Flavor Characteristics of Navel Orange Wine Fermented by Saccharomyces cerevisiae SC-125 and Angel Yeast SY

Abstract

This research utilized Jintang navel oranges as the primary raw material, and employed two distinct yeast strains, Saccharomyces cerevisiae SC-125 and Angel yeast SY, for a dual fermentation approach. Employing single-strain fermentation as the control, this study aims to ascertain the physicochemical markers, alterations in organic acids and amino acids, alongside the antioxidant properties throughout the fermentation process, all within an optimized environment. The characterization of flavor compounds in the navel orange wines subjected to diverse yeast strains and mixed fermentation was conducted using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HP-SPME/GC-MS). This method facilitated the identification of flavor compound types and concentrations. Moreover, electronic sensory systems including electronic noses and electronic eyes were harnessed to discern distinctions among various navel orange wines. Through these techniques, the research aimed to elucidate the variances induced by different yeast strains during both individual and mixed fermentation processes, shedding light on their impacts on the ultimate quality of navel orange wines.

1. Introduction

China is a significant region for navel orange production, with these delectable fruits being primarily sold in both fresh fruit and processed juice forms. Renowned for their crispy flesh, and delightful sweetness, navel oranges have garnered immense popularity and affection from consumers [1]. However, the susceptibility of navel oranges to damage during transportation and storage, coupled with the relatively limited precision processing technology, calls for a need to delve into the realm of heavily processed navel orange food. This endeavor aims to enhance storage life, prolong shelf life, and ultimately increase the overall added value. Enhancing the deep processing of fruits through techniques like brewing and other processes can significantly boost their added value for consumption. By employing such methods, it becomes possible to augment the levels of polyphenols, amino acids and other bioactive compounds with powerful antioxidant properties. These substances play a pivotal role in maintaining human health and well-being [2]. Consequently, this approach not only addresses the issue of fruit production and sales imbalances but also fosters the sustainable development of the fruit industry [3].

Saccharomyces cerevisiae, a conventional choice in the alcoholic wine industry, is favored for its exceptional fermentation capabilities and robust tolerance [4,5]. Aroma stands as a paramount factor in gauging both the quality of fruit wine and its reception among consumers. However, the use of a single starter can only obtain a stable aroma, which will result in an insufficient and monotonous aroma of fruit wine, which cannot fully reflect the aroma characteristics of fruit. Hence, studies endeavor to enrich microbial diversity as a means of modulating flavor attributes, consequently enhancing color, aroma, and overall quality [6,7]. With the rise of the fruit wine market, S. cerevisiae and commercial yeast are increasingly widely used in fermented drinks, but there are few studies on co-fermentation between commercial yeasts and S. cerevisiae.

Currently, the technique of headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC-MS) holds widespread recognition for its utility in quantitatively analyzing volatile compounds [8]. The progressive advancements in electronic and multi-sensor technology have made it feasible to achieve objective and precise discrimination outcomes [9]. The electronic nose (E-Nose) and electronic eye (EE) represent efficient instruments that have been developed to rapidly detect food flavors and simulate the human visual system. These technologies offer a comprehensive representation of volatile flavor compounds (VFC) present in tested samples. They have the capacity to compare and analyze VFC profiles among samples, mitigating the inherent subjectivity and inconsistent repeatability associated with human olfaction and vision [10]. The incorporation of HS-SPME/GC-MS and electronic nose/electronic eye analyses holds significant importance in both fruit wine production and quality control processes.

The objective of this study is to optimize the process conditions for producing navel orange fruit wine through mixed fermentation of brewing yeast S. cerevisiae SC-125 and Angel yeast SY. The aim is to determine the most suitable fermentation conditions. The analysis of process parameter indicators for navel orange fruit wine prepared under the identified optimal fermentation conditions, involving the mixed fermentation of S. cerevisiae SC-125 and Angel yeast SY, will be carried out. By employing techniques like electronic nose analysis, the sensory attributes of the wine will be evaluated. Additionally, an analysis of the in vitro antioxidant capacity of the fermented fruit wine will be conducted. This research presents novel insights contributing to the advancement of aromatic and nutritionally enriched navel orange fruit wine.

