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Brazilian Journal of Microbiology logoLink to Brazilian Journal of Microbiology
. 2024 Sep 25;55(4):3215–3228. doi: 10.1007/s42770-024-01525-5

Naringenin and caffeic acid increase ethanol production in yeast cells by reducing very high gravity fermentation-related oxidative stress

Berna Kavakcıoğlu Yardımcı 1,2,✉
PMCID: PMC11711439  PMID: 39320639

Abstract

Very high gravity (VHG) fermentation is an industrial-scale process utilizing a sugar concentration above 250 g/L to attain a significant ethanol concentration, with the advantages of decreased labor, production costs, water usage, bacterial contamination, and energy consumption. Saccharomyces cerevisiae is one of the most extensively employed organisms in ethanol fermentation through VHG technology. Conversely, high glucose exposure leads to numerous stress factors that negatively impact the ethanol production efficiency of this organism. Here, the impact of various phytochemicals added to the VHG medium on viability, glucose consumption, ethanol production efficiency, total antioxidant-oxidant status (TAS and TOS), and the response of the enzymatic antioxidant system of yeast were investigated. 2.0 mM naringenin and caffeic acid increased ethanol production by 2.453 ± 0.198 and 1.261 ± 0.138-fold, respectively. The glucose consumption rate exhibited a direct relationship with ethanol production in the naringenin-supplemented group. The highest TAS was determined as 0.734 ± 0.044 mmol Trolox Eq./L in the same group. Furthermore, both phytochemical compounds exhibited robust positive correlations with TAS (rnaringenin = 0.9986; rcaffeic acid = 0.9553) and TOS levels (rnaringenin = -0.9824; rcaffeic acid = -0.9791). While naringenin caused statistically significant increases in glutathione reductase (GR) and thioredoxin reductase (TrxR) activities, caffeic acid significantly increased TrxR and superoxide dismutase (SOD). Both phytochemicals seem to impact the ethanol production ability by regulating the redox status of the cells. We believe that the incorporation of particularly cost-effective antioxidants into the fermentation medium may serve as an alternative way to enhance the efficiency of bioethanol production using VHG technology.

Keywords: Very high gravity fermentation, Yeast, Ethanol production, Phytochemicals, Oxidative stress

Introduction

Very high gravity (VHG) fermentation refers to the procedure of utilizing a medium with a sugar concentration greater than 250 g/L in order to attain a significant ethanol concentration on an industrial scale [1, 2]. VHG technology provides several significant advantages at once. Utilizing this technology in bioethanol production leads to decreased labor requirements, decreased production expenses, conservation of water, decreased bacterial contamination and environmental impact, as well as decreased energy consumption, all while concurrently raising the ethanol concentration [3]. The eukaryotic yeast Saccharomyces cerevisiae is indisputably one of the most extensively employed organisms in the process of ethanol fermentation through VHG technology because of its multiple favorable characteristics for industrial applications [4, 5]. These characteristics involve rapid proliferation, efficient anaerobic glucose metabolism, elevated ethanol production, outstanding yield, and an impressive capacity to withstand diverse environmental stressors, such as high ethanol levels, acidic conditions, and low oxygen levels [6]. Undoubtedly, it is important to consider that the high amounts of glucose used in VHG fermentation expose this resistant organism to numerous stress factors that negatively impact the efficiency of ethanol production. The literature demonstrates that a variety of strategies are employed to improve the organism’s ability to cope with fermentation-related stress factors and, as a result, increase its ethanol production capacity. For instance, scientists used genome shuffling and adaptive evolution to boost S. cerevisiae’s ethanol yield during VHG fermentation [7–11]. The classical approach is to improve the fermentation conditions and provide nutritional support to facilitate microbial growth [12–14]. The addition of osmoprotectants into the fermentation medium is another prevalent and long known approach that appears to enhance the organism’s capacity to tolerate high osmotic stress [15–17]. Aside from the aforementioned approaches, another alternative is the addition of antioxidant substances to the fermentation medium, as fermentation-related stress factors also induce oxidative stress [18]. The literature review identified a study that examined the effect of introducing antioxidants into the S. cerevisiae fermentation medium on the efficiency of ethanol production, which reports supplementation with Reactive Oxygen Species (ROS) scavengers like N-acetyl-L-cysteine alleviates oxidative stress and enhances fermentation performance [19] It has been shown that dipeptides, which are antioxidants derived from plants, improve the ability of Saccharomyces pastorianus to withstand osmotic stress. This improvement is achieved by strengthening the integrity of the cell membrane and lowering oxidative damage. Additionally, these biomolecules enhanced the concentration of volatile compounds and antioxidant activities, hence improving the quality of beer during high gravity brewing [20]. In a similar vein, the study that investigated the influence of wheat-gluten hydrolysates and their ultrafiltration fractions, which contained antioxidant peptides, on yeast growth and ethanol production during VHG fermentation, reported that these supplements significantly enhanced yeast growth, viability, and stress tolerance, resulting in increased mitochondrial membrane potential and ethanol production [21].

