Controlled Aeration Driven by Oxidation-Reduction Potential Affects Ester Profile in Wine Alcohol Fermentation with Different Starter Cultures

Xue, S. J.; Wang, L.; Chen, S.; Liu, X. H.; Hu, K.; Jiang, J.; Tao, Y. S.; Jin, G. J.

doi:https://doi.org/10.1155/2023/5667458

Australian Journal of Grape and Wine Research

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Abstract Introduction Materials and Methods Results and Discussion Conclusions Data Availability Conflicts of Interest Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 5667458 | https://doi.org/10.1155/2023/5667458

Controlled Aeration Driven by Oxidation-Reduction Potential Affects Ester Profile in Wine Alcohol Fermentation with Different Starter Cultures

S. J. Xue,¹L. Wang,¹S. Chen,¹X. H. Liu,²K. Hu,¹J. Jiang,¹Y. S. Tao,^1,3and G. J. Jin^1,3

Academic Editor: Renata Ristic

Received26 Sept 2022

Revised14 Mar 2023

Accepted17 Mar 2023

Published31 Mar 2023

Abstract

Background and Aims. Aeration, an important operation in winemaking, cannot be controlled accurately based on dissolved oxygen for ester production in wine alcohol fermentation. The following study describes an aeration control method with oxidation-reduction potential (ORP) in alcohol fermentation and investigates its effect on ester production in fermentations with different starter cultures. Methods and Results. The proposed method is based on using ORP as a switch for aeration timing. Different aeration levels driven by ORP were performed in wine alcohol fermentation with different starter cultures including Saccharomyces cerevisiae (Sc), Pichia fermentans (Pf), Hanseniaspora uvarum (Hu), and their mixes (Pf/Sc and Hu/Sc). The accumulated aeration volume, residual sugar concentration, viable cell number, and ester concentration were analyzed. Results showed that aeration levels could be controlled effectively with an ORP value, and aeration with higher ORPs triggered faster sugar utilization in Sc and Hu/Sc fermentation. Pf and Hu survived one day less in their respective cofermentation with aeration when ORP was −100 mV or −50 mV compared to the natural ORP (−150 mV∼−105 mV in Pf/Sc and −141 mV∼−107 mV in Hu/Sc, respectively). Aeration driven by ORP changed ester profiles in cofermentations. With the aeration levels increasing, the proportion of medium-chain fatty acid ethyl esters in the concentration of total esters first increased and then decreased in Hu/Sc fermentation. When ORP was −100 mV in Pf/Sc fermentation, the proportion of higher alcohol acetates to total esters was highest (8.14%), while that of ethyl acetate to total esters was lowest (87.35%). Conclusions. Aeration driven by ORP improved wine ester profiles by increasing the proportion of medium-chain fatty acid ethyl esters or reducing that of ethyl acetate in cofermentations. Significance of the Study. The present study will allow wineries and researchers to optimize the aeration process in alcohol fermentation and develop a scientific aeration strategy to improve the wine ester profile and aroma quality.

1. Introduction

Esters, including acetate esters (ethyl acetate and higher alcohol acetates) and fatty acid ethyl esters (mainly medium-chain fatty acid ethyl esters, MCFAEEs), are the main volatile compounds that contribute to the fruity, floral, and sweet aromas in beer, wine, and liquor [1–4]. These esters synergize one another, and different ester profiles can affect the aroma perception of wine [5]. In winemaking, several parameters such as the inoculation strategy [6] and esterase activity [7] could influence the ester profile. For instance, the esterase activity was enhanced by overexpressing the gene (IAH1) in wine yeast, which resulted in the significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate, and 2-phenylethyl acetate [7]. Besides, grape nitrogen and lipid composition might be related to red wine ester profiles [8].