2. Materials and Methods

2.1. Yeast Strains and Materials

Two distinct brewing yeast strains were employed in this study. One of these strains, a commercial brewing yeast known as Angel yeast SY, was procured from Angel Yeast Co., Ltd. (Yichang, China). The other strain, designated as S. cerevisiae SC-125, was isolated from fragmented fruit and vegetable samples [11]. These yeast strains were all provided by the Key Laboratory of Sichuan Province for Food and Biological Engineering at Xihua University, Sichuan, China. Prior to inoculation, activation was performed for 48 h in a potato glucose medium (200 g/L yeast extract, 20 g/L glucose, pH 7). The solid culture medium was supplemented with 20 g/L agar.

The navel oranges were sourced from the Jintang Navel Orange Orchard in Sichuan, with a diameter ranging from 75 to 80 mm. Sodium hydroxide (Guangzhou Ciswater Technology Co., Ltd., Guangzhou, China), Folin-Ciocalteu reagent, 1,1-Diphenyl-2-picrylhydrazyl (DPPH), n-hexanol (Chengdu Kelong Chemical Reagent Factory, Chengdu, China), phenol (Jinan Zesheng Chemical Co., Ltd., Jinan, China), sulfuric acid (Meishan Xinghongsheng Chemical Co., Ltd., Meishan, China), gallic acid, citric acid, pectinase, potassium metabisulfite, and phenolphthalein (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) were all of analytical grade. DNS (3,5-Dinitrosalicylic acid) was obtained from Fuchun Chemical Reagent Co., Ltd. (Tianjin, China).

2.2. Vinification Process

The procedure for preparing the inoculum started by peeling and removing the cores from freshly purchased navel oranges. The fruit’s flesh was then processed using a juicer (JYL-C19V, Zhejiang Red Juice Industry Co., Ltd., Ningbo, China) to extract the pulp, creating a pulpy mixture. To ensure proper preservation, we added potassium metabisulfite to the pulp to achieve an effective SO₂ content of 60 mg/L. Next, we introduced pectinase enzyme to the mixture, achieving a final concentration of 150 mg/L, and performed enzyme hydrolysis at a constant temperature of 40 °C for 2 to 3 h.

After the enzymatic treatment, the mixture underwent centrifugation at 3000 rpm for 30 min, followed by filtration through a 120-mesh sieve to obtain a clarified solution. This clarified solution was then transferred into 250 mL fermentation containers. To ensure sterility, the containers were subjected to a 15 min pasteurization process at 60 °C. For the mixed yeast fermentation, we used a combination of S. cerevisiae SC-125 and commercial Angel yeast SY. Both yeast strains were activated with a seed liquid concentration of 10⁶ CFU/mL and added in a 4% (v/v) ratio to the pulp mixture. The fermentation process took place at a constant temperature of 30 °C, and we established three parallel sets for monitoring purposes.

To track the fermentation progress, we employed a handheld refractometer (V201, Shanghai Smart Instrument Manufacturing Co., Ltd., Shanghai, China). Fermentation continued until soluble solids reached a stable level, signifying the completion of the primary fermentation phase. Throughout this fermentation period, samples were collected every 12 h. These samples of navel orange fermentation supernatant were collected under aseptic conditions on a clean bench and subsequently frozen at −50 °C for preservation.

2.3. Single-Factor Experimental Design for Co-Fermentation Conditions

The total esters were determined using the reflux saponification method [12]. In order to explore the effects of inoculation proportion, fermentation temperature, and fermentation time on the fermentation process, various factors were examined. The inoculation proportions were set at 1:4, 1:2, 1:1, 2:1, and 4:1, respectively (30 °C, 72 h). Fermentation temperatures were set at 26 °C, 28 °C, 30 °C, and 32 °C, respectively (1:4, 72 h). Fermentation times were set at 24 h, 48 h, 72 h, 96 h, and 120 h, respectively (1:4, 30 °C). The total ester content was measured after each of these variations. Using the combined results of the Box–Behnken response surface design and the single-factor experiments, the variables of inoculation proportion (v/v), fermentation temperature (°C), and fermentation time (h) were considered as independent variables. The total ester yield was considered as the response value (Table S1).