Although there are a limited number of studies on the subject, the use of compounds or fractions with antioxidant properties as supplements is a relatively new and effective strategy that is employed to enhance the microbial growth and production efficiency of yeast in VHG fermentation. The primary benefit of this strategy, in contrast to alternative methodologies, lies in its straightforward implementation and cost-effective alternatives. Based on this rationale, the impact of various phytochemicals added to the VHG fermentation medium on the efficiency of ethanol production and parameters linked to oxidative stress were examined in this report. We used gallic acid, quercetin, naringenin, caffeic acid, catechin, hesperidin, and epicatechin in the final concentration range of 0–2.0 mM. Each of these substances is classified as a polyphenol and exhibits potent antioxidant properties [22]. We discovered that naringenin and caffeic acid greatly increased the production of ethanol in yeast cells by lowering the oxidative stress caused by VHG fermentation. Hence, we believe that it is crucial to conduct research and evaluate the utilization of cost-effective antioxidant sources in VHG technology.

Materials and methods

Materials

Unless otherwise stated, all of the chemicals used were analytical or of higher purity, obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The S. cerevisiae BY4741 wild-type strain (MATa his3Δ1 leu2Δ0 met15Δ0ura3Δ0) was obtained from Horizon Discovery Ltd. The measurements were done using a microplate photometer (Multiskan FC 357, Thermo Fisher, USA) and a UV-Vis spectrometer (T80 & T80+, PG Instruments Ltd., UK).

Methods

Routine maintenance conditions of S. cerevisiae cells and VHG fermentation of ethanol

The wild S. cerevisiae BY4741 strain was routinely maintained on a yeast extract peptone dextrose (YPD) agar medium that contained 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 20 g/L Bacto-agar for 5 days at 30 °C (pH: 5.6). We allowed the cells to reach the early exponential phase at an optical density (OD660nm) of 0.5 in agar-free YPD broth at 30 °C and 180 rpm. Agar-free YPD medium containing 30 g/100 mL glucose (YPD30) was used as the VHG fermentation medium. Yeast cells in the early exponential growth phase were inoculated into 3 mL of YPD30 medium in 6 well plates, starting with a cell density of 107 cells/mL. To establish a semi-aerobic system, the plates were covered with parafilm [19]. The ethanol fermentation process was conducted for 48 h at 30 °C and 100 rpm. We utilized dimethyl sulfoxide (DMSO, 99.9%) as the solvent because of its high solubility to prepare stock solutions of gallic acid, quercetin, naringenin, caffeic acid, catechin, hesperidin, and epicatechin at a concentration of 200 mM. These solutions were subsequently introduced into the YPD30 medium at final concentrations of 0.1, 0.2, and 2.0 mM. The groups that had antioxidants added were called test groups. The groups that did not have antioxidants added but had the same amount of DMSO (1%, 0.1%, 0.05%) as the test groups were called control groups. At the concentrations specified, DMSO did not show toxicity to yeast cells. All samples were prepared in three replicates in three independent experiments.

Cell proliferation assay

At the end of the fermentation process, the OD values of all samples were spectrophotometrically measured at 660 nm, and the results were expressed as cell proliferation percentages compared with the respected controls, the proliferation percentages of which were accepted as 100%. The formula used is given below.

graphic file with name M1.gif

Quantification of remaining glucose in the VHG fermentation medium

The residual glucose content in the medium after fermentation was quantified using the dinitrosalicyclic acid (DNS) method [23]. Initially, the DNS reagent was prepared by dissolving 1 g of DNS in 20 mL of 2 M sodium hydroxide (NaOH). The resulting solution was gradually added to a 50 mL solution containing 30 g of sodium potassium tartrate while stirring. The final volume of the mixture was completed to 100 mL by adding distilled water. Then, 500 µL of cell-free medium was mixed with 500 µL of DNS reagent, and the mixture was boiled for 10 min. After cooling, 5 mL of distilled water was added to the mixture, and the absorbance was measured at 546 nm against a blank containing 500 µL of distilled water instead of the cell-free medium. The glucose level was expressed as ppm by using a calibration curve prepared in the range of 0–200 ppm glucose.

