Chemical Fate of Ascorbic Acid in Wheat Flour Extract: Impact of Dissolved Molecular Oxygen (O₂), Metal Ions, Wheat Endogenous Enzymes and Glutathione (GSH)

Abstract

Ascorbic acid (AH₂) is a commonly used additive in food products. In wheat breadmaking, it is, for example, added to flour for its dough strengthening and bread volume-enhancing effects. While these bread property-enhancing effects are well known, the final chemical fate of AH₂ in breadmaking applications remains nearly undocumented. This study tries to shed light on the chemical fate of AH₂ in wheat breadmaking by investigating the chemical and enzymatic conversion of AH₂ and its reaction products using ¹³C NMR spectroscopy in combination with AH₂ labelled with ¹³C on the C₃ carbon. Following the chemical conversion of AH₂ as function of time, in ultra-pure water, tap water, and wheat flour extracts, in the presence and absence of dissolved O₂ and glutathione (GSH), the specific impact of the presence of trace metal ions, dissolved oxygen and endogenous GSH on the oxidation of AH₂ could be elucidated.

1. Introduction

Ascorbic acid (AH₂), also known as vitamin C, plays a crucial role in human health due to its involvement in various physiological processes [1,2,3,4]. Its importance was first recognized in 1932 by Nobel Laureate, Albert Szent-Györgyi, who discovered this water-soluble micronutrient. Since then, ascorbic acid has been the subject of extensive research, initially for scurvy prevention and later also for its immune system boosting properties, its role in antioxidant defense mechanisms and collagen synthesis-enhancing effects. Besides being a micronutrient, AH₂ has applications in a variety of processes in the food industry. In breadmaking, 20 to 150 ppm of AH₂ is commonly dosed to wheat flour for its dough strengthening and bread volume-enhancing effects [5,6,7]. The action of AH₂ involves oxidation to dihydroascorbic acid (DHA), a process facilitated by AH₂ oxidases and metal ions, with O₂ serving as substrate [8]:

2AH₂ + O₂ ↔ 2DHA + 2H₂O (Reaction I)

AH₂ oxidation is critical in breadmaking, where it promotes disulfide bond formation between gluten proteins. This in turn strengthens the gluten network, which improves its ability to trap fermentation gases and contributes to bread quality. During breadmaking, the oxidation process is predominantly catalyzed enzymatically, with ascorbic acid oxidase delivering about 75% of the DHA production [9,10]. The level of AH₂ oxidase activity in flour is known to vary, depending on factors such as wheat genotype, agronomic conditions, post-harvest storage, and flour extraction rate [8,9,10,11]. Non-enzymatic oxidation of AH₂ also occurs, not only in dough, but also in aqueous solutions. This process is mediated primarily by Cu²⁺ and Fe³⁺ metal ions [8,12], either acting directly as a catalyst or serving as co-factor for AH₂ oxidase (e.g., Cu²⁺) [13]. The non-enzymatic oxidation of AH₂ involves transition metal ions, the formation of O₂^−•, and monodehydroascorbic acid. While the latter undergoes disproportionation to AH₂ and DHA, O₂^−• generates thiyl radicals from glutathione (GSH) or other thiol-containing molecules. This may participate in reshuffling disulfide bonds within glutenin molecules [14]. Regardless of the oxidation pathway, the availability of dissolved O₂ governs the production and activity of DHA in the dough matrix, underscoring the importance of AH₂ oxidation for bread quality.

Aside from interacting with the gluten network, DHA can degrade under certain conditions. By prolonged exposure to an oxidative environment or by interactions with other reactive species, DHA can convert into less active or inactive compounds. This occurs by initial conversion to 2,3-diketogulonic acid (DKGA), which further decomposes into gluonic acid, threonic acid, oxalic acid, etc. (Figure 1). DHA can, however, also be regenerated into AH₂ by glutathione (GSH), endogenously occurring in wheat flour in a range from 18 to 144 nmol GSH/g [11,15]. As GSH reacts with DHA, the latter is reduced to AH₂, while GSH is dimerized (GSSG) by formation of a disulfide bridge.