2.4. Determination of Colony Count and Basic Physicochemical Indicators during the Fermentation Process

After activating S. cerevisiae SC-125 and Angel yeast SY (with seed liquid concentrations of 10⁶ CFU/mL each), they were left to ferment at a constant temperature under the optimal conditions for mixed fermentation. The fermentation was carried out for 72 h. Samples were collected every 12 h during this period. The navel orange fermentation supernatant was collected in a sterile environment on a clean bench and then frozen at −50 °C for preservation. The colony count of the mixed fermentation of S. cerevisiae SC-125 and Angel yeast SY in navel orange juice at different fermentation stages was determined using the Potato Dextrose Agar (PDA) plate counting method. Meanwhile, both yeast strains were individually fermented with navel orange juice as controls. The colony count was expressed as colony-forming units per milliliter (CFU/mL). Soluble solids were determined using a handheld refractometer [13], pH was measured using a pH meter [14], and reducing sugar content was determined using the DNS (3,5-dinitrosalicylic) method [15].

2.5. Chemical Analysis

2.5.1. Determination of Alcohol Content, Organic Acids and Amino Acids

The alcohol content of the samples was determined using gas chromatography with an external standard method [16]. Organic acid content in the samples was measured following the method outlined by Chen et al. [17]. The amino acid content in the samples was determined using an automated amino acid analyzer [18].

2.5.2. Quantification of Volatile Compounds

Based on the HS-SPME/GC-MS technique [19], the analysis of volatile compounds was conducted. Semi-quantitative analysis was performed using the internal standard method, with n-hexanol at a concentration of 0.4175 mg/L chosen as the internal standard. For the extraction, 3.8 mL of the fermentation sample was mixed with 0.2 mL of diluted n-hexanol (diluted 105 times with ultrapure water) and placed in a headspace vial. One gram of NaCl was added, the vial was sealed, and then it was placed in a 60 °C constant-temperature water bath for 20 min to achieve equilibrium. A SPME fiber was inserted into the vial for adsorption for 20 min, and then it was removed and introduced into the gas chromatograph inlet. Desorption was performed for 3 min at 220 °C before analysis using GC-MS. Gas chromatography conditions included a DB-Wax column (30 mm × 0.25 mm × 0.25 μm), an injection temperature of 240 ℃, and a temperature program: initial temperature of 40 °C held for 2 min, followed by an increase at a rate of 6 °C/min to 240 °C, held for 4 min. The carrier gas was helium with a linear velocity of 1.0 mL/min and no split flow. Mass spectrometry conditions utilized an electron ionization (EI) source with an electron energy of 70 eV. The filament current was set at 0.20 mA, the ion source temperature was 200 °C, and the interface temperature was 250 °C.

2.6. Determination of Total Phenols, Total Flavonoids, and Antioxidant Activity

The determination of total phenol content was carried out using the Folin–Ciocalteu reagent (FCR) method via UV–vis spectrophotometry [20]. Rutin and gallic acid were employed as standard compounds. The content of total flavonoids in the samples was determined using UV–vis spectrophotometry following the procedure outlined in reference [19]. For the assessment of antioxidant activity, the DPPH· radical scavenging activity was determined according to the method described by Kaewkod et al. [21], the hydroxyl radical scavenging activity was evaluated following the method by Hui et al. [22], and the ABTS⁺ radical scavenging activity, often used to assess the antioxidant capacity of substances in vitro, was determined by referencing the approach of Wu et al. [23].

2.7. Electronic Nose and Electronic Eye Measurements of Fermented Navel Orange Wine

To investigate the flavor changes during the fermentation process of navel orange juice and analyze the variations in aroma, the headspace air-sampling method [20] was employed. The performance of the 10 sensors in the electronic nose system is outlined in Table S2. The electronic eye equipment used in this study comprised a system for capturing images of the navel orange wines and another system for collecting color signals of the wines. The measurement principle involved converting RGB data into the CIE system using an image acquisition device within a standard light source box. This method enabled non-contact image acquisition and supported the measurement of mixed color samples with non-solid, irregular shapes.

2.8. Statistical Analyses

A comparison of odor differences was conducted, and principal component analysis (PCA) was carried out using Origin 2018 to compare the aroma characteristics. Chemical data was analyzed using IBM SPSS software (version 26) through one-way analysis of variance (ANOVA) and least significant difference (LSD) test (p = 0.05). TBtools software was used for heat map drawing. The distribution of important volatile substances is visualized through Circos software (http://circos.ca/), accessed on 18 July 2023.