Quantification of ethanol production by VHG fermentation

The ethanol concentrations produced by yeast cells in a VHG fermentation medium were assessed using the Ethanol Colorimetric Assay Kit from Elabscience Biotechnology, following the manufacturer’s instructions. In the procedure, 40 µL of either a sample or a standard were introduced onto the 96-well plate, followed by the addition of 160 µL of a reaction working solution (prepared by mixing buffer solution A, enzyme working solution, buffer solution B, substrate working solution, and chromogenic agent in the ratios specified in the kit) to each well. The OD value of each well at 450 nm was measured with a microplate reader and recorded as A1. Then, the plate was incubated at 37 °C with shading light for 10 min and A2 values were recorded. By subtracting the absorbances, the ethanol amounts were converted to µmol/mL using the calibration equation.

Cell lysis procedure

The commercial CelLytic Y Cell Lysis Reagent (Sigma, USA) was used to lyse yeast cells. The cell pellet was resuspended in lysis buffer at a concentration of 1 g wet weight of cells per 5 mL buffer and incubated at room temperature for 30 min with moderate shaking. Cell lysates were centrifuged at 3,000 rpm for 5 min, and the supernatants were taken for biochemical analysis.

Total antioxidant and oxidant status

The total antioxidant and oxidant status (TAS and TOS) of the yeast cells after VHG fermentation process were determined by using TAS and TOS colorimetric assay kits, respectively (Elabscience Biotechnology), and the protocols were followed according to the manufacturer’s recommendations.

In the TAS assay, 10 µL samples or standards with varying concentrations were combined with 200 µL of reagent 1, and OD values were determined at a wavelength of 660 nm (A1). Subsequently, 20 µL of reagent 2 was introduced into the mixtures, followed by an incubation period at 37 °C for 5 min. Following the incubation period, OD values of the samples were reevaluated at a wavelength of 660 nm (A2). The results were obtained by performing linear regression analysis on the difference in absorbance (ΔA) between the blank and the standard (A2-A1), plotted against the standard concentration. The results were then represented as mmol of Trolox Equivalent per L (mmol Trolox Equiv./L).

During the TOS experiment, 20 µL samples or standards with varying concentrations were combined with 200 µL of reagent 1. OD values were then measured at a wavelength of 590 nm, denoted as A1. Subsequently, 50 µL of reagent 2 was introduced into the mixtures, followed by an incubation period at 37 °C for 5 min. Following the incubation period, the samples were reevaluated by measuring their OD values at a wavelength of 590 nm (A2). The results were derived by doing linear regression analysis on the difference in absorbance (A2-A1) vs. the standard concentration. The results were then reported as µmol of hydrogen peroxide equivalent per g of protein (µmol H2O2 Equiv./g protein).

Antioxidant enzyme activities

The activities of glutathione reductase (GR), glutathione peroxidase (GSHPx), glutathione-S-transferase (GST), thioredoxin reductase (TrxR), and superoxide dismutase (SOD) in the yeast cells were measured using colorimetric kits from Cayman Chemicals, and the protocols were followed according to the manufacturer’s recommendations.

The determination of GR activity is based on the monitoring of the oxidation of NADPH to NADP + through a reaction with GR, which catalyzes the reduction of oxidized glutathione (GSSG) to its reduced form (GSH). In the assay, 20 µL of sample was combined with 100 µL of assay buffer and 20 µL of GSSG. Subsequently, the enzymatic reaction was initiated by adding 50 µL of NADPH, and the absorbance change was measured at 340 nm once every min to obtain a minimum of five-time intervals. The NADPH extinction coefficient of 0.00373 µM-1 was employed to compute the results. One unit was defined as the required enzyme quantity for the oxidation of 1.0 nmol NADPH per min at 25 °C.