Aeration, a common step in the winemaking process, changes the yeast growth and redox state, which has been investigated to affect ester production in wine [9]. However, excessive aeration resulted in the increase of ethyl acetate and the decrease of ethyl esters, which might have a negative effect on wine aroma quality [10, 11]. Recently, more and more researchers focused on improving the aeration strategy in wine alcohol fermentation. Canonico et al. [12] found that low aeration during the early stages of fermentation decreased ethyl acetate production and was beneficial to better ester profiles compared to the conditions of no aeration and high aeration. Yan et al. [13] found that aeration for a short time during the cell growth stage increased acetate ester concentration and decreased ethyl ester concentration. However, the oxygen demand of yeasts is dynamic and strain-dependent during wine alcohol fermentation [14], which caused an accurate aeration strategy which would be difficult to be realized by controlling the intensity and timing of aeration. Dissolved oxygen concentration was normally used as an indicator to monitor aeration in wine fermentation in recent studies [15–17]. However, the very low levels of dissolved oxygen, often less than 0.08 mg/L [18–20], are infeasible to be determined with a conventional oxygen electrode during the wine fermentation process. An indicator that can accurately reflect the redox status during wine alcohol fermentation needs to be developed for appropriate aeration strategies.

Oxidation-reduction potential (ORP) is the tendency of a compound to acquire or release electrons and is directly related to the redox status, which is a sensitive indicator to delicate changes of dissolved oxygen concentration under microaerobic and anaerobic conditions [21, 22]. Liu et al. [14] found that aeration driven by ORP could affect the fermentation performance and ethanol production of yeasts in very high gravity fermentation. Holman and Wareham [23] demonstrated that ORP could be a monitoring tool in a low dissolved oxygen wastewater treatment process. However, few studies have focused on the effect of controlled aeration driven by ORP on ester profiles in wine alcohol fermentation.

The present study evaluated the effect of controlled aeration driven by ORP on yeast growth, ester profile, and ester production in single fermentation of S. cerevisiae (Sc), Pichia fermentans (Pf), Hanseniaspora uvarum (Hu), and the cofermentation with Pf/Sc and Hu/Sc. Three aeration strategies based on ORP including natural ORP (especially −150 mV∼−105 mV in Pf/Sc and −141 mV∼−107 mV in Hu/Sc, respectively), −100 mV, and −50 mV were performed and understood in term of the evolution of ORP and accumulated aeration volume. Then, residual sugar concentration, ethanol concentration, and viable cell number were assayed during fermentation. Ester composition in final wines and the dynamic of esters and their precursors during fermentation have been investigated under different aeration.

2. Materials and Methods

2.1. Yeasts and Media

Saccharomyces cerevisiae ACTIFLORE® F5 (Sc) was obtained from LAFFORT Co. (Bordeaux, France). Pichia fermentans Z9Y-3 (Pf) was isolated from a Chinese liquor pit [24]. Hanseniaspora uvarum Yun268 (Hu) was screened from spontaneous fermentation of Blue French (Vitis vinifera L.) grape [25]. Yeasts were collected after growing them in the YPD broth (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract, and unadjusted pH) at 28°C and 170 rpm for 48 h on a platform shaker.

The synthetic grape juice [26] was used in microvinification. It was prepared with 100 g/L glucose, 100 g/L fructose, 0.3 g/L citric acid, 5 g/L malic acid, 5 g/L tartaric acid, 1 g/L yeast extract (Aoboxing, Beijing, China), 2 g/L (NH₄)₂SO₄, 0.4 g/L MgSO₄, 5 g/L KH₂PO₄, 0.2 g/L NaCl, and 0.05 g/L MnSO₄. pH was adjusted to 3.3 with 10 M NaOH and 37% v/v HCl. Yeast extract and other ingredients were sterilized separately by autoclaving for 10 min at 110°C and then mixed under sterile conditions before inoculation.

2.2. Chemicals

All the chemical standards used for aroma identification and quantification were purchased from Sigma-Aldrich (Shanghai, China). The chemicals were as follows: ethyl acetate (≥99.9%), isobutyl acetate (>99.8%), isoamyl acetate (≥99.7%), phenylethyl acetate (≥97.0%), ethyl butyrate (≥99.5%), ethyl isovalerate (≥99.7%), ethyl hexanoate (≥99.0%), ethyl octanoate (≥98.5%), ethyl decanoate (≥99.0%), isobutyl alcohol (≥99.8%), isoamyl alcohol (≥98.5%), 2-phenylethanol (≥99.0%), 2-octanol (≥99.5%), hexanoic acid (≥99.0%), octanoic acid (≥99.5%), and decanoic acid (≥99.5%). Water was purified by using a Milli-Q system (Millipore, Bedford, USA).