3. Results and Discussion

3.1. Optimization of the Response Surface of Fermented Navel Orange Wine

From Figure 1, it is evident that the total ester content of the co-fermented wines is consistently higher than that of the control group with single-strain fermentation. As the inoculation amount of Angel yeast SY increases, the total ester content also increases. This trend could be attributed to the commercial Angel yeast SY being more adapted to the fermentation medium of navel orange wines. In the co-fermentation system, Angel yeast SY, as the dominant strain, has a greater impact on the total ester content.

Ultimately, based on the experimental results, a Box–Behnken response surface design was employed in conjunction with the outcomes of single-factor experiments. The analysis center point was set at an inoculation proportion of 1:4, fermentation temperature of 30 ℃, and fermentation time of 72 h (as indicated in Table S3). A response surface experiment was carried out according to this setup. Analyzing the regression model’s variance through analysis of variance (ANOVA) (as shown in

Table 1. Analysis of Variance of Regression Model.

Source	Sum of Squares	df	Mean Square	F Value	p Value
Model	0.3	9	0.033	29.62	<0.0001	significant
A	2.00 × 10−4	1	2.00 × 10−4	0.18	0.6851
B	1.80 × 10−3	1	1.80 × 10−3	1.61	0.2452
C	8.00 × 10−4	1	8.00 × 10−4	0.72	0.4257
AB	1.23 × 10−3	1	1.23 × 10−3	1.1	0.3301
AC	2.25 × 10−4	1	2.25 × 10−4	0.2	0.6673
BC	4.23 × 10−3	1	4.23 × 10−3	3.78	0.0931
A2	0.098	1	0.098	87.25	<0.0001
B2	0.032	1	0.032	28.66	0.0011
C2	0.13	1	0.13	118.26	<0.0001
Residual	7.83 × 10−3	7	1.12 × 10−3
Lack of Fit	2.55 × 10−3	3	8.50 × 10−4	0.64	0.6262
Pure Error	5.28 × 10−3	4	1.32 × 10−3
Cor Total	0.31	16

A—Inoculation proportion; B—Fermentation temperature; C—Fermentation time.

), it is evident that the model is highly significant. Furthermore, with an R-squared value of 0.9744 and a well-matching adjusted R-squared value (R²_Adj = 0.9415), the results indicate that the model fitting is quite satisfactory. The mutual analysis among individual factors is depicted in Figure S1. These results collectively demonstrate the good fit of the model and its potential for experimental predictions.

Through the analysis of the regression equation, the optimal conditions for mixed co-fermentation were determined as follows: an inoculation proportion of 1:4 (v/v), a fermentation temperature of 30.16 °C, and a fermentation time of 71.49 h. Under these optimized conditions, the total ester yield was measured at 3.84 ± 0.07 g/L, which is quite close to the predicted value of 3.92 g/L. Notably, the total ester yield in the co-fermentation process significantly outperformed the individual fermentation of S. cerevisiae SC-125 (2.30 g/L) by 1.7 times, and also surpassed the individual fermentation of Angel yeast SY (2.92 g/L) by 1.3 times.

3.2. Changes in Fundamental Indicators during the Fermentation Process

3.2.1. Changes in Microbial Colony Counts and pH during Fermentation

Cell viability is a critical factor in evaluating functional products [24]. Figure 2a,b depicts the changes in viable cell counts and pH during single-strain fermentation of S. cerevisiae SC-125, single-strain fermentation of Angel yeast SY, and their co-fermentation. Prior to introducing the microorganisms into the juice, under identical and suitable conditions, the strains in all three fermentation methods were activated to approximately 6.0–7.0 Lg CFU/mL. These microbial strains exhibited robust growth in the orange juice with a pH range of 3.4–3.6. These findings suggest that navel orange juice, as a fermentation substrate, is conducive to the growth of S. cerevisiae SC-125 and Angel yeast SY. The cell concentrations consistently remained above the minimal concentration (6.0 Lg CFU/mL) necessary to sustain healthy growth [25].

