One-Pot Enzymatic Bioconversion of Native Whey for the Simultaneous Production of Galacto-Oligosaccharides and Antioxidant Peptides

Abstract

The integrated valorization of whey into multifunctional food ingredients is constrained by sequential processing routes and the need for purified lactose and protein fractions. The simultaneous enzymatic conversion of lactose and whey proteins in a single reactor remains underexplored despite the frequent co-formulation of galacto-oligosaccharides (GOS) and whey protein hydrolysates in functional foods. This study evaluated the feasibility of a one-pot enzymatic system using native whey as the sole substrate for the concurrent production of GOS and antioxidant peptide fractions. A batch process combining β-galactosidase from Aspergillus oryzae and Alcalase^® was assessed through a 3² factorial design, analyzing the effects of pH (4.5–6.5) and temperature (40–60 °C) on GOS yield and degree of protein hydrolysis. The system enabled simultaneous transgalactosylation and proteolysis under mildly acidic conditions without significant mutual enzyme inhibition. Multi-response optimization identified pH 6.0 and 59.5 °C as the optimal conditions, yielding 25.7 ± 0.2% GOSs and 10.5 ± 0.3% protein hydrolysis. The antioxidant capacity and emulsifying and foaming properties were strongly dependent on pH, temperature, and reaction time. The results demonstrate that native whey can be directly transformed into a multifunctional ingredient through a one-pot enzymatic strategy, offering a simplified valorization approach.

1. Introduction

Whey has been the most extensively researched food by-product over the past decade within the domains of food science, biotechnology, and allied fields. It is the primary by-product of cheese, yogurt, and casein production, and contains approximately 50% of the milk solids and 20% of the total milk proteins. This composition makes whey a rich source of lactose, whey protein, vitamins, and dairy minerals [1]. Due to its nutrient-rich profile, whey has become a focal point for the application of innovative bioprocesses aimed at the valorization of by-products into high-value compounds for the food, chemical, and pharmaceutical industries [1,2].

Depending on the coagulation process, whey can be classified as sweet or acid whey, which differ mainly in mineral content, acidity, and protein composition [3,4]. At the industrial level, whey is commonly further processed into derivatives such as whey protein concentrate (WPC), whey protein isolate (WPI), and whey protein hydrolysates (WPHs), which are obtained through combinations of membrane separation and enzymatic hydrolysis [5]. As a result, peptides with enhanced digestibility and reduced allergenicity, as well as bioactivities such as antioxidant and angiotensin-converting enzyme (ACE) inhibition activities, are typically generated from whey protein hydrolysates (WPHs) obtained through enzymatic hydrolysis of WPC or WPI fractions [6,7]. In parallel, lactose recovered from whey ultrafiltration processes is frequently used as a substrate for the synthesis of galacto-oligosaccharides (GOS), a well-established class of prebiotics produced by β-galactosidase-catalyzed transgalactosylation reactions [8,9,10]. GOS selectively stimulate beneficial gut microbiota and contribute to improved mineral absorption and immune function [9,11].

From an industrial perspective, the production of food-grade lactose necessary for GOS synthesis and the process of generating WPHs involve multiple sequential stages, including separation, desalination, and drying. These steps are essential for recovering, purifying, and concentrating these fractions, which are typically produced as by-products from other dairy processes. In this regard, previous studies on whey valorization have predominantly relied on sequential processing strategies, where lactose is first purified and converted into galacto-oligosaccharides (GOS) through β-galactosidase-catalyzed transgalactosylation, followed by independent enzymatic hydrolysis of whey protein concentrates or isolates to obtain bioactive peptides [8,12,13]. This often results in prolonged processing times and significant consumption of water and energy. These ingredients are frequently combined in food formulations such as infant formula, clinical nutrition and sports products; thus, milk fractionation technologies for process intensification are being developed. For instance, microfiltration of skim milk allows for the recovery of native whey, a permeate that is free of added salts, starter cultures, rennet, and fermentation by-products, while the concentrate retains the casein micelles that are needed for cheese manufacturing [14,15]. Native whey preserves lactose and whey proteins in their native state, making it an attractive substrate for direct enzymatic valorization without extensive upstream purification [16].

On the other hand, one-pot multi-enzyme systems in which multiple enzymatic reactions occur in a single reactor represent an appealing strategy for enhancing whey valorization by reducing the number of processing steps and resource consumption [17,18]. However, their implementation is hindered by the need to reconcile the operational requirements of different enzymes. For instance, β-galactosidases typically favor mildly acidic conditions to maximize transgalactosylation activity [12,19], whereas proteases such as Alcalase exhibit optimal activity at neutral to alkaline pH and elevated temperatures [20]. Therefore, it is crucial to characterize the kinetics of these enzymes to determine their activity and stability within appropriate operational windows [17]. Moreover, the concurrent use of a protease may compromise the integrity of β-galactosidase due to its potential hydrolysis by the protease, posing a risk to GOS production.