2.3. Aeration Strategies Controlled by ORP

Three different aeration strategies were performed according to the ORP levels of the synthetic medium to evaluate the effects of controlled aeration driven by ORP on the ester profile during wine alcohol fermentation. These strategies were (i) aeration I (natural ORP without air feeding), (ii) aeration II (the ORP value was −100 mV), and (iii) aeration strategy III (the ORP value was −50 mV). An aeration system controlled by ORP was constructed using a 1.3 L gas-tight bottle with a working volume of 650 mL (Figure 1). The bottle was sealed with a plastic cap and an airlock (1) and was placed in a thermostatic shaker. The ORP value (mV) in the medium was monitored using a rechargeable ORP composite electrode (type 501, Leici, Shanghai, China) submerged into the medium (2). To decrease the potential risk of grape must oxidation and mimic oxygen feeding in the winemaking industry, single-sterile air (21% v/v oxygen) was sprayed through a gas pipe (3) on the surface of the fermenting medium according to the real-time ORP value. When the ORP value of the fermenting medium fell to its lowest value and remained stable, aeration began until the value rose to the set value. The airflow rate was controlled at 60 mL/min with a rotameter (LZB-2, Yinhuan, Yuyao, China) (4). The rotameter was calibrated in the factory, so we adjusted its knob to make sure that the float of the rotameter was on the scale corresponding to the desired flow rate. 10 mL of the fermenting samples (5) was taken with a disposable syringe through the sampling pipe (6) submerged in the medium. The gas pipe and the sampling pipe were closed except for air feeding or sampling.

2.4. Analytic Determinations

Three aeration strategies driven by ORP were performed in three single fermentations (Sc, Pf, and Hu) and two cofermentations (Pf/Sc and Hu/Sc) in our work. Fermentations were performed in triplicate using the aeration system incubated at 22°C with 110 rpm of agitation. In single fermentation, the initial concentration of Sc or non-Saccharomyces (NS) was 3 × 10⁶ cells/mL. In cofermentation, 2 × 10⁶ cells/mL of Sc and 1 × 10⁶ cells/mL of NS were inoculated simultaneously in accordance with our previous studies reporting wine aroma enhancement compared to single Sc fermentation [24, 27].

Sugar concentration was measured using the copper reduction assay at intervals of 24 h until it dropped below 2 g/L [28]. The procedure was performed as follows: The fermentation sample was diluted to a sugar concentration of about 2−4 g/L. Then, 5 mL of this diluted sample was mixed with 5 mL Fehling A reagent (34.7 g CuSO₄·5H₂O dissolved in 500 mL distilled water), 5 mL Fehling B reagent (173 g potassium sodium tartrate and 50 g NaOH in 500 mL distilled water), and 50 mL distilled water. The mixture was heated to a boil and kept for 2 minutes. Then, 2 drops of the methylene blue indicator were added, and the mixture was titrated with 5 g/L glucose standard solution until blue disappeared. In the controlled test, the sample was replaced with distilled water and the other test conditions were the same as above. The samples (10 mL) were taken every 12 hours in the first 48 hours of fermentation and subsequently every 24 hours. The sugar concentration of the samples was calculated based on the difference in the amount of the glucose standard solution between the two titration experiments. The viable cell number was checked and counted using the Wallerstein nutrient (WLN) agar that allowed the growth of differential colonies. The fermenting medium was sampled at the selected time points to assay the dynamic changes of aroma profiles. All the samples were centrifuged at 4°C and for 5 min, then filtered using a 0.22 μm membrane, and stored at −20°C until further analysis.

2.5. Volatile Analysis

The yeast-derived volatile compounds were analyzed using headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS) as described by Hu et al. [29]. Two grams of sodium chloride, 6 mL water, 2 mL sample, and 20 μL of the internal standard (16 mg/L in ethanol and 2-octanol) were placed in a 20-mL vial. The vial was tightly capped and heated at 40°C with 600 rpm agitation for 15 min. A 50/30 μm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was then exposed to extract volatiles for 30 min with continuous heating and agitation as mentioned above and subsequently desorbed at 230°C in the GC injector for 5 min. Shimadzu QP2020 GC-MS (Shimadzu Corporation, Kyoto, Japan) combined with a DB-WAX column (60 m × 0.25 mm × 0.25 μm, Agilent J & W, USA) was used for volatile analysis. The volatiles were injected in splitless inlet mode and carried by helium (99.999%) at a constant flow rate of 1.5 mL/min. The initial temperature of the GC oven was set at 40°C for 3 min, then increased to 160°C at 4°C/min, raised to 220°C at 7°C/min, and held at 220°C for 8 min. The temperatures of the transfer line and ion sources were at 220°C and 200°C, respectively. The MS was operated in an electron ionization mode at 70 eV and scanned over a mass range of m/z 35–350 with a scan interval of 0.2 s.