The assessment of GPx activity relies on monitoring the oxidation of NADPH to NADP + through a linked reaction with GR. This reaction catalyzes the regeneration of GSH from GSSG, which is produced by the GPX activity. During the test, a 20 µL sample was combined with 50 µL of assay buffer, 50 µL of co-substrate mixture, and 50 µL of NADPH. Next, the enzymatic reaction was started by adding 20 µL of cumene hydroperoxide, and the change in absorbance was measured at 340 nm per min to collect a minimum of 5 data points. The results were determined using the NADPH extinction coefficient of 0.00373 µM-1. One unit was defined as the amount of enzyme needed to oxidize 1.0 nmol of NADPH per minute at 25 °C.

GST activity is determined by monitoring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH. The assay involved mixing 20 µL of sample with 150 µL of assay buffer and 20 µL of GSH. To initiate the enzymatic reaction, 10 µL CDNB was added. The absorbance change at 340 nm was measured every min for at least 5 time points. The findings were computed using the CDNB extinction coefficient of 0.00503 µM-1, and one unit was expressed as the needed enzyme amount for conjugating 1.0 nmol CDNB with GSH per min at 25 °C.

The assessment of TrxR activity relies on the reduction of 5,5’-dithio-bis-(2-dinitrobenzoicacid) (DTNB) to 5-thio-2-nitrobenzoicacid (TNB) in the presence of NADPH. This reaction yields a yellow product that can be measured by its maximum absorbance at 405–414 nm. During the test, a 20 µL sample was combined with 140 µL of assay buffer. The reaction was then started by adding 20 µL of NADPH and 20 µL of DTNB. The absorbance change was measured at 340 nm every min to acquire a minimum of 5 time points. The enzyme activity was assessed in the presence of aurothiomalate, which is a particular inhibitor of TrxR, in order to determine the activities of additional enzymes that can reduce DTNB. The results were determined using the DTNB extinction coefficient of 0.00792 µM-1. One unit was defined as the NADPH-dependent production of 2 µmol of product per minute at a temperature of 22 °C.

SOD activity is determined by detecting the formazan dye formed by the interaction of tetrazolium salt with superoxide radicals produced by xanthine oxidase. In the experiment, 10 µL sample was combined with 200 µL radical detector, and the reaction was triggered by adding 20 µL xanthine oxidase. After 30 min of incubation at room temperature with gentle shaking, the absorbance was measured at 450 nm. The results were computed using a linear regression of linearized rate (LR) versus SOD activity, with one unit being the quantity of enzyme necessary for 50% superoxide radical dismutation per min at 25 °C.

LR = The absorbance of blank/The absorbance of standard or sample.

Total protein concentrations

The Bradford method was used to measure the total protein concentrations of the yeast cells [24].

Statistical analysis

The data are presented as the mean ± S.E.M from three dependent and three independent experiments. The differences in variance were analyzed statistically using a two-way analysis of variance (ANOVA) test. Tukey’s test was used as a post hoc. GraphPad prism 5.0 statistics software (GraphPad, La Jolla, CA) was utilized.

Results and discussion

VHG fermentation technology that is currently employed for large-scale bioethanol production as a biofuel has significant advantages including reduced process water needs, lower energy expenses, more productivity, and higher ethanol levels in the final product [25]. But a significant factor to bear in mind during this technology is that the yeast cells are exposed to critical osmotic stress, which results in a reduction of both the growth and viability of this organism. Yeast cells also encounter heat and elevated ethanol concentrations as stressors throughout the process. Indeed, ethanol holds significant importance among all the stresses associated with fermentation. Because of its solubility in both water and lipids, ethanol can enter cells and enhance the flexibility of cell membranes. The accumulation of this substance in cells impairs growth, modifies metabolism, and triggers an oxidative burst, mainly caused by the formation of ROS in mitochondria [26–29]. Numerous studies have demonstrated the impact of ROS on cell proliferation and metabolism [30–33]. Hence, including antioxidants into the VHG fermentation medium could serve as an economical and efficient way to enhance ethanol production efficiency by reducing oxidative stress. From this point of view, the objective of this research was to investigate the impact of different phytochemicals added to the fermentation medium on ethanol production via alterations in oxidant and antioxidant systems.