To date, multi-enzyme systems have been explored for lactose-derived oligosaccharide synthesis or for protein hydrolysis independently, but reports addressing the concurrent application of β-galactosidase and proteases in whey-based matrices remain scarce. Most existing studies have focused either on sequential processing strategies or on combinations of glycosidases targeting carbohydrate modification [18,21], while the concurrent use of β-galactosidase and proteases in a single reactor remains largely unexplored, particularly when native whey is the sole substrate. A comparative overview of representative whey-based GOS and whey protein hydrolysate production strategies reported in the literature is presented in Table S1 [3,12,13,18,20,21,22,23] in the Supplementary Materials. This review of the literature shows that several fundamental questions remain unresolved: (i) Can β-galactosidase from Aspergillus oryzae and Alcalase operate concurrently under compatible pH and temperature conditions without significant mutual inhibition? (ii) How do pH and temperature affect GOS yield and protein hydrolysis when both enzymes are applied simultaneously? (iii) Does the one-pot configuration compromise reaction performance relative to individual enzymatic processes? (iv) How does the extent of protein hydrolysis and the resulting peptide size distribution influence antioxidant activity? (v) How do these structural changes, together with lactose conversion, modulate technological properties such as emulsifying and foaming?

The objective of this study was to evaluate the feasibility of a one-pot enzymatic system for the simultaneous production of GOSs and antioxidant peptide fractions using concentrated native whey as the sole substrate. This strategy differs from previous research by evaluating enzyme compatibility, process performance, and functional properties within a unified operational framework, thus generating multifunctional food ingredients in a single processing step.

2. Materials and Methods

2.1. Materials

Monohydrated lactose and biPro Whey Protein Isolate were purchased from Saputo Ingredients (Montreal, QC, Canada) and Davisco Foods International (Savage, MN, USA), respectively. Monobasic monohydrate phosphate (NaH₂PO₄·H₂O), sodium dibasic heptahydrate phosphate (Na₂HPO₄·7H₂O), potassium dibasic phosphate (K₂HPO₄), citric acid, and o-nitrophenyl-β-D-galactopyranoside (o-NPG) were purchased from Merck (Darmstadt, Germany). For the determination of the concentration of released α-amino groups, the reagents sodium borate decahydrate (borax), sodium dodecyl sulfate (SDS), o-phthaldialdehyde (OPA), absolute ethanol, and dithiothreitol (DTT) were used for the preparation of the OPA reagent, and serine was employed to create the calibration curve.

The proteolytic enzyme used was subtilisin, derived from the commercial preparation Alcalase^® 2.5 L, an endoproteinase from Bacillus licheniformis supplied by Novozymes (Bagsvaerd, Denmark), with a declared activity of 2.5 [AU/g] and a density of 1.08 [g/mL]. The enzyme exhibits an operating pH range of 7 to 10 and an operating temperature range of 30 to 65 °C. For the synthesis of GOS, a commercial β-galactosidase from Aspergillus oryzae (Enzeco^® Fungal Lactase) was used, which was kindly provided by the Enzyme Development Corporation, EDC (New York, NY, USA). This is a monomeric enzyme with an average specific activity of 138,552 ± 17,526 [IU/g] under optimal conditions, and it has been shown to operate at pHs of 4.5–5.5 and temperatures of 40 to 60 °C [13,24].

2.2. Assessment of the Operational Window

To define the operational window, the effect of pH and temperature on the activity of each enzyme was determined under non-reactive conditions (50 mM McIlvaine buffer solution). As outlined in Section 2.1, a combination of pH values (4.5, 5.5, and 6.5) and temperatures (40, 50, and 60 °C) was used in a 3² experimental design. For each condition, the enzymes were conditioned for a period of one minute before proceeding with the determination of enzymatic activity. For β-galactosidase, the reaction was performed by mixing 180 μL of a 45 mM o-NPG solution with 20 μL of the enzymatic solution at 40 °C for 1 min with automatic stirring in a 96-well microplate. The activity was expressed as international units (IU) per g of catalyst, i.e., the amount of enzyme that hydrolyzes 1 μmol of o-NPG per min.