Standard calibration curves were developed using volatile compound standards diluted continuously in a synthetic wine (11% v/v ethanol, 6 g/L tartaric acid, and pH 3.3). The standard mixture was blended with 20 μL internal standard (16 mg/L, 2-octanol) and analyzed according to the same HS-SPME/GC-MS procedure as described above.

2.6. Statistical Analysis

Data were presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) with the Duncan test (α = 0.05) was conducted to compare volatile compound concentrations, and a two-way ANOVA test was carried out to reveal the effect of the two tested factors (aeration strategy and starter culture) on ester production using SPSS statistical package version 20.0 (SPSS Inc. Chicago, IL, USA). The target product yields were calculated as μg of compounds produced/g of sugar consumed [30]. The dynamic changes in ester concentrations throughout fermentation were analyzed with polynomial fitting using Origin 2019 (OriginLab Cooperation, Northampton, MA, USA).

3. Results and Discussion

3.1. Aeration Controlled by ORP

The evolution of ORP and accumulated aeration volume during fermentation is shown in Figure 2. The change in ORP showed an approximate “L” shape with a rapidly decreasing trend from >150 mV at the beginning of fermentation due to the consumption of oxygen by the yeasts and then remained close to stable under the natural ORP without aeration, except for Hu fermentation, which was consistent with previous studies [31, 32]. The ORP value dropped to lowest <−100 mV at about 30 h, and then, aeration was activated to maintain ORP at −50 mV and −100 mV, respectively. During the fermentations of Sc, Pf/Sc, and Hu/Sc, ORP could be detected and controlled effectively at the set points by aeration. Especially from day 1.5 to day 3, ORP values had significant differences for different aeration strategies, which showed that ORP was a good indicator for aeration in the fermentations of Sc, Pf/Sc, and Hu/Sc.

(a)

(b)

(c)

(d)

(e)

The accumulated aeration volume increased first and then kept close to stable during fermentation, reflecting the difference in oxygen demand of yeasts to maintain specific redox status at different fermentation stages. The accumulated aeration volume in Pf-aeration III (Figure 2(b)) fermentation was highest after 2 d, followed by that in Hu-aeration III (Figure 2(c))fermentation, which suggested that Pf and Hu needed more oxygen to maintain the redox status of aeration III than Sc. However, the accumulated aeration volume did not increase any more after 2.5 d in Hu-aeration III fermentation. It might be because the accumulation of ethanol inhibited the activity of Hu cells with weak tolerance to ethanol, leading to the cessation of oxygen demand. In Sc-aeration III fermentation (Figure 2(a)), the accumulated aeration volume was lower than that in Hu/Sc-aeration II (Figure 2(e)) and Pf/Sc-aeration II fermentations (Figure 2(d)). The difference in accumulated aeration volumes reflected the different oxygen demands of different starter cultures and the operability of aeration strategies driven by ORP in wine alcohol fermentation. Supplemental Figure S1 shows that the dissolved oxygen concentrations (DO) dropped rapidly from the initial value of <2 mg/L and stabilized around zero after 30 h even in the condition of high aeration (aeration III), except for Hu fermentation, which showed that it was very difficult to distinguish all aeration strategies in the view of DO [22].

3.2. Sugar Consumption, Ethanol Production, and Yeast Growth

The residual sugar concentration in final wines from Sc, Pf/Sc, and Hu/Sc fermentations was below 2 g/L (Table 1). However, for wines from Pf and Hu fermentations, there was still a lot of sugar but very little ethanol, which indicated that Pf or Hu alone could not complete fermentation under the winemaking conditions. Besides, no significant difference was found in ethanol concentration and the yield of ethanol/sugar with different aerations. This indicated that the three aeration strategies investigated did not change ethanol synthesis in winemaking. As shown in Figure 3(a), sugar was exhausted in Sc fermentation with aeration II within 7 d, followed by aeration III within 8 d and aeration I within 9 d. Varela et al. [10] proved that the lack of oxygen inhibited sugar uptake and cell growth resulting in incomplete or even “stuck” fermentation, while they also found that excessive oxygen did not increase the rate of fermentation [11]. No significant difference in sugar consumption was observed in Pf/Sc fermentations (Figure 3(b)). There was a more sluggish decrease in sugar concentration when coculturing Sc with Hu in aeration I than in other aeration strategies (Figure 3(c)). It supported that appropriate oxygen (aeration II) improved sugar consumption between Sc and Hu/Sc fermentations [33].