3.2.2. Changes in Alcohol Content and Reducing Sugars

The utilization of reducing sugars signifies the yeast’s ability to conduct alcoholic fermentation and convert substrates [26]. Moreover, microbial tolerance and utilization of sugars are important indicators for determining the endpoint of fermentation. As illustrated in Figure 2c, the alcohol content of navel orange wines produced via different fermentation methods displays an initial increase and then gradually declines, possibly due to insufficient nutrients in the culture medium. The co-fermentation group exhibits higher alcohol content than the single-strain fermentation groups, with the S. cerevisiae SC-125 fermentation group having higher alcohol content than the Angel yeast SY group. Throughout the fermentation process, the alcohol content of the co-fermentation group remains around 0–6%, reaching an endpoint alcohol content of 5.2%, which aligns with the standard for low-alcohol wines below 6%. As evident from Figure 2d, within the first 36 h, the reducing sugar content of all three fermentation methods rapidly decreases from 70.31 g/L. Notably, the co-fermentation process demonstrates the highest sugar utilization rate. During the subsequent 36 to 72 h, the reducing sugar content remains below 3 g/L for all groups, gradually stabilizing and indicating the near completion of fermentation.

3.2.3. Changes in Organic Acids

The changes in organic acid content during the fermentation process of single- and mixed-strain navel orange wines are depicted in Figure 2e–h. Organic acids have a role in inhibiting pathogenic microorganisms in fruit wines and can directly influence the pH and overall taste of navel orange wines [27]. Over the 0 to 72 h fermentation period, the contents of citric acid, acetic acid, and malic acid exhibit an increasing trend, while the content of tartaric acid initially decreases and then gradually rises. Citric acid is the main organic acid in navel orange wines, with a natural concentration of 3.5 g/L in navel orange juice. After fermentation, the co-fermented wine displays the highest citric acid content, reaching 4.33 g/L.

3.3. Changes in Amino Acids before and after Fermentation

The amino acids in navel orange wines mainly originate from microbial metabolism and their raw materials. Amino acids contribute to the high nutritional value of navel orange wines, and many flavors in navel orange wines come from free amino acids. As shown in Figure 3, among the three fermentation methods, the co-fermented wine exhibits the highest total amino acid content, followed by Angel yeast SY, and the lowest is observed in S. cerevisiae SC-125 wine. After fermentation of navel orange juice, most amino acid contents increase to varying degrees compared to navel orange juice, except for significant reductions in aspartic acid, serine, glutamate, and isoleucine. Particularly notable increases are observed in phenylalanine, lysine, histidine, arginine, and proline. This increase could be attributed to brewing yeast utilizing the sugar sources and amino acids in navel orange juice to synthesize both functional and structural proteins [28].

3.4. Analysis of Volatile Compounds before and after Fermentation

After analyzing post-fermentation samples using HS-SPME/GC-MS, the obtained results resembled those presented in Figure 4 and Table S1. In the strain SC-125 monoculture fermentation group, a total of 38 substances were identified, while the Angel yeast SY monoculture fermentation group detected 33 substances. Comparatively, the co-fermentation group revealed the presence of 40 volatile compounds, surpassing the monoculture fermentation counterparts. The co-fermentation process led to an increased variety and quantity of aromatic compounds, contributing to a more enriched aroma profile in the navel orange wine. Noticeable discrepancies in volatile compound type and concentration were evident across all fermented fruit wines. These encompassed 14 alcohols, 20 esters, 4 acids, and 2 other types of compounds (Figure 4 and Figure 5b,c).

Based on the observations from Figure 5a, it is evident that various volatile compounds exhibit significant differences in both their types and concentrations. To further analyze the distinctions among the SC-125 group, SY group, and the volatile flavor compounds during co-fermentation (SC-SY), we employed SIMCA 14.1 software to conduct Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). The OPLS-DA model decomposes the information in the X-axis matrix into two categories as follows: those that are related to Y (grouping) and those that are unrelated, thus effectively filtering out variables that are irrelevant to the groupings. Additionally, by considering the Variable Importance in Projection (VIP) scores, it enhances the reliability of discriminating the differential metabolites obtained [29]. As depicted in Figure 5c, it illustrates the correlation between different volatile compounds and the three groups of fermentation samples. Compounds located further from the coordinate center point indicate a greater contribution of that compound to the differentiation among the samples.