Subtilisin activity was determined using an azocasein assay, which was adapted from [25]. Briefly, 1 mL of azocasein solution (in 50 mM sodium phosphate buffer, pH 7.5) was incubated at the assay temperature, and 0.1 mL of diluted Alcalase was added to initiate the reaction. After 10 min, 1 mL of 10% (w/w) trichloroacetic acid (TCA) was added to stop the reaction. The mixture was vortexed and left to stand for 5 min. The mixture was then centrifuged at 8000× g for 6 min, and 1.0 mL of the supernatant was mixed with 1.0 mL of a 1 N NaOH solution. The absorbance at 440 nm was measured against a blank prepared by adding protease after the reaction was stopped with TCA. The assays were performed in triplicate. One unit of proteolytic activity was defined as the amount of protease that produced an increase of 1.0 absorbance unit per minute of reaction under the assayed conditions. Specific activity (U/g) was calculated from the plot of protease activity (U/mL) vs. protease concentration (g/mL).

2.3. Simultaneous Production of GOS and Bioactive Peptides in One-Pot Reactor

2.3.1. Substrate Preparation

Native whey was obtained by skim milk microfiltration as previously reported in [13]. This whey was not subjected to enzymatic coagulation and therefore does not correspond strictly to conventional sweet or acid whey, but rather to a serum phase free of starter cultures, rennet, and fermentation by-products. The native whey had a pH of 6.3 ± 0.3, total solid content of 94 ± 6 g/L, total protein content of 5.92 ± 0.98 g/L, lactose content of 81 ± 4 g/L, and ash content of 0.43 ± 0.2%. The native whey solutions were concentrated to 40° Brix and a 300 mM McIlvaine buffer solution was used to adjust the pH to 4.5, 5.5, and 6.5. The mixtures were stirred until a homogeneous mixture was obtained.

2.3.2. Effect of pH and Temperature Under Reactive Conditions

Once sufficient enzyme activities within the operational window were confirmed, we assessed the effect of pH and temperature under the reactive conditions for the simultaneous synthesis of GOSs and protein hydrolysates in a one-pot reactor. A 3² experimental design with one central point in two blocks was used, resulting in a total of 20 experimental runs. Control experiments were conducted under identical pH and temperature conditions using either β-galactosidase alone (control GOS) or Alcalase alone (control DH) to enable direct comparisons with the one-pot system.

For all reactions, a batch reactor with mechanical stirring and pH and temperature sensors (Mettler-Toledo G20S compact titrator) was used, and a thermostatically controlled bath was utilized to maintain a constant temperature. If necessary, prior to initiating the reaction, the pH was adjusted using a 1 [M] HCl solution; during protein hydrolysis, a 0.5 [M] NaOH solution was used in the titrator as needed due to the release of protons during protein hydrolysis.

For protein hydrolysis, an enzyme-to-substrate ratio (E/S) of 0.07 [U/g of protein] was used, whereas for the synthesis of GOSs, an enzyme-to-substrate ratio of 100 [U/g of lactose] was employed since under these reaction conditions, the product peak is reached within 120 min [13]. In the case of the simultaneous reaction with both enzymes, the protease was added first to initiate controlled protein hydrolysis and to allow for stabilization of the pH-stat system before the addition of β-galactosidase. The short delay (~90 s) was for operational reasons rather than mechanistic ones and did not result in measurable differences in lactose conversion or degree of hydrolysis, as confirmed by the control experiments. Once the reaction was initiated, 400 [µL] samples were taken at 0, 2, 4, 10, 20, 40, 60, 90, and 120 [min] and added to an Eppendorf tube containing 400 [µL] of 10% [v/v] trichloroacetic acid (TCA) to inactivate the enzymes and stop the reaction. The samples were then centrifuged at 10,000 [rpm] for 10 [min], and the supernatant was transferred to a new Eppendorf tube.

2.3.3. Quantification of Protein Hydrolysis Products

To measure protein hydrolysis, the concentration of released α-amino groups was quantified throughout the reaction using the o-phthaldialdehyde (OPA) method. This method is based on the reaction of amino groups with o-phthaldialdehyde (OPA) in the presence of dithiothreitol (DTT), which forms a compound detectable by spectrophotometry at 340 [nm] [26,27]. The concentration of released α-amino groups, along with the number of hydrolyzed peptide bonds (h), volume of the mixture (0.05 [L]), and percentage of protein in the native whey, were used to calculate the degree of hydrolysis (DH) (Equation (1)).

$$h_{tot}={\displaystyle \frac{1}{6.38}}\left[{\displaystyle \frac{g_N}{g_{proteín}}}\right]·{\displaystyle \frac{1}{14}}\left[{\displaystyle \frac{{mol}_N}{g_N}}\right]·1000\left[{\displaystyle \frac{mmol}{mol}}\right]=\mathrm{11,196}\left[{\displaystyle \frac{mmol}{g_{proteín}}}\right]$$

(1)

It should be noted that peptide fractions were not isolated or purified in this study, as the objective was to generate a multifunctional matrix rather than discrete ingredients. Therefore, DH can also be interpreted as the yield of the protein hydrolysis reaction.