(a)

(b)

(c)

As shown in Figures 3(a)–3(c), the viable cell number of Sc reached about 1 × 10⁸ CFU/mL, 8.7 × 10⁷ CFU/mL, and 5 × 10⁷ CFU/mL, respectively, in the Sc, Pf/Sc, and Hu/Sc fermentations at 1.5 d; meanwhile, aeration began to be performed. In Sc and Hu/Sc fermentations, the highest cell number of Sc was found in aeration II, followed by aeration III and aeration I, but there was no significant difference of the Sc cell number in Pf/Sc fermentations with different aeration strategies. Appropriate oxygen feeding could enhance lipid production, promoting additional growth of yeast cells during fermentation [34], but an excessive amount of oxygen might trigger lipid degradation of yeasts [35], which supported the highest cell number of Sc in aeration II. In cofermentation, both Pf and Hu survived one day less in aeration II and III than in aeration I, which suggested that an increase in aeration was beneficial to the dominance of S. cerevisiae over NS yeasts in wine alcohol fermentation. A recent study also reported that NS yeasts had a long survival time under anaerobic conditions during mixed culture fermentation with S. cerevisiae [12].

3.3. Ester Compositions and Profiles

The concentrations of ethyl acetate (EA), higher alcohol acetates (HAAs), and MCFAEEs found in the final wines are reported in Table 2. EA can contribute fruity aroma properties and have favorable effects on wine aroma at concentrations below 80 mg/L [36]; however, it can also bring nail polish flavor to wines at high levels and can be hydrolyzed into acetate acid, which can cause wine aroma quality defects [13]. Aeration decreased the EA concentration in Sc, Pf/Sc, and Hu/Sc fermentations. There were 13.1% and 41.7% decrements of EA in aeration II and aeration III in comparison with aeration I in Sc fermentation, respectively, while aeration II and aeration III resulted in 22.2% (12.8%) and 32.4% (9.6%) decreases of EA in Pf/Sc (Hu/Sc) fermentation, respectively. Hu/Sc fermentation generated higher amounts of EA (85.5 mg/L) than other starter cultures. More HAAs were produced in cofermentations relative to single fermentation and decreased when aeration increased. HAA concentration decreased by 16.1% (35.8%) and 20.8% (46.6%) in aeration II (aeration III) compared to aeration I in Pf/Sc and Hu/Sc fermentations, respectively. Accumulating studies have reported that the gene of alcohol acetyltransferase (AATase, EC 2.3.1.84), a key enzyme of EA and HAAs synthesis, was repressed by aeration, resulting in reduced AATase activity [37, 38]. So the decrease in EA and HAAs may be caused by lower AATase activity under higher aeration. Pf/Sc fermentation has been proved to improve MCFAEE concentration [39]. In our study, the total MCFAEE concentration in Pf/Sc fermentation was higher than that in Sc and Hu/Sc fermentations in all aeration strategies and reached the top (2248 μg/L) in aeration I. With an increase in aeration, the total MCFAEE concentration decreased in Pf/Sc fermentation, while it remained unchanged and then decreased in Sc fermentation. However, in Hu/Sc fermentation, it showed no significant change.