The changes in cell proliferation, glucose consumption and ethanol production depend on the addition of phytochemicals to VHG fermentation media

The haploid S. cerevisiae BY4741 strain was grown in VHG fermentation media under the specified conditions. Phytochemicals were added into the media to achieve a concentration range of 0.1–2 mM. Subsequently, cell growth percentages, residual glucose levels, and ethanol production were assessed at the end of the VHG fermentation process. The results showed that while all phytochemicals studied partially stimulated cell proliferation at least at one concentration, only quercetin and hesperidin at 2.0 mM led to substantial changes in cell proliferation compared to the respective DMSO control (Fig. 1). Previous studies have additionally documented that quercetin enhances the lifespan of yeast cells by promoting resistance to oxidative stress. For instance, it was showed that about 0.03 mM quercetin increases oxidative stress resistance and longevity in S. cerevisiae. The viability of yeast cells, which were pre-incubated with quercetin for 15 min prior to exposure to 1.5 mM H2O2 for 1 h, increased from 13 to 40% according to the colony forming unit method [34]. Vilaça et al. (2012) found similar findings with 0.3 mM quercetin under the same stress conditions [35]. They reported that in the presence of this polyphenol, oxidative stress resistance increased 2.5-fold in the wild type BY4741 cells. Bayliak et al. (2016) demonstrated by methylene blue staining that 100 µM of quercetin enhanced live yeast cell population by 22%, 20% and 30% under exposure to H2O2, copper ions, and heat shock, respectively [36]. However, we have only encountered a single study that indicates that hesperidin was unable to statistically significantly enhance the cell survival of yeast across a diverse dose range of 0 µM to 100 µM based on monitoring of the cell population by OD660nm [37]. As a matter of fact, we found significant increases in cell proliferation only at the highest quercetin and hesperidin concentrations.

Fig. 1.

Fig. 1

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The cell proliferation percentages of the yeast cells cultured in VHG fermentation media containing different phytochemicals at the final concentration range of 0.1-2.0 mM. The whiskers show the minimum to maximum values of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 denote significant differences between control and test groups or indicated test groups by two-way ANOVA with Tukey’s post-test (The groups exhibiting significant differences from the control were marked with a line in the same color as the control)

The residual glucose concentrations are illustrated in Fig. 2. In the quercetin-supplemented groups, the greatest amount of glucose was consumed. The aforementioned level exhibited a progressive decline as the concentration of quercetin increased. A study on fermentation using Lactobacillus plantarum also found that quercetin promotes more rapid sugar consumption [38]. The second phytochemical to increase glucose consumption was caffeic acid, which followed the same pattern as quercetin. It was determined that glucose consumption in the 0.1 mM naringenin-supplemented group increased when compared to the corresponding DMSO control, however the increase was not significant. On the other hand, a statistically significant decrease was observed when the highest concentration of the phytochemical was used in comparison to the DMSO control. No statistically significant alterations were observed in the groups that received gallic acid as opposed to the control group. In contrast, compared to their respective controls, catechin, hesperidin, and epicatechin significantly decreased glucose consumption at all concentrations examined. No research has been found in the literature that investigate the impact of these phytochemicals, except for quercetin, on the glucose consumption of microbial cells. Notably, extensive clinical in vivo investigations have been conducted on the subject, with a particular emphasis on examining the regulatory impact of phytochemicals on glucose metabolism in diverse metabolic disorders, mainly diabetes [39, 40]. On the other hand, we came across an in vitro study which reports that 100 µM naringenin pre-treatment for 15 min caused the statistically significant decreases in glucose uptake of basal and insulin stimulated human breast cancer cells [41]. It was also stated in the same study that the p44/p42 mitogen-activated protein kinase pathway appears to contribute significantly to insulin-stimulated glucose uptake in these cells. Another paper reported that the presence of 10 nM caffeic acid resulted in a reduction in glucose uptake in endothelial cells, which is likely due to the activation of survival mechanisms through the modulation of NF-κB-related signaling pathways and the activation of anti-apoptotic proteins [42]. However, due to the distinct regulation of glucose metabolism in human cells by insulin and in yeast cells by glucose availability, oxygen levels, and the presence of fermentable versus non-fermentable sugars, the literature findings do not provide any indication about the probable mechanism by which phytochemicals affect glucose consumption in yeast cells.

Fig. 2.