2.3.4. Quantification of Reaction Products from GOS Synthesis

In this study, all carbohydrates produced from reactions utilizing native whey were quantified using a High-Performance Liquid Chromatography (HPLC) system equipped with a refractive index detector, autosampler, and isocratic pump. The chromatographic separation was conducted on a Benson Polymeric BP-100 Ca column, which was maintained at 80 °C, while the detector temperature was set to 40 °C. Milli-Q water, at a constant flow rate of 0.4 mL/min, was used as the mobile phase for elution. For each analysis, 20 μL of the sample was injected.

The chromatograms were processed and integrated using Jasco’s ChromNAV software. Quantitative results were expressed as the total sugar concentration (% w/w), which was based on the assumption that the peak area is directly proportional to the mass percentage of each sugar type [28]. Calibration standards (galactose, glucose, and galacto-oligosaccharides (GOSs), including tri- and tetrasaccharides) were employed to determine their retention times and to confirm the linear range of the detector for accurate quantification.

The progress of this reaction was expressed in terms of the reaction conversion rate (X) and reaction yield (Y), defined as follows:

$$X={\displaystyle \frac{L_0-L}{L_0}},$$

(2)

$$Y={\displaystyle \frac{GOS}{L_0}},$$

(3)

where L₀ is the initial mass of lactose (g), L is the lactose mass at different time points, and GOS is the total amount of GOS produced (g).

2.4. Determination of Bioactive and Technological Properties

2.4.1. Antioxidant Capacity

Under the identified suitable reaction conditions, the antioxidant capacity of the reaction products was monitored using the ABTS and DPPH methods. The ABTS method was performed according to the protocol in [29] with slight modifications. The ABTS⁺ radical cation was generated by mixing equal volumes (1:1 ratio) of two aqueous solutions: (1) 7 mM ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and (2) 2.45 mM potassium persulfate (K₂S₂O₈). The mixture was stored in the dark at room temperature for 16 h before use. The working ABTS⁺ solution was prepared by diluting the stock solution with deionized water to obtain an absorbance of 0.70 ± 0.02 at 734 nm. For the assay, 20 µL of sample was mixed with 980 µL of the ABTS⁺ working solution, vortexed, and incubated at room temperature for 7 min. The mixture was then centrifuged at 8000 rpm for 5 min, and the absorbance was measured at 734 nm using a spectrophotometer.

The DPPH assay was conducted following the method in [30] with minor modifications. A 20 µL aliquot of the sample was mixed with 800 µL of a 0.036 mM methanolic DPPH (2,2-diphenyl-1-picrylhydrazyl) solution, vortexed, and allowed to react in the dark at room temperature for 16 min. The mixture was then centrifuged at 8000 rpm for 5 min, and absorbance was measured at 515 nm using a spectrophotometer. Trolox was used as the calibration standard for both assays, and antioxidant capacity was expressed as milligrams of Trolox equivalents per milliliter of reaction volume (mg TE/mL).

2.4.2. Polyacrylamide Gel Electrophoresis and Silver Staining of Low-Molecular-Weight Peptides

The electrophoretic profiles of the low-molecular-weight peptide products were analyzed using denaturing polyacrylamide gel electrophoresis (tricine–SDS-PAGE), a technique widely employed to study peptide fragments ranging from 2 to 20 kDa in size [31]. This system shows enhanced sharpness of low-molecular-weight bands compared with conventional Tris–glycine systems [32] and is widely recommended for separating oligopeptides and bioactive peptides due to its low lateral diffusion coefficient and high resolving power [33]. Discontinuous 16% Tris-tricine gels (29:1 acrylamide:bisacrylamide) with a 5% stacking gel were prepared. Polymerization was initiated with freshly prepared 0.05% (v/v) TEMED and 0.05% (w/v) ammonium persulfate (APS). Peptide samples (30 μg total protein) were mixed with 2X loading buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.02% bromophenol blue, and 10% β-mercaptoethanol) and heated at 95 °C for 5 min to ensure complete denaturation. Samples were then loaded into the wells along with a specific 2–40 kDa molecular weight marker (Spectra™ Multicolor Low Range Protein Ladder, ThermoFisher^®, Waltham, MA, USA). Electrophoresis was carried out using a commercial Tris–tricine buffer system (Invitrogen, Novex Tricine SDS Running Buffer 1X, Waltham, MA, USA) at 70 V during stacking and 90 V during separation until the dye front reached the bottom of the gel. Following electrophoresis, the gels were fixed for 30 min in a solution containing 50% methanol and 10% glacial acetic acid, followed by three washes with ultrapure water (15 min each). For silver staining, an adapted protocol was used to ensure high sensitivity and minimal background. Recent improvements in silver staining methods have significantly increased detectability and uniformity, resulting in enhanced visualization of low-molecular-weight peptides [34].