In wine, the increased proportion of MCFAEEs to total esters (TEs, including EA, HAAs, and MCFAEEs) is beneficial to aroma quality since the risk of acetic acid production from EA and HAAs is reduced. Acetic acid is generally considered to be associated with undesirable sensory descriptors in wine [38]. To further investigate the ester profile regulated by aeration and starter cultures in wine alcohol fermentation, the proportions of EA to TEs, HAAs to TEs, and MCFAEEs to TEs were analyzed. Compared to the corresponding ester proportion in wines from Sc fermentation, a specific enhancement of the MCFAEEs proportion was observed in wines obtained from Pf/Sc fermentation, in contrast to the EA proportion that was specifically increased in Hu/Sc fermentation, which supported that the production of secondary metabolites was considered an individual strain characteristic in winemaking [40]. With an increase in aeration in Sc fermentation, the proportion of MCFAEEs to TEs increased from 3.53% to 4.36%, while that of HAAs to TEs decreased from 7.18% to 6.09%. In Pf/Sc fermentations, the MCFAEE proportion had no significant difference in three aerations, but the EA proportion was slightly lower in aeration II (87.35%) than that in other aeration strategies. In Hu/Sc fermentation, the MCFAEE proportion first increased and then decreased with an increase in aeration. Thus, aeration II was beneficial to decreasing the EA proportion in Pf/Sc fermentation and increasing the MCFAEE proportion in Hu/Sc fermentation.

To evaluate the effects of different starter cultures and aeration strategies on the ester profile of the final wines and identify the chemical and volatile compounds that discriminate between wines, principal component analysis (PCA) was applied (Figure 4). PC1 (52.1%) showed that MCFAEEs and MCFAs were specific for the wines in aeration I and aeration II in Sc and Pf/Sc fermentations. With regard to PC2 (22.7%), it differentiated more in relation to the starter cultures. Ethyl acetate, isobutyl alcohol, and isoamyl alcohol were specific for wines produced by Hu/Sc fermentation. Phenethyl alcohol had a stronger correlation with aeration III than aeration II in Hu/Sc fermentation, indicating that the production of phenethyl alcohol required more oxygen in Hu/Sc fermentation. Besides, isobutyl acetate, isoamyl acetate, and phenethyl acetate, located on the top right-hand side, seemed not to be specific for wines obtained under any aeration strategies. In addition, the two-way ANOVA of the effect of aeration strategies, starter cultures, and their interaction on wine ester profiles was performed (Table 3). Generally, the concentrations of individual and total esters were significantly modified by both the aeration strategy and the starter culture. However, phenethyl acetate production was only affected by the starter culture. Notably, a significant interaction was found between these two factors in the production of specific esters, such as EA , isobutyl acetate , ethyl hexanoate , ethyl octanoate , and total MCFAEEs . These results not only highlighted the response of ester profiles to aeration and starter cultures but also suggested that the regulation of aeration on ester profiles could be significantly conditioned by starter cultures [41].

Figure 4

PCA of major aroma compounds in wines with different aeration strategies and starter cultures. Aeration I (green), aeration II (red), and aeration III (blue). Sc (circle) represented single fermentation with S. cerevisiae F5, Pf/Sc (triangle) represented cofermentation with Pichia fermentans Z9Y-3 and S. cerevisiae F5, and Hu/Sc (square) represented cofermentation with H. uvarum Yun268 and S. cerevisiae F5. EA, ethyl acetate; IBE, isobutyl acetate; IAE, isoamyl acetate; PEE, phenethyl acetate; EH, ethyl hexanoate; EO, ethyl octanoate; ED, ethyl decanoate; IBA, isobutyl alcohol; IAA, isoamyl alcohol; PEA, phenethyl alcohol; HA, hexanoic acid; OA, octanoic acid; DA, decanoic acid.