Fig. 2

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The remaining glucose levels in VHG fermentation media containing different phytochemicals at the final concentration range of 0.1-2.0 mM. Data with error bars show the mean ± S.E.M of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 denote significant differences between control and test groups or indicated test groups by two-way ANOVA with Tukey’s post-test (The groups exhibiting significant differences from the control were marked with a line in the same color as the control)

We found that naringenin and caffeic acid were the only phytochemicals that significantly increased the ethanol production in yeast cells compared to the respected controls among the investigated antioxidants (Fig. 3). While naringenin supplementation significantly increased the ethanol production levels by 1.338 ± 0.041, 1.764 ± 0.243, and 2.453 ± 0.198-folds, the addition of caffeic acid caused 1.092 ± 0.043, 1.247 ± 0.099, and 1.261 ± 0.138-fold increases at concentrations of 0.1, 0.2, and 2 mM compared to the respected DMSO controls, respectively. Conversely, Wu et al. discovered that the addition of 200 ppm caffeic acid did not increase the lychee wine fermentation capacity of the S. cerevisiae DV10 strain [43] It was observed that there were generally significant increases with increasing concentrations of the phytochemicals. On the contrary, these levels significantly decreased in the quercetin and epicatechin-supplemented groups for all studied concentrations compared to the respected controls.

Fig. 3.

Fig. 3

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The ethanol levels produced by yeast cells cultured in VHG fermentation media containing different phytochemicals at the final concentration range of 0.1-2.0 mM. Data with error bars show the mean ± S.E.M of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 denote significant differences between control and test groups or indicated test groups by two-way ANOVA with Tukey’s post-test (The groups exhibiting significant differences from the control were marked with a line in the same color as the control)

Glucose consumption is a crucial metric for assessing the metabolic processes of cells, and it exhibits a strong correlation with both the rate of proliferation and the dynamics of the cell population [44]. In a glucose-rich medium, S. cerevisiae cells proliferate rapidly, metabolize glucose, synthesize ATP through glycolysis, and release ethanol into the medium in the process of fermentation [45]. Consequently, one can anticipate an increase in both proliferation percentages and ethanol production amounts as the cells consume more glucose. Nevertheless, our findings did not always demonstrate such a correlation. While it is generally true that the quantity of ethanol produced increased with the increase in glucose consumption in caffeic acid-supplemented groups, no concentration-dependent correlation was observed. Unsurprisingly, the catechin, hesperidin, and epicatechin-supplemented groups had lower ethanol production efficiency than the controls due to significantly lower glucose consumption levels. On the other hand, glucose consumption was dramatically reduced in the 2.0 mM naringenin-supplemented group, which produced the highest level of ethanol when compared to the control. In fact, a similar situation was also observed in the quercetin-supplemented group. We found that ethanol production efficiency decreased significantly despite increased glucose consumption in quercetin-supplemented groups. From these findings, the product efficiency appears to be significantly influenced by the type and concentration of antioxidants used, despite the absence of studies on the ethanol production efficiency of these phytochemicals. Furthermore, it is important to consider that antioxidants may have pro-oxidant effects and influence various metabolic pathways based on their concentration [46–48].

Naringenin and caffeic acid increase ethanol production in yeast cells by reducing VHG fermentation-related oxidative stress

After the VHG fermentation process with the addition of naringenin and caffeic acid, we analyzed the overall antioxidant and oxidant levels of the yeast cells compared to a control group. The findings are displayed in Fig. 4. We observed statistically significant variations in relation to phytochemical concentration and all test groups showed substantial differences from their respective controls (p < 0.05). The group that was administered 2 mM naringenin exhibited the highest TAS level of 0.734 ± 0.044 mmol Trolox Eq./L. In contrast to the progressive increase in TAS levels that accompanied the phytochemical concentrations, the opposite trend was observed in the case of TOS levels, with strong positive correlations (rnaringenin = -0.9946; rcaffeic acid = -0.9975). Additionally, strong positive correlations were found between ethanol and TAS (rnaringenin = 0.9986; rcaffeic acid = 0.9553), as well as strong negative correlations between ethanol and TOS levels (rnaringenin = -0.9824; rcaffeic acid = -0.9791) for both phytochemical compounds. Thus, it is evident that both phytochemicals within the tested concentration range regulate cellular redox status, which seems to impact the ethanol production ability of the yeast cells. Supportingly, several studies have demonstrated that both phytochemicals have the ability to protect cell viability or reduce cell toxicity by reducing oxidative stress. Gerçek et al. showed that 0.2–0.8 mg/mL naringenin, which corresponds to approximately 0.075–0.3 mM, protected yeast cells against toxicity induced by the organophosphorus pesticide malathion [49]. 1-100 ppm caffeic acid promoted viability against stress induced by 1.0 mM H2O2 for 1 h, with a 106% increase in S. cerevisiae BY4741 cells [50].