DHA + 2GSH → AH₂ + GSSG (Reaction II)

While AH₂ oxidation in bread dough can be catalyzed both by enzymes and metal ions, DHA reduction by Reaction I almost exclusively occurs enzymatically [12,16]. Controlled regeneration of DHA by reaction with GSH, a reaction which is facilitated by endogenous GSH dehydrogenase, can therefore assist in protecting AH₂ against over-oxidation. This potentially mitigates the effects of DHA degradation. An excess of GSH relative to DHA can, however, disrupt the positive effect of DHA on the gluten network by reducing the DHA availability. The interplay between enzymatic and non-enzymatic oxidation, DHA degradation, and GSH-mediated DHA regeneration underscores the complexity of the ascorbic acid chemistry occurring in bread dough. Targeting optimal bread quality, the delicate balance between AH₂ oxidation and DHA regeneration and degradation mechanisms highlights the need for in situ and ex situ studies investigating the fate of AH₂ in dough and dough components (e.g., dough liquor) in various conditions, to ultimately achieve precise control of the AH₂/DHA cycle.

Analysis of AH₂ and DHA in complex systems is challenging, because of their limited stability. In particular, the spontaneous oxidation of AH₂ and hydrolysis of DHA are known to be affected by a series of factors including temperature, light, pH, amount of dissolved O₂, ionic strength of the solvent, and the presence of oxidizing enzymes or divalent cations such as Cu²⁺. This causes problems in the determination of AH₂ and DHA concentrations [17]. To address these challenges, it is critical to investigate the oxidation–reduction dynamics of AH₂ in in situ conditions that mimic actual dough matrices with non-destructive techniques providing identification and quantification of the individual species involved. While previous studies had proposed roles for DHA and GSH in dough strengthening, the molecular-level kinetics and transformation pathways of AH₂ and its oxidation products in aqueous flour systems remain poorly quantified. It is, furthermore, unclear whether endogenous components such as GSH primarily act by directly reducing DHA or by modulating the oxidative environment indirectly; for example, through O₂ scavenging. The present study employed high-field ¹³C nuclear magnetic resonance (NMR) spectroscopy to monitor the chemical and enzymatic interconversion of AH₂ and DHA in dough liquor and in synthetic aqueous systems under various conditions—including ultra-pure water, tap water, and pristine and enzyme-deactivated flour extracts (unheated and heated). Adding GSH, metal ions, and modulating the dissolved oxygen concentration assisted in elucidating the respective contributions of enzymatic and non-enzymatic processed. Starting with AH_2, labelled with ¹³C on the C₃ position (¹³C₃ AH₂) (

Table A1. Overview of the samples and the media used.

Sample	Solvent Used	Prepared Under Atmospheric Conditions or Flushed with N2
AH2 in Ultra-pure water	Ultra-pure water (MilliQ, Millipore)	Atmospheric conditions
AH2 in Tap water	Tap water	Atmospheric conditions
AH2 in Ultra-pure water—N2 treated	Ultra-pure water (MilliQ, Millipore)	Flushed with N2
AH2 in Tap water—N2 treated	Tap water	Flushed with N2
AH2 in Flour extract	1 Flour extract in tap water	Atmospheric conditions
AH2 in Heated flour extract	1 Heat-treated (70 °C—30 min) flour extract	Atmospheric conditions
AH2 in Flour extract—N2 treated	1 Flour extract in tap water	Flushed with N2
AH2-Flour + 0.04 GSH-Atm	0.04 mg/mL GSH added to the flour extract	Atmospheric conditions
AH2-Flour + 0.44 GSH-Atm	0.44 mg/mL GSH added to the flour extract	Atmospheric conditions
AH2-Flour + 0.87 GSH-Atm	0.87 mg/mL GSH added to the flour extract	Atmospheric conditions

¹ Wheat flour extract was obtained by suspending 10.0 g wheat flour in 20.0 mL tap water, shaking (10 min, 150 rpm), and centrifuging (10 min; 5000 g). Wheat flour extracts were heat-treated to inactivate endogenous wheat flour AH₂ oxidase and GSH dehydrogenase. Following this, the extracts were kept at 70 °C for 30 min and then allowed to cool to room temperature.

and Scheme A1), ¹³C NMR spectroscopy allows us to simultaneously determine DHA and AH₂ concentrations, as well as the concentrations of the DHA degradation products. The approach revealed distinct kinetics for the enzymatic and non-enzymatic pathways, shedding light on the possible regeneration of AH₂ from DHA* during wheat breadmaking.

