all present in the co-inoculation group, while in the other three groups, only acetaldehyde was detected. Relative to the S. cerevisiae and W. anomalus groups, the aldehyde and ketone compounds in longan wines significantly increased under co-inoculation and sequential inoculation. The application of co-inoculation increases the variety and amounts of aldehyde and ketone compounds.

Other compounds

Table 6 shows that among the four groups of fermented longan wine, a total of five other compounds were detected, in which two, five, four, and three compounds were separately found in the S. cerevisiae, W. anomalus, co-inoculation, and sequential inoculation groups, respectively. Compared with the S. cerevisiae and W. anomalus groups, the other compounds in the co-inoculation group significantly increased, while that in the sequential inoculation group did not vary. Therefore, the application of co-inoculation can effectively increase the varieties and amounts of other compounds in longan wine.

Above all, co-inoculation can enrich the variety of aldehyde and ketone aromatic compounds, while sequential inoculation can enhance the variety of aromatic ester compounds in longan wine (Figure 2A). Relative to the S. cerevisiae group, the amount of ester compounds significantly decreased, while those of acid, aldehyde, ketone, and other compounds in fermented longan wine significantly increased in the co-inoculation group (Figure 2B). Moreover, the acid, aldehyde, and ketone compounds in the longan wine fermented by sequential inoculation significantly increased. Compared with inoculation using W. anomalus alone, co-inoculation significantly decreased the ester compounds while increasing the alcohol, aldehyde, ketone, and other compounds in longan wine. Similarly, sequential inoculation enhanced the amounts of alcohol, aldehyde, and ketone compounds. Thus, mixed inoculation (co-inoculation and sequential inoculation) can regulate the aromatic characteristics of longan wine, including the variety and amount of aromatic compounds.

Main aromatic compounds

The odor activity value (OAV) can be used to further evaluate the influence of the main aromatic compounds on the aromatic characteristics of fruit wines. An OAV larger than 1 indicates that the compounds make a great contribution to the aroma of wines. On the contrary, an OAV lower than one implies that the compounds contribute little to the aroma (Borren and Tian, 2020). The OAVs of 14 main aromatic compounds in longan wine are displayed in Table 7, and the OAVs of 10 compounds were greater than 1, while those of four compounds were less than 1. The OAVs of isoamyl acetate, linalool, and ethyl laurate were large among the four groups of fermented longan wine, which indicates that they greatly contribute to the aroma of longan wine.

The influence of the main aromatic compounds in longan wine on the characteristics of wines was further evaluated by applying PCA. As shown in Figure 3A), a total of three principal components (PC1 [48.05%], PC2 [26.91%], and PC3 [25.04%]) were extracted, and their cumulative contribution rate was 100%.

The four groups of fermented longan wines show a large distribution difference in PCA. The sequential inoculation group is more closely related to many ethyl ester aromatic compounds (such as ethyl laurate, ethyl caprylate, ethyl caproate, and phenethyl acetate) (Figure 3B). The aromatic characteristics of the W. anomalus group are related to some acetic ester compounds (ethyl acetate and isobutyl acetate), and the characteristic aroma of the co-inoculation group is possibly derived from linalool. There was a lack of typical aromatic compositions in the S. cerevisiae group.

Discussion

Yeasts play an important role in the fermentation of fruit wines. The non-Saccharomyces yeasts generally refer to a class of yeasts that includes W. anomalus, Hanseniaspora uvarum, and Metschnikowia pulcherrima, etc., and does not include S. cerevisiae (Wang et al., 2016). Many non-Saccharomyces yeasts secrete multiple glycosidases, can improve various qualities (e.g., aroma, color, and taste) of fermented fruit wines, and contribute to increasing the richness and specificity of fruit wines (Morata et al., 2019). Therefore, the wines fermented by mixing S. cerevisiae with selected non-Saccharomyces yeasts not only retain a certain alcohol content but also exude improved quality characteristics (aromatic and sensory characteristics).