The gels were sensitized with 0.02% (w/v) sodium thiosulfate for 1 min, rinsed with ultrapure water (2 × 30 s), and incubated in an ice-cold solution of 0.1% (w/v) silver nitrate with 0.075% (v/v) formaldehyde for 20 min under gentle agitation and protected from light. After removing any excess reagent (10 s wash in ultrapure water), the gels were developed using 2% sodium carbonate (w/v) containing 0.05% (v/v) formaldehyde until bands were visible (approximately 5–15 min). The reaction was stopped using 5% glacial acetic acid for 5 min, and the gels were stored in ultrapure water at 4 °C. Band densitometric analysis was performed using a high-resolution scanner and dedicated software for relative quantification, allowing for comparisons of peptide distribution and abundance between samples. These methods are compatible with the subsequent mass spectrometry analyses.

2.4.3. Determination of Emulsifying Activity Index (EAI) and Emulsion Stability Index (ESI)

The emulsifying activity index (EAI) and emulsion stability index (ESI) were determined according to the method described in [35] with minor modifications. Emulsions were prepared by mixing 3 mL of vegetable oil with 12 mL of diluted sample (1 g/L), followed by homogenization using a dispersing unit (OV 625 Digital Disperser, VELP Scientifica) at 10,000 rpm for 1 min. Immediately after homogenization, a 100 µL aliquot was withdrawn from the bottom of the freshly prepared emulsion and diluted in 10 mL of a sodium dodecyl sulfate (SDS) solution (0.1% w/v) (Merck). The mixture was vortexed for 20 s, and absorbance was measured at 500 nm using a UV–Vis spectrophotometer (6715, JENWAY, Staffordshire, UK). To determine the ESI, the same procedure was repeated after 10 min using an identical volume of the emulsion.

The emulsifying activity index (EAI) was calculated using the following equation:

$$AI({\mathrm{m}}^2/\mathrm{g})={\displaystyle \frac{2\times 2303\times A_0\times D}{C\times \phi \times {10}^3}},$$

(4)

where A₀ is the initial absorbance (0 min), D is the dilution factor, c is the protein concentration (~0.029 g/mL), and φ is the volumetric fraction of oil in the emulsion (0.2 in this case).

The ESI was calculated using the following equation:

$$SI(\%)={\displaystyle \frac{A_{10}}{A_0}}\times 100,$$

(5)

where A₁₀ is the absorbance after 10 min of emulsification and A₀ is the initial absorbance.

2.4.4. Determination of Foaming Capacity Index (FCI) and Foam Stability Index (FSI)

The foaming capacity and foam stability indices of the samples were determined according to the methodology described in [22] with minor modifications. A 20 mL volume of sample solution (2% w/v) was homogenized using the same homogenizer described above at 10,000 rpm for 2 min. Immediately after homogenization, the volume of the foam-containing solution was measured in a graduated cylinder (0 min). The sample was then allowed to stand undisturbed, and the volume was measured again after 30 min.

The foaming capacity index (FCI) and foam stability index (FSI) were calculated using Equations (6) and (7), respectively:

$$FCI\left(\%\right)={\displaystyle \frac{V_1}{V_0}}\times 100,$$

(6)

$$FSI\left(\%\right)={\displaystyle \frac{V_2}{V_1}}\times 100,$$

(7)

where V₀ is the initial volume before homogenization, V₁ is the volume immediately after homogenization, and V₂ is the volume measured after standing for 30 min.

2.5. Statistical Analysis

All results were expressed as the mean ± standard deviation (SD) of three replicates. The effect of the reaction conditions (pH and temperature) on Y and DH was assessed using the surface response methodology, while the effect on bioactive and technological properties was assessed using two-factor ANOVA tests (α = 0.05). All computations were performed with Excel from Microsoft 365^® and Statgraphics 18-X64 (Statpoint Inc., Warrenton, VA, USA, 2020).