Fig. 4.

Fig. 4

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TAS and TOS levels of yeast cells cultured in VHG fermentation media containing different amounts of naringenin and caffeic acid phytochemicals. As there were no notable variations in the outcomes with varying DMSO concentrations, a single control value is provided as the average. Data with error bars show the mean ± S.E.M of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 denote significant differences between control and test groups or indicated test groups by two-way ANOVA with Tukey’s post-test (The groups exhibiting significant differences from the control were marked with a line in the same color as the control)

The functions of primary antioxidant enzyme activities in yeast during ethanol production in VHG fermentation media supplemented with naringenin and caffeic acid

Glutathione-related enzyme activities

In the present study, it was aimed to examine the impact of enzyme activity variations in GR, GSHPx, and GST on the levels of ethanol production. The enzyme GR facilitates the transformation of GSSG to GSH. The GLR1 gene encodes both cytosolic and mitochondrial isoforms of the GR enzyme in yeast cells; however, the cytosolic enzyme primarily regulates the glutathione-related redox state in the intermembrane space [51]. By utilizing GSH as an electron donor, GSHPx facilitate the reduction of H2O2 or organic hydroperoxides to water or the corresponding alcohols. S. cerevisiae has been found to possess GSHPx activity, and three genes encoding GSHPx 1–3 were discovered using genome sequencing [52]. The enzyme superfamily GST is an umbrella term for members that facilitate the conjugation of GSH with an extensive range of xenobiotic substances [53]. GTT1 and GTT2-encoded enzymes account for the vast majority of cellular GST activity in yeasts [54]. Figure 5 displays the alterations in the activities of these GSH-associated enzymes.

Fig. 5.

Fig. 5

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GR, GSHPx, and GST enzyme activities of yeast cells cultured in VHG fermentation media containing different amounts of naringenin and caffeic acid phytochemicals. As there were no notable variations in the outcomes with varying DMSO concentrations, a single control value is provided as the average. Data with error bars show the mean ± S.E.M of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 denote significant differences between control and test groups or indicated test groups by two-way ANOVA with Tukey’s post-test (The groups exhibiting significant differences from the control were marked with a line in the same color as the control)

The results showed that the only significant increase in GR enzyme activity relative to the control was found in the medium supplemented with 2 mM naringenin (p < 0.01). On the other hand, significant decreases were observed in the groups that received lower amounts of naringenin compared to the control group. While the groups supplemented with caffeic acid exhibited a comparable pattern, no substantial alterations were detected in comparison to the control group. The activities of the GSHPx and GST enzymes were found to generally decrease substantially as the concentration of both phytochemicals increased. In addition, it was found that as a result of both applications, the activity of the relevant enzymes declined in comparison to the control groups. A slight increase in GSHPx activity was only seen in the group that received 0.1 mM caffeic acid compared to the control group.

Thioredoxin reductase activity

Yeast cytoplasmic TrxR is a crucial enzyme that controls the redox status of the thioredoxin system, serving as a disulfide reductase system that safeguards cells from oxidative and reductive stress [55]. Figure 6 illustrates the alterations in the activity of the TrxR enzyme in cells subsequent to the VHG fermentation process, in which varying concentrations of naringenin and caffeic acid were added. TrxR activity significantly increased with higher concentrations of both phytochemical applications. Despite the fact that the enzyme activity was significantly lower than the control levels in 0.1 mM of naringenin group, it was significantly higher than the control levels in the conditions that contained 0.2 and 2.0 mM of naringenin supplementation. In the case of caffeic acid, a significant increase in TrxR activity was observed only at the highest dose of phytochemical in comparison to the control group. In contrast, the enzyme activities in the remaining two groups were considerably lower than the control.

Fig. 6.