Mixed fermentation has become a commonly accepted production mode (Johnson, 2013). There have been reports on fermenting and producing various wines based on fruits—Vitis vinifera L. (Rossouw and Bauer, 2016), Diospyros kaki L.f (Jing et al., 2018), Lycium barbarum L. (Ju et al., 2017), and Rosa roxburghii (Zhao et al., 2020)—by mixing non-Saccharomyces yeasts with S. cerevisiae. However, research on the influence of non-Saccharomyces yeasts on the characteristics of longan wine is somewhat limited. Using sequential inoculation and co-inoculation based on W. anomalus, the influences of mixed fermentation on basic physiochemical parameters, electronic sensory characteristics, and aromatic characteristics of longan wine can be analyzed.

Our results showed that the longan wine fermented either through co-inoculation or sequential inoculation exhibited lower alcohol content than the control group, that is, the S. cerevisiae group (Table 1). The possible reason for this is that the two fungi interact during the fermentation, which influences their metabolism for carbohydrates, thus leading to the reduction in the alcohol content. Research results indicate that W. anomalus, which secretes the virulence factor with broad-spectrum resistance, can effectively inhibit the pollution caused by infectious microbes (Padilla et al., 2018). Nevertheless, the influence of W. anomalus on S. cerevisiae during the production of longan wine has not yet been clarified, and remains to be further explored.

Our research results also reveal that mixed fermentation can regulate the aromatic characteristics of longan wine. As for the variety of aromatic compounds, co-inoculation can increase the variety of aromatic aldehyde and ketone compounds in longan wine, while sequential inoculation can enrich the variety of aromatic ester compounds (Figure 2A). In terms of aromatic compounds, ester compounds significantly decreased, while those of acids, aldehydes, ketones, and other compounds in longan wine fermented through co-inoculation significantly increased (Figure 2B). The longan wine produced based on sequential inoculation delivered significantly greater amounts of acid, aldehyde, and ketone compounds. Therefore, the longan wine produced by different methods of mixed fermentation presents diverse aromatic characteristics. Hence, it is feasible to apply different mixed inoculation methods according to different production demands.

The proportion of yeasts in mixed inoculation also influences the quality characteristics of fruit wines. Ju et al. (2019) suggested that wolfberry wine fermented by mixing H. uvarum with S. cerevisiae in the inoculation ratio of 3:1 presents the highest sensory score, and its aromatic characteristics greatly differ. Wine with a low alcohol content fermented by mixing H. uvarum with S. cerevisiae in a 2:1 ratio exhibits suitable acidity and sweetness, complex aroma and flavor, and highly stable microbes (Cui et al., 2020). Only the influence of different inoculation methods (co-inoculation and sequential inoculation) on basic physiochemical properties and aromatic characteristics of longan were analyzed in this study. Although the influence of different inoculation ratios on the quality of longan wine was not analyzed, it will be further examined in subsequent research.

Above all, the influences of mixed fermentation (co-inoculation and sequential inoculation) of W. anomalus and S. cerevisiae on basic physiochemical properties, electronic sensory characteristics, and aromatic characteristics of longan wine were analyzed. Our results indicate that mixed fermentation can reduce the alcohol content in longan wine, and it also can decrease the acidity and the electronic sensory richness of the wine. Thus, different inoculation methods greatly affect the aromatic characteristics of longan wine. Co-inoculation can increase the variety of aldehyde and ketone aromatic compounds, decrease the amounts of esters, and increase the amounts of acids, aldehydes, ketones, and other compounds. Sequential inoculation can increase the variety of ester aromatic compounds and amounts of acid, aldehyde, and ketone compounds. Therefore, mixed fermentation can regulate the quality characteristics (including basic physiochemical parameters, electronic sensory characteristics, and aromatic characteristics) and enrich the variety of longan wine.