2. Results and Discussion

2.1. Identification of Ascorbic Acid and Its Reaction Products in Water at Ambient Conditions

In ultra-pure water (MilliQ), the chemical shift of the C₃-atom of freshly dissolved AH₂ was 162.9 ppm (Appendix A Figure A1a). This is higher than the values reported in the literature, ranging from 155 to 157 ppm [18,19,20]. Dissolved in tap water, the chemical shift of the C₃ carbon of freshly dissolved AH₂ was 178.3 ppm (Appendix A Figure A1b). Chemical shifts for the C₃-atom of AH₂ solutions derived from Na, Zn or Cd ascorbate salts were previously reported as 176.34, 174.37 and 174.15 ppm, respectively [19]. The difference observed between the ultra-pure- and tap water-based solutions was attributed to the presence of metal ions (Appendix A Table A1) in tap water. Since the other carbon atoms of AH₂ were not labeled with ¹³C, their resonances remained below the detection limit.

The transformation of ascorbic acid (AH₂) either dissolved in tap water or in ultra-pure water is depicted as function of time in Figure 2. In the absence of metal ions (lab water type 1; Figure 2a), DHA* (108.3 ppm) was first detected after four hours. After 24 h, an additional resonance at 97.2 ppm (attributed to DKGA.2H₂O) was observed. In the tap water-based sample (Figure 2b), oxidation of AH₂ to DHA* and its subsequent transformations occurred much faster. While in the tap water sample, after 2 h, 15.2% of the AH₂ was already converted into DHA*, in ultra-pure water the conversion after 2 h only amounted to 0.6%. DKGA is formed by irreversible ring-opening of DHA* (Figure 1). Irrespective of whether DHA* and DKGA were present in ultra-pure or in tap water, the chemical shifts of the C₃-atoms of both components were identical, implying that only the chemical shift of the C₃ atom of AH₂ is affected by the presence of metal ions (Appendix A Figure A1). The ¹³C chemical shift observed for the C₃-atom in DHA* was comparable to what is reported in the literature: 108.8 ppm in H₂O [21] or 106.3 ppm in D₂O [22]. For DKGA.2H₂O, reported values of 94.4 ppm (pH = 7.0) and 97.4 ppm (pH = 7.4, phosphate buffer) have been reported [23,24].

After 24 h, 55.0% of the initial AH₂ concentration was present as DKGA in the tap water sample, as compared to 4.2% in the AH₂-pure water sample. In the AH₂-tap water sample, aside from DKGA, degradation products with ¹³C chemical shifts 83.9, 176.8 and 180.8 ppm were also observed from 4 to 6 h onwards. These species could be related to multiple degradation pathways. The resonance at 83.9 and 176.8 ppm may arise due to the formation of 2-carboxy-L-lyxonolactone and 2-carboxy-L-xylonolactone via benzylic acid rearrangement of DKG-lactone (Scheme A2) [25]. Another pathway is the decarboxylation of DKG to 3,4,5-trihydroxy-2-keto-L-valeraldehyde (TKVA) (Scheme A3) [24]. Transition metal ions such as Cu²⁺ and Fe³⁺ have been reported to accelerate AH₂ oxidation, thus decreasing the stability of AH₂ [19,26,27,28,29,30]. Both Cu²⁺ and Fe³⁺ are more abundant in tap water than in pure water (Appendix A 

Table A2. Concentrations (µg/L) of the different transition metal ions in pure and tap water determined by inductively coupled plasma mass spectrometry.

Transition Metal Ion	Concentration (µg/L)
	Pure Water	Tap Water
Zn	5.71	89.76
Fe	2.91	50.88
Cu	0.30	23.54
Cd	0.30	1.17
Ni	0.14	1.20
Mn	0.05	0.78
Mo	0.03	0.96
Co	0.01	−0.01

). This could explain the different oxidation kinetics observed between both solutions. Simultaneously, AH₂ transformation kinetics also could be affected by the presence of trace compounds resulting from tap water chlorination [31]. Similar effects have previously also been reported for the oxidation of GSH in tap water versus ultra-pure water. These observations support the validity of the employed NMR methodology using ¹³C-labeled AH₂ at the C₃ position for monitoring oxidative transformations. The results also highlight the importance of controlling metal ion content in water to balance AH₂ oxidation and degradation pathways.