3.4. Dynamic of Esters and Their Precursors

The dynamic changes of esters and their corresponding precursors during single Sc, Pf/Sc, and Hu/Sc fermentation were analyzed to further explain the effect of aeration on wine ester profiles (Figure 5). In Figure 5, for each compound, its concentrations were standardized in fermentation with different starter cultures and different times. The color changes from blue to white to red, and correspondingly, the concentration of the compound gradually increases. For example, during Sc-aeration I fermentation, the color of EA changed from dark blue to light blue and finally gray as the fermentation time increased, which showed a continuous increase in EA concentration. Similarly, the increase in EA was also found under aeration II during Sc fermentation and under aeration III during Hu/Sc fermentation. During Hu/Sc-aeration I fermentation, the color of EA was first kept light pink and then changed to dark pink, which indicated that the EA concentration stayed stable and then increased over time. While the EA concentration increased first and then decreased under aeration II during Hu/Sc fermentation. Besides, the EA concentration was higher in Hu/Sc fermentation than that in Sc and Pf/Sc fermentations at all the time points examined. Isoamyl acetate accounted for >80% of the total HAAs, and only its concentration exceeded its threshold value among the three kinds of HAAs detected in this study. The isoamyl acetate concentration increased gradually under aeration I during Sc fermentation, aeration II during Pf/Sc fermentation, and aeration III during Hu/Sc fermentation, while it increased first and then decreased under aerations I and III during Pf/Sc fermentation and under aeration II during Hu/Sc fermentation. It increased first, then decreased, and finally increased under aeration I during Hu/Sc fermentation. Meanwhile, isoamyl acetate did not always change over time in the same way as its precursor-isoamyl alcohol, which was consistent with the studies of Farina et al. [42] and Shekhawat et al. [43]. It might be caused by different activities of AATase responsible for the synthesis of isoamyl acetate from isoamyl alcohol and acetic acid under different aerations [37]. Ethyl hexanoate and ethyl octanoate were the main MCFAEEs in Pf/Sc fermentation. Their change over time was similar to the change of their corresponding MCFAs during Pf/Sc fermentation under different aerations except Day 2. In the synthesis of MCFAEEs, the supply of medium-chain fatty acyl CoA, reflected by the concentration of the corresponding MCFAs, was the limiting factor instead of acyltransferase activity, such as EEB1 and EHT1 [44].

Figure 5

Heatmap indicating the dynamic changes in the concentrations of major esters and their precursors during wine alcohol fermentation with different aeration strategies and starter cultures. Sc represented single fermentation with S. cerevisiae F5, Pf/Sc represented cofermentation with Pichia fermentans Z9Y-3 and S. cerevisiae F5, and Hu/Sc represented cofermentation with H. uvarum Yun268 and S. cerevisiae F5. I represented aeration I, II represented aeration II, and III represented aeration III.

4. Conclusions

In this work, the wine ester profile in response to controlled aeration driven by ORP was investigated in fermentation with different starter cultures. Our results showed that the accumulated aeration volume increased with an increase in ORP and that aeration intensity was effectively regulated by ORP. Aeration is beneficial to the dominance of Sc over NS yeasts in cofermentations and accelerates the sugar consumption in Sc and Hu/Sc fermentations. The EA and HAA concentrations decreased with an increase in aeration and reached highest in Hu/Sc-aeration I fermentation. The MCFAEE concentration reached the top in Pf/Sc-aeration I (natural ORP without air feeding) fermentation and had the lowest values under aeration III in Sc fermentation. The ester profile was regulated by both aeration and starter cultures. Aeration II could lead to a lower proportion of EA to TEs in Pf/Sc fermentation and a higher proportion of MCFAEEs to TEs in Hu/Sc fermentation compared with other aerations. Our study provided an effective aeration strategy driven by ORP in wine alcohol fermentation and would be of considerable value for improving wine ester profiles. However, in this work, only three aeration conditions were set based on the representative ORP values, which resulted in limited information about ester change in response to aeration. In further research, more aeration conditions need to be set up in order to obtain a clearer ester change rule. Besides, membrane filtration should be used for the sterilization of media instead of autoclaving, better simulating the composition of grape juice to obtain more reliable yeast performance.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (31801528 and 31972199), the Basic Research Program of Natural Science in Shaanxi Province (2023-JC-YB-145 and 2021JQ-147), the Chinese Postdoctoral Science Foundation (2019M663833), the Key Science and Technology Project of Xinjiang Uygur Autonomous Region (2022A02002-4), and the Key Research and Development Project of Xinjiang Uygur Autonomous Region in the 14th Five Year Plan (2020B01005-2). The authors would like to thank Hong-Yan Zhang from the College of Plant Protection, Northwest A & F University, for the excellent technical assistance of instrumental analysis and thank Associate Professor David William Jeffery from the University of Adelaide for proofreading.

Supplementary Materials

Supplemental figure 1: dynamic of dissolved oxygen concentrations during fermentation with different aeration strategies and starter cultures. Sc, Pf, Hu, Pf/Sc, and Hu/Sc: fermentations with single S. cerevisiae F5, single Pichia fermentans Z9Y-3, single H. uvarum Yun268, Pichia fermentans Z9Y-3 and S. cerevisiae F5, and H. uvarum Yun268 and S. cerevisiae F5, respectively. I represented aeration I, II represented aeration II, and III represented aeration III. (Supplementary Materials)

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