Ester compounds play a crucial role in shaping the predominant aroma of fruit wines, constituting essential components of their aromatic profiles. Co-fermentation demonstrated higher levels and a greater diversity of ester compounds compared to the strain SC-125 and Angel yeast SY monoculture fermentations. Aromas primarily generated by co-fermentation included compounds such as phenylethyl acetate, ethyl laurate, and ethyl acetate. Phenylethyl acetate imparts a strawberry-like fragrance [30], ethyl laurate exudes an oily fruity scent, while ethyl acetate carries a fruity aroma. Co-fermentation also produced aromas that were not generated in the single-strain fermentations, such as isovalerate, propionate, and butyrate esters. These aromatic compounds contributed notes of fruitiness and rose blossom to the navel orange wine. Alcohol compounds are predominant aroma constituents in fruit wines, typically originating from the breakdown of sugars or amino acid conversions within the wine. Among the three fermentation methods, alcohol compounds exhibited variation in both type and concentration, with co-fermentation yielding the highest diversity and content. Carbonyl compounds, secondary metabolites generated by yeast metabolism, were also present. In this study, acid compounds included lauric acid, isobutyric acid, normal decanoic acid, and caproleic acid. Acid compounds were relatively less abundant compared to alcohols and esters under all three fermentation conditions. This discrepancy might be attributed to esterification reactions between acids and alcohols, leading to the generation of more ester compounds within the fermentation medium.

3.5. Changes in Total Phenols and Total Flavonoids and In Vitro Antioxidant Activity

Due to yeast cells’ adsorption and desorption effects on phenolic substances, changes in the fermentation environment lead to desorption as well [31]. Polyphenols and tannins are the primary antioxidant-active compounds, while total flavonoids are one of the key quality factors in navel orange fruits, possessing multiple functions such as anti-inflammatory, antioxidant, regulation of fruit color changes, and flavor development. Figure 6a,b illustrates the variations in total phenol and total flavonoid contents in navel orange wines under different fermentation methods. Across all three fermentation methods, total phenol content showed an increase, with co-fermented wine consistently having higher levels than monoculture fermentation. Total flavonoid content exhibited a rising trend, with minimal difference between the strain SC-125 and Angel yeast SY groups. The co-fermentation group had the highest total flavonoid content, showing a 24% increase compared to before fermentation. This was followed by the strain SC-125 and Angel yeast SY groups, which saw increases of 13.6% and 14.4%, respectively. The rise in total phenol and flavonoid contents might be attributed to factors such as alcohol and pH in the fermentation environment, prompting microorganisms like yeast to decompose and transform large molecular phenolic compounds [32].

From Figure 6c, it is evident that the co-fermentation of mixed strains exhibited the highest free radical scavenging capacity, with DPPH·, hydroxyl, and ABTS⁺ free radical scavenging rates reaching 79.5%, 80.4%, and 84.8%, respectively. The antioxidant capacity of navel orange wines showed a positive correlation with total phenol content, affirming that the synergistic co-fermentation of strains contributes to increased antioxidant activity. Moreover, co-fermentation exhibited the most significant enhancement in antioxidant effectiveness.

3.6. Electronic Nose and Electronic Eye Results Analysis

Using an electronic nose for rapid assessment of odor variations in navel orange wines under different fermentation methods yields a radar plot that showcases the response of electronic nose sensors to the distinct fermentation approaches (Figure 7a). It is evident from Figure 7a that the peak areas for W1S and W5S are most prominent among the ten sensors, showing sensitivity to nitrogen compounds and short-chain alkanes, respectively. The peak areas for the other sensors are relatively smaller. Specifically, in terms of peak area on the W1S sensor, co-fermented beverages exhibit the largest peak area, followed by Angel yeast SY beverages, and lastly, SC-125 yeast wines. In the context of the peak area on the W1S sensor, the order of peak area magnitude is co-fermented > SC-125 yeast > Angel yeast SY. To determine the statistical significance of these differences, an analysis of variance (ANOVA) was performed. When considering the peak areas across all ten sensors, it was found that co-fermented wines exhibited higher aroma sensitivity compared to those from single-culture fermentation.