3. Results and Discussion

3.1. Determination of Enzymes’ Operational Window

Figure 1A shows the specific activity profiles of β-galactosidase from Aspergillus oryzae under non-reactive conditions. Peak activity was observed at 50 °C, with comparable values at pH 5.5 and 6.5, indicating a plateau behavior. However, at 60 °C, a decline in activity was evident, probably due to enzyme denaturation. Although the optimal pH for A. oryzae β-galactosidase has been reported to be around 4.5 [36,37], the results here suggest a shift in the operational window under the conditions used in this study. This deviation might be attributed to differences in enzyme formulation, assay conditions, or buffer composition.

Regarding Alcalase, the maximum activity has been reported to occur at pH 7.5–9 and temperatures of 50–60 °C [20,23]. In this study, a minimal but appreciable level of proteolytic activity was still observed at pH 4.5 and 50 °C (Figure 1B). Although Alcalase exhibits reduced catalytic efficiency under acidic conditions, measurable hydrolysis still occurred. Even low degrees of hydrolysis can generate bioactive peptide fractions, as previously reported for whey proteins, with moderate DH levels. Furthermore, in applications where a low degree of hydrolysis is sufficient, such as those aimed at reducing antigenicity or improving sensory attributes, Alcalase has proven to be effective even at moderately acidic pHs, particularly when the reaction time is optimized or when used in combination with other enzymes [38]. These findings support the feasibility of applying Alcalase with other enzymes such as Aspergillus oryzae β-galactosidase under suboptimal conditions; however, studies under reactive conditions are needed to validate their simultaneous activity.

3.2. Effect of pH and Temperature on Protein Hydrolysis

The comparison of the hydrolysis of whey proteins using Alcalase vs. Alcalase and β-galactosidase from Aspergillus oryzae is shown in Figure 2. The performance of Alcalase was similar to its specific activity profile in its range of operating conditions. The higher the pH and temperature, the higher the protease performance. The extent of the hydrolysis reaction can be evaluated based on the degree of hydrolysis after 120 min. The DH reached between 1.2% and 3.6% at pH 4.5, and increased to 2.6–7.7% and 5.8–14.1% at pH 5.5 and 6.5, respectively. Alcalase exhibited the same reaction profile under the two operating conditions.

The simultaneous addition of Alcalase and β-galactosidase did not affect the performance of Alcalase: the DH values of the control and one-pot treatments after 120 min were not significantly different (p ≥ 0.05). The performance of Alcalase under acidic conditions is dependent on its intrinsic catalytic activity and the solubility of the substrate proteins. Whey proteins such as α-lactalbumin and β-lactoglobulin may exhibit partial insolubility around their isoelectric points (pI 4.2–5.3), particularly at pH 4.5 and 5.5, which could limit enzyme–substrate interactions. This solubility effect or the reduced enzymatic activity at low pH could explain the observed hydrolysis behavior, but it is difficult to isolate the contribution of each factor [39]. Despite its suboptimal activity below pH 6.5, Alcalase still demonstrated measurable hydrolytic capacity at pH 4.5, suggesting residual but functional enzymatic activity in acidic reactive environments, which aligns with previous findings [40]. This compatibility allows for process optimization in “one-pot” systems where synergistic or sequential hydrolysis could be achieved.

3.3. Effect of pH and Temperature on GOS Synthesis

To evaluate the influence of pH and temperature on GOS production using native whey as the substrate, reactions were carried out at three pH levels (4.5, 5.5, and 6.5) and three temperatures (40, 50, and 60 °C). As shown in Figure 3, GOS synthesis was affected by pH and temperature, with the highest yield obtained at pH 4.5 and 50 °C (27.6%), followed by a progressive decline at higher pHs and temperatures (25.2% at pH 5.5 and 60 °C), with a pronounced effect at pH 6.5 where the maximum Y_GOS ranged from 10 to 20.8%. These results highlight a preference of the β-galactosidase from Aspergillus oryzae for slightly acidic conditions when catalyzing transgalactosylation reactions. This is consistent with the behavior of this enzyme when it catalyzes transgalactoslylation at high lactose concentrations (i.e., its activity is stable at pHs of 3 to 6) [12].