2.2. Impact of Molecular Oxygen on the Chemical Conversion of AH₂

From Reaction I, it can be derived that AH₂ oxidation should be influenced by the availability of dissolved O₂ [32]. This was confirmed by flushing ¹³C₃-AH₂ solutions with N₂ gas during preparation, and sealing the samples immediately after. I Both in ultra-pure and in tap water flushed with N₂, AH₂ remained much more stable (Figure A2). After 24 h, negligible conversion of AH₂ was observed in either solution, while only 75.5% and 6.4% of AH₂, respectively, remained in comparable samples equilibrated with ambient air. These observations confirm that molecular oxygen is a key driver of AH₂ oxidation, regardless of the aqueous matrix. The pronounced stabilization of AH₂ under O₂-lean conditions indicates that oxygen control during dough preparation and conditioning could offer technological opportunities in experimental and industrial settings.

2.3. Enzymatic Oxidation of Ascorbic Acid

AH₂ is often used in wheat breadmaking. owing to its dough strengthening and bread volume-enhancing effects [5]. While its bread property-enhancing effects ware well known, the final chemical fate of AH₂ in breadmaking applications remains nearly undocumented. To compare the enzymatic oxidation of AH₂ with the chemical oxidation, ¹³C₃-AH₂ was dissolved in unheated and in heat-treated aqueous wheat flour extract prepared using tap water. Heat treatment was performed to deactivate the endogenous enzymes. Following its dissolution, the transformation of AH₂ was monitored over time (Figure 3). The accompanying ¹³C NMR spectra are shown in the Appendix A as Figure A3.

Comparing samples based on untreated (Figure 3a) and heat-treated (Figure 3b) extracts, the heat-induced deactivation of AH₂ oxidase clearly lowered the rate of AH₂ oxidation to DHA*. While only 61.1% of AH₂ remained in the AH₂-flour extract sample after two hours (Figure 3a), 85.9% AH₂ was still present in the AH₂-heated flour extract sample after the same time (Figure 3b). In addition, it was striking that some unidentified components with ¹³C chemical shifts 68.7 and 116 ppm were formed after some time. The latter was formed only in the AH₂-flour extract sample (Figure 3a), and not in the AH₂-heated flour extract sample (Figure 3b), nor in the AH₂-tap water sample (Figure 2b). Figure 3a reveals that once DHA* is formed, it is quickly converted to DKGA. This may indicate that the regeneration of AH₂ from DHA* by GSH dehydrogenase is far less efficient than that previously assumed by Grosch and Wieser [8]. Peculiarly, after 4 h, the AH₂ concentration in samples based on heat-treated wheat flour extract (80%) remained significantly higher than that in the plain tap water samples (52.7%). This implies that components in the wheat flour extract, for example endogenous GSH, either prevent AH₂ oxidation or induce chemical regeneration of DHA* into AH₂.

2.4. The Effect of Glutathione on the Conversion of Ascorbic Acid

In presence of AH₂ oxidase, GSH, and GSH dehydrogenase, three components endogenously present in wheat flour, AH₂ could be produced by regeneration of DHA (Reaction IV) [8]. In addition, DHA might also be non-enzymatically reduced to AH₂ by GSH [33,34]. But GSH is also known to rapidly decrease the available O₂ concentration in water, as it swiftly oxidizes into GSSG, especially in tap water [35]. The detected AH₂ in the AH₂-flour extract sample may thus not only contain initially added, unreacted AH₂, but also regenerated AH₂. In addition, overall, the presence of GSH can, therefore, stabilize AH₂ [36].

To specifically evaluate the impact of these reactions, both AH₂ and GSH were added to unheated wheat flour extract at concentrations of 0.04, 0.44 or 0.87 mg GSH/mL extract. The two highest GSH concentrations resulted in (1) equimolar levels of GSH and AH₂ added: GSH = 0.44 mg GSH/mL (1.4 µmol/mL extract); AH₂ = 0.25 mg/mL (1.4 µmol/mL extract), and (2) a molar ratio of GSH:AH₂ = 2: GSH: 0.87 mg/mL (2.8 µmol/mL extract); AH₂: 0.25 mg/mL (1.4 µmol AH₂/_mL extract). The latter was chosen based on Reaction IV, which shows that one DHA molecule (formed from AH₂) requires two GSH molecules for its regeneration. In neither of these two samples was AH₂ oxidation observed, even after 16 h.