Principal component analysis (PCA) was employed to analyze the electronic nose data collected during the fermentation of navel orange wine using diverse techniques. As depicted in Figure 7b, the first and second principal components represented 83.9% and 11.7% of the variance, respectively, leading to a cumulative variance contribution rate of 95.6%, surpassing the 85% threshold. The SC-125 single-culture-fermented navel orange wine is situated on the positive side of PC1 and PC2, while the co-culture fermentation is on the negative side of both PC1 and PC2. Although the SY single-culture fermentation falls on the positive side of PC2, it is positioned near the zero point on PC1. The WIS short-chain alkanes are located in the lower left corner of the score plot (negative side of PC1). This underscores PCA’s effectiveness in capturing comprehensive information from the original dataset and its potential for establishing correlations with the impact on each nasal sensor [33]. The distribution of distinct navel orange wine samples across various quadrants is evident in the graph. Moreover, no overlapping of samples from different categories is observed. Samples from the strain SC-125 and Angel yeast SY groups are positioned closely, indicating a pronounced similarity between the two. Consequently, it can be inferred that the three samples stand apart and can be aptly grouped and classified using the electronic nose. Different yeast strains and fermentation methods lead to varying degrees of alteration in the aroma components of navel orange wine.

As indicated in

Table 2. Chroma of Navel Orange Wine under Different Fermentation Methods.

Navel Orange Wine	L	a	b	h	C
Saccharomyces cerevisiae SC-125	74.63	4.40	40.29	83.77	24.09
Angel Yeast SY	73.68	7.88	41.01	79.12	41.76
Co-fermentation	75.34	4.13	44.88	84.75	25.13

, following the fermentation of navel orange wines, their brightness levels are notably higher. Particularly, co-fermented wines exhibit the highest brightness. Following the formula in the CIELAB color space: ΔEab = sqrt((ΔL)² + (Δa)² + (Δb)²), and applying it to the Lab coordinates as follows: SC-125: Lab (74.63 4.40 40.29); SY: Lab (74.63 4.40 40.29); co-fermentation: Lab (74.63 4.40 40.29); We obtain the following ΔE × ab values: ΔE × ab (ΔE between SC-125 and SY) ≈ 3.20; ΔE × ab (ΔE between SC-125 and co-fermentation) ≈ 5.29; ΔE × ab (ΔE between SY and co-fermentation) ≈ 3.35. Typically, ΔE values greater than one are considered visible color differences, while ΔE values greater than three are regarded as significant color differences. Therefore, we can observe that significant differences exist between both the individual fermentations and the mixed fermentation [34].

Additionally, co-fermented wines possess a greater yellowness and chroma angle, rendering them more inclined towards a yellow hue and brighter appearance compared to the monoculture-fermented counterparts. While the saturation of the Angel yeast SY group surpasses that of the co-fermentation, it is accompanied by a decrease in both yellowness and brightness. Overall, the co-fermentation process has a more pronounced positive impact on the color attributes. This finding is consistent with literature reports, indicating that mixed fermentation can enhance the color intensity of fruit wines [35].

4. Conclusions

This study revealed that compared to monoculture fermentation, mixed-strain fermentation of navel orange wine exhibited enhanced fermentation capabilities. The reduction of reducing sugars was more rapid, resulting in higher alcohol content. Notably, the concentrations of citric acid, malic acid, acetic acid, and tartaric acid all increased. Amino acids such as aspartic acid, glutamic acid, valine, isoleucine, and leucine also experienced an increase. In terms of volatile compounds, both the variety and quantity of volatile compounds in the mixed-strain-fermented wine exceeded those of monoculture-fermented wines, leading to heightened aromatic qualities in the navel orange wine. Total phenol and total flavonoid contents showed an upward trend in mixed-strain fermentation. Antioxidant capacity analysis indicated that the mixed-strain-fermented wine exhibited the highest scavenging rates for DPPH·, hydroxyl radicals, and ABTS⁺ radicals. Sensory analysis conducted using an electronic nose demonstrated that the mixed-strain-fermented wine displayed greater sensitivity to W5S and W1S sensors, primarily responding to nitrogen compounds and short-chain alkanes. Electronic eye measurements indicated that the color of the mixed-strain-fermented wine was the brightest. Utilizing a mixed fermentation of brewing yeast and commercial yeast in the production of orange grape wine has increased the richness of flavor compounds. This led to the development of a flavorful and nutrition-rich low-alcohol navel orange wine, overcoming the flavor limitations of single-yeast fermentation. The study provides a theoretical reference for the development of mixed fermentation navel orange wines utilizing brewing yeasts, thus enhancing the overall quality and sensory experience.