Temperature also played a key role in modulating GOS production. An increase from 40 to 60 °C generally enhanced GOS yield, likely due to improved solubility of native whey components, which helps prevent lactose precipitation at high concentrations [41]. However, this beneficial effect was diminished at pH 6.5, particularly at 60 °C, as GOS synthesis slowed down. This can be attributed to partial unfolding of the β-galactosidase tertiary structure, leading to decreased catalytic activity. However, it is known that this enzyme from Aspergillus oryzae has good stability when concentrated native whey is used as the substrate, even when physical stress is applied [13]. Therefore, an alternative hypothesis for this behavior could be due to the co-activity of proteases under these conditions. At higher pHs and temperatures, the hydrolytic activity of Alcalase may preferentially target native whey proteins, but after prolonged exposure, partial degradation of β-galactosidase itself might occur, compromising its ability to catalyze lactose conversion (Figure 4). Supporting this, Figure S1 in the Supplementary Materials shows that in the one-pot co-reaction with Alcalase, after 2 h, normalized β-galactosidase activity decreased by approximately 10% in most cases, likely due to thermal denaturation, and dropped significantly (≈30%) at pH 6.5 and 60 °C. This suggests that Alcalase may lose substrate selectivity under such conditions, potentially affecting β-galactosidase integrity. In addition, it is well known that the Maillard reaction occurs rapidly between sugars like lactose and free lysine residues or N-terminal amino groups in proteins (pH > 6), especially at temperatures ≥ 60 °C, which can lead to advanced Maillard reaction products (MRPs). Figure S2 shows the release of monosaccharides (glucose and galactose), which is typical for this reaction. These mono- and disaccharides can covalently bind to reactive amino acids in enzymes, including aggregated enzyme–protein complexes [42]. This suggests that A. oryzae β-galactosidase could also be structurally inhibited through interactions with MRPs, which has been observed under similar conditions [43], but the underlying mechanism will need to be examined in future work.

Figure 3 and Figure 4 indicate that while pH and temperature modulate the transgalactosylation profile, the overall catalytic performance of β-galactosidase remains largely consistent in the presence of Alcalase when compared with control conditions without protease. This demonstrates the feasibility of simultaneous production of protein hydrolysates and GOS derived from native whey using a one-pot strategy.

3.4. One-Pot System Optimization and Antioxidant Functionality

Optimization of the one-pot biocatalytic system was conducted using GOS yield (Y_GOS) and degree of hydrolysis (DH) after 2 h as the response variables.

Table 1. ANOVA for response surface methodology applied to Y_GOS and DH.

Variable	ANOVA YGOS		ANOVA %DH		Multi-Response Optimization
	F-Ratio	p-Value	F-Ratio	p-Value
A: pH	1.52	0.2416	457.7	0
B: Temperature	4.22	0.0697	199.11	0	Desirability: 0.694
AA	3.97	0.0697	33.46	0.0001
AB	1.65	0.2231	52.02	0
BB	14.69	0.0412	1.17	0.3018
Adjusted R2	67.2		97.74
Mean	1.19		0.37
Durbin–Watson	p = 0.9712		p = 0.6838
Optimal conditions	pH 5.3 and 51 °C		pH 6.5 and 60 °C		pH 6 and 59.5 °C
Predicted value					YGOS: 22.09%
	24.49%		13.01%		DH: 9.46%
Experimental validation	26.41 ± 0.43%		13.5 ± 0.55%		YGOS: 25.67 ± 0.17%
					DH: 10.5 ± 0.30%

shows a summary of the ANOVA for both response variables. The response surface model for Y_GOS showed an adjusted R² of 67.20%, with pH 5.3 and 51 °C as the optimal conditions. In this model, only the quadratic component of temperature significantly influenced Y_GOS (opposite effect). In contrast, the model for DH exhibited a high degree of fit (adjusted R² = 97.74%), with pH 6.5 and 60 °C as the optimal conditions. Temperature, pH, and their interaction had significant positive effects on hydrolysis extent. The lower adjusted R² for Y_GOS compared with that of DH likely reflects the intrinsic kinetic complexity of transgalactosylation reactions, which involve dynamic competition between hydrolysis and oligosaccharide formation, as well as product re-hydrolysis. Unlike proteolysis, which follows more monotonic kinetics, GOS formation depends on transient concentration equilibria. Additional variables such as enzyme ratio, lactose concentration, and reaction time could further refine predictive accuracy, but they were intentionally fixed in this study to isolate the effects of pH and temperature to determine a feasible operational window.