Figure 4 shows the evolution of the dosed AH₂ concentration, as a function of time, for a sample with added AH₂:GSH molar ratio of 10 (0.04 mg GSH/mL extract). The corresponding ¹³C NMR spectra are shown in Appendix A Figure A4. In this last sample, DHA* and DKGA were detected, indicating the oxidation of AH₂ (Figure 4). Comparing the time evolution of the AH₂ levels in AH₂-flour extract samples, with (Figure 4) and without (Figure 3a) added GSH, it was observed that the addition of GSH in a 1/10 GSH/AH₂ ratio nearly doubled the remaining AH₂ concentration after 4 h (84% vs. 46%, respectively). In addition, after four hours, only 4.6% DKGA was detected in this sample, compared to 37% in the AH₂-flour extract sample. When high GSH levels were added, 100% AH₂ was still present after 16 h (Appendix A Figure A4) compared to only 17.2% in the AH₂-flour extract sample (Figure 3a). Overall, these results indicate that, rather than serving as electron donor for the regeneration of DHA* to AH₂, GSH primarily acts on decreasing the availability of dissolved O₂. This mechanistic insight may inform optimized antioxidant strategies in food formulations. It also highlights the need to consider GSH levels in commercial flour as a variable influencing dough behavior.

3. Materials and Methods

3.1. Materials and Chemicals

Additive-free wheat flour (Crousti) was provided by Puratos NV (Groot-Bijgaarden, Belgium) [7,37]. Its moisture (14.6%) and protein (13.1% of dry matter) contents were determined in triplicate, according to the AACC method 44-15-02 (1999) and an adaptation of the AOAC method 990.03 (1995) to an automated Dumas protein analysis system (VarioMax Cube N, Elementar, Hanau, Germany), respectively. According to the supplier, the ash content was 0.57% ± 0.03. All chemicals, reagents, and solvents were purchased from Merck Life Science (Overijse, Belgium) and were of analytical grade. Pure water with a resistivity of 18.2 MΩ.cm at 25 °C was obtained using a Millipore Milli-Q lab water system (Merck MilliPore, Burlington, MA, USA).

3.2. Preparation of the Different Aqueous Solutions

AH₂’s oxidation to DHA* and its possible regeneration were studied in media, the codes of which are listed in Table A1. Cations in pure water and tap water were quantified by inductively coupled plasma mass spectrometry, using an Agilent 7700× (Agilent Technologies, Santa Clara, CA, USA), the results of which are given in Appendix A Table A2.

Wheat flour extract was obtained by suspending 10.0 g wheat flour in 20.0 mL tap water, shaking (10 min, 150 rpm), and centrifuging (10 min; 5000× g). Wheat flour extracts were heat-reated to inactivate endogenous wheat flour AH₂ oxidase and GSH dehydrogenase. Following this, the extracts were kept at 70 °C for 30 min and then allowed to cool to room temperature. Pfeilsticker and Roeung observed minimal AH₂ oxidase activity after heating wheat flour extract for 60 min at 60 °C, and Every et al. reported full inactivation of GSH dehydrogenase when flour extract was heated for 20 min at 70 °C [12,38].

The different media (Table A1) were adjusted to pH 7.2 using 0.1 M sodium phosphate buffer (pH 7.4) prepared with pure water. One volume of D₂O was added to nine volumes of pure water, tap water, or (heat-treated) wheat flour extract.

Where appropriate (Table A1), O₂ was eliminated from the NMR tube and different media before and after AH₂ addition by flushing with dry nitrogen (N₂) gas. GSH was added to certain wheat flour extract samples (Table A1) after pH adjustment and D₂O addition, and immediately before ¹³C₃-AH₂ was added. To obtain 0.04 mg GSH/mL flour extract, 0.40 mg GSH was first dissolved in 10.0 mL 10% D₂O containing aqueous flour extract, while 0.44 or 0.87 mg GSH wasdissolved in 1.00 mL 10% D₂O containing aqueous flour extract.