Fig. 6

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TrxR enzyme activity of yeast cells cultured in VHG fermentation media containing different amounts of naringenin and caffeic acid phytochemicals. As there were no notable variations in the outcomes with varying DMSO concentrations, a single control value is provided as the average. Data with error bars show the mean ± S.E.M of three experiments. *p < 0.05; **p < 0.01; ****p < 0.0001 denote significant differences between control and test groups or indicated test groups by two-way ANOVA with Tukey’s post-test (The groups exhibiting significant differences from the control were marked with a line in the same color as the control)

Superoxide dismutase activity

The metalloenzyme SOD catalyzes the dismutation of molecular oxygen to H2O2 and superoxide anion radical. S. cerevisiae contains two SOD genes, SOD1 and SOD2. SOD1 encodes Cu/Zn SOD, while SOD2 gene produces Mn-SOD. Cu/Zn SOD is cytoplasmic, whereas Mn-SOD is mitochondrial, similar to the majority of other eukaryotic organisms [56]. A study demonstrated that a portion of the Cu/Zn SOD enzyme and its copper chaperone (CCS) are found together in the mitochondrial intermembrane space. This enzyme variant plays a role in safeguarding the mitochondrion from oxidative damage in yeast [57]. It was aimed to assess the effect of varying concentrations of naringenin and caffeic acid phytochemicals introduced into the fermentation medium on SOD activity in comparison to the control group, and the obtained results are presented in Fig. 7. The SOD enzyme activity in yeast cells cultured in naringenin-supplemented VHG fermentation media exhibited a substantial decrease as the content of the phytochemical increased. It is noteworthy that the enzyme activity was significantly lower than control levels at all tested naringenin concentrations. In contrast, it was seen that there were generally considerable increases in SOD activity depending on the increasing concentration of caffeic acid. The group that received 2 mM caffeic acid had considerably higher enzyme activity (2.843 ± 0.12) compared to the control group (p < 0.01).

Fig. 7.

Fig. 7

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SOD enzyme activity of yeast cells cultured in VHG fermentation media containing different amounts of naringenin and caffeic acid phytochemicals. As there were no notable variations in the outcomes with varying DMSO concentrations, a single control value is provided as the average. Data with error bars show the mean ± S.E.M of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 denote significant differences between control and test groups or indicated test groups by two-way ANOVA with Tukey’s post-test (The groups exhibiting significant differences from the control were marked with a line in the same color as the control)

The results of the combined analysis of antioxidant enzyme activities indicate that naringenin and caffeic acid phytochemicals enhance the activity of certain antioxidant enzymes while decreasing the activity of others. It can be noted that these increases and decreases are generally in accordance with the phytochemical concentration. Despite the absence of research on the direct impact of naringenin and caffeic acid phytochemicals on the yeast antioxidant enzymes under investigation, there are conflicting findings regarding the impact of polyphenol treatment on antioxidant enzyme activities, which may vary depending on the cell type or stress condition [58–60]. On the other hand, recent discoveries indicate that polyphenols may exert a variety of potential mechanisms of action, including interacting with cell signaling and affecting gene expression, in cytoprotection against oxidative stress, which may be distinct from their traditional activities, which in turn modulates particular enzyme activities that help the cells respond to oxidative stress [61]. Regardless, comprehending the precise underlying mechanism would undoubtedly aid in the development of novel strategies to enhance the efficiency of ethanol production achieved by VHG fermentation.

Conclusion

VHG fermentation exposes cells to endogenously produced oxidative stress besides fermentation-related stressors resulting from the high levels of glucose and ethanol. This study examined the impact of various phytochemicals, including gallic acid, quercetin, naringenin, caffeic acid, catechin, hesperidin, and epicatechin, added to the VHG fermentation medium of S. cerevisiae, on cell viability, glucose consumption, ethanol production efficiency, total antioxidant-oxidant status, and the response of the enzymatic antioxidant system. Naringenin and caffeic acid significantly increased the efficiency of ethanol production among the phytochemicals investigated. We could not find any clear correlation between ethanol concentration and cell proliferation or glucose consumption. Rather, it was showed that naringenin and caffeic acid induce certain antioxidant enzyme activities and regulate cellular antioxidant– oxidant status. Most importantly, our results showed that the product efficiency appears to be significantly influenced by the type and concentration of antioxidants used. This insight implies that the optimization of antioxidant supplementation could enhance ethanol yield under VHG conditions for large-scale industrial applications. We believe that more cost-effective antioxidant substances and natural sources should be evaluated to boost the efficiency of industrial bioethanol production in VHG fermentation.

Acknowledgments

This study was supported by the Scientific Research Projects Unit of Pamukkale University (PAUBAP 2023FEBE006).

Declarations

Competing interests

Berna Kavakcıoğlu Yardımcı declares that she has no conflict of interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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