¹³C₃-AH₂ aliquots (0.25 mg) were accurately weighed in Eppendorf tubes. Immediately before the start of the NMR measurements, 1.0 mL of the different 10% D₂O-containing samples was added to the Eppendorf tube, of which 0.5 mL was transferred to a 5 mm NMR tube, which was then closed with a polyethylene cap.

3.3. ¹³C Liquid-State Nuclear Magnetic Resonance Measurements

¹H decoupled ¹³C NMR spectra were acquired on an 800 MHz Bruker Avance Neo spectrometer (Bruker Belgium NV, Kontich, Belgium) equipped with a 5 mm multinuclear BBO probe (1H/2D/X). ¹³C direct excitation NMR spectra were acquired using an excitation pulse with 30-degree flip angle at a 25 kHz radio frequency, recycle delay of 4 s, and 720 transients. The spectra were referenced to tetramethylsilane, using the secondary reference sodium trimethylsilyl-propanesulfonate in D₂O at 0.0173 ppm [39]. The experiments were performed by following procedures for absolute quantitative NMR [40,41].

Phase correction, baseline correction, and integration of the ¹³C NMR spectra were performed using the Bruker TopSpin 4.0.9 software. For each time point, the integrals of the C₃-carbon signals corresponding to AH₂, DHA*, DKGA, and other identifiable degradation products were quantified. The relative abundance of each component was calculated by normalizing its integrated signal area to the total integrated area of all detected ¹³C signals. This allowed time-resolved tracking of AH₂ transformation pathways under the investigated experimental conditions.

4. Conclusions

Ascorbic acid (AH₂) is often used in wheat breadmaking. owing to its dough strengthening and bread volume-enhancing effects. While these bread property-enhancing effects are well known, the final chemical fate of AH₂ in breadmaking applications remains nearly undocumented. This study investigated the chemical fate of AH₂ in relevant wheat flour extract, evaluating the specific impact of dynamic processes which could be involved in the oxidation of AH₂. Using various aqueous media, including oxygenated, as well as deoxygenated, tap water and ultra-pure water, the impact of dissolved oxygen, trace metal ions and trace reaction products of chlorine-based water disinfection were evaluated. These observations underscore the validity of the NMR approach employing ¹³C-labeled AH₂ and the critical importance of monitoring metal ion content in aqueous systems when aiming to balance oxidation and degradation of AH₂. To discriminate between enzymatic and non-enzymatic processes, the fate of AH₂ in heat-treated and in pristine wheat flour extract was evaluated as a function of time. Finally, both glutathione (GSH) and AH₂ were added to pristine wheat flour extracts in molar ratios of GSH:AH₂ = 2, 1 and 0.1, to elucidate the impact of endogenous GSH on the oxidation and/or regeneration of AH₂. Remarkably, reducing the dissolved O₂ concentration in both media by purging with N₂ gas significantly enhanced the AH₂ stability, as evidenced by the limited formation of DHA* and hydrolysis to DKGA. While the oxidation of AH₂ in water was strongly enhanced by the presence of trace metal ions and/or trace reaction products of water disinfection, surprisingly, the impact of these components in solutions based on wheat flour extract was reduced. By introducing different ratios of both GSH and AH₂ into wheat flour extract, it was shown that the dominant impact of endogenous GSH on AH₂ availability must be to decrease the availability of dissolved oxygen, rather than to assist in the regeneration of DHA* in AH₂ by GSH dehydrogenase. This points to the potential for regulating dough redox balance by managing GSH levels and oxygen exposure during mixing and fermentation.. These insights into the stability and transformation of AH₂ could spark new strategies for optimizing dough conditioning by managing the balance between oxidation and regeneration of ascorbic acid, not only through redox additives, but also by modulating flour composition or by controlling the process atmosphere. Future research is needed to translate these findings to technologically relevant modifications to dough and breadmaking processes. Coupling NMR-based techniques with functional dough assays may help bridge molecular-level transformations with macroscopic bread quality outcomes. In situ monitoring of AH₂ transformations during dough processing, enabled by the advent of high-pressure MAS rotors [42] capable of containing CO₂ release, in combination with resolution enhancement techniques [43], represents an exciting scientific avenue.