Despite the fitted response for Y_GOS being low, a desirability function analysis (D = 0.68) was able to yield a compromise optimum at pH 6.0 and 59.5 °C that simultaneously optimizes both responses. Experimental validation under this condition achieved a Y_GOS of 25.67 ± 0.17% and DH of 10.5 ± 0.3%, confirming the effectiveness of this one-pot strategy for simultaneous native whey bioconversion. Normally, acidic conditions are preferred by β-galactosidase from Aspergillus oryzae for the production of GOS, resulting in a Y_GOS of 25 to 28% under similar reaction conditions in other studies [12,21,44]. In addition, since compounds with specific functional and bioactive properties are usually obtained at a DH between 5 and 10%, the enzymatic hydrolysis of whey proteins in our experiments can be considered successful [45,46,47,48]. Therefore, the results obtained in the one-pot system under multi-response optimization can be considered adequate. Next, the evolution of the bioactive and technological properties of the products from this system over a longer reaction duration was investigated. For exploration and comparison purposes, the reaction conditions that maximize only Y_GOS or DH were also assessed.

3.5. Effect of One-Pot Reaction Conditions on the Bioactive and Technological Properties

3.5.1. Antioxidant Evolution Under Optimized Conditions

To assess the functional potential of the bioconverted whey, antioxidant capacity was evaluated over time under the three conditions identified during process optimization using DPPH and ABTS assays (Figure 5A,B). Both assays showed an overall increase in antioxidant capacity throughout the 120 min reaction, indicating the continuous generation of antioxidant-active compounds during enzymatic processing. The results reflected significant differences in antioxidant profiles depending on temperature and pH. In the DPPH assay (Figure 5A), the highest initial activity (~4800 mg TE/mL) was observed at pH 6.5 and 60 °C; however, this value decreased over time, likely due to the formation of transient or unstable antioxidant compounds. In contrast, at pH 5.3 and 51 °C, a slower but sustained increase in DPPH activity was recorded, suggesting gradual production and accumulation of more stable antioxidant species.

The ABTS assay (Figure 5B) presented a distinct kinetic pattern, with the highest and most consistent antioxidant capacity also observed at pH 6.5 and 60 °C. Under these conditions, a rapid increase was observed during the first 20 min, followed by a plateau, implying efficient release of soluble antioxidant compounds early in the reaction. This divergence between assays can be attributed to their different sensitivities: ABTS detects both hydrophilic and lipophilic compounds, while DPPH is more selective for lipophilic antioxidants [49,50]. Moreover, the enzymatic hydrolysis of milk proteins releases low-molecular-weight peptides containing antioxidant residues (e.g., tyrosine and tryptophan), while the presence of reducing sugars such as lactose and monosaccharides may favor Maillard reactions under higher temperatures and mildly alkaline pHs. These reactions probably generate Maillard reaction products (MRPs), which also contribute to antioxidant activity [51]. Thus, both enzymatic and non-enzymatic pathways are likely involved in the generation of antioxidant potential in the one-pot system under the tested conditions.

3.5.2. Effect of Reaction Conditions on Bioactive Peptide Profile

The extent and specificity of protein hydrolysis under each reaction condition were confirmed via SDS-PAGE analysis (Figure 5C), which revealed progressive degradation of whey proteins such as β-lactoglobulin and α-lactalbumin, with visible changes in band intensity and migration patterns beginning at 90 min and continuing through to 200 min. Across all conditions, the enzymatic action of Alcalase produced peptide fractions mainly between 2 and 10 kDa in size, consistent with the size range of many documented antioxidant peptides [50]. Notably, the pH 6.5/60 °C treatment (lanes 5–6) exhibited the most extensive degradation and densest peptide bands, while the pH 5.3/51 °C condition (lanes 8–9) showed the lowest degree of hydrolysis. Intermediate profiles were observed at pH 6.0/60 °C (lanes 3–4), but the peptide band patterns indicated a narrower peptide spectrum compared with that of the pH 6.5/60 °C condition.

The differences in peptide profiles closely matched the ABTS assay results, reinforcing the link between peptide diversity and antioxidant potential. For instance, ABTS activity nearly doubled under pH 6.5/60 °C compared with pH 6.0/60 °C, highlighting the impact of enzymatic conditions on functional outcomes. These observations suggest that peptide richness, molecular weight distribution, and specific amino acid content (e.g., tyrosine, histidine, and tryptophan contents) all contribute to antioxidant functionality. In addition, the presence of multiple reaction pathways (proteolysis, transgalactosylation, and maybe Maillard reaction products) creates a complex bioactive matrix with synergistic effects.

The molecular size distribution and antioxidant behavior of the peptides generated in this study align with the reported characteristics of bioactive peptides derived from milk proteins. Most peptides obtained under optimal conditions (particularly pH 6.5 and 60 °C) had molecular weights ranging between 2 and 10 kDa, a range commonly associated with antioxidant activity [50]. Peptides of this size are typically generated through microbial fermentation or enzymatic digestion, either in vitro or as part of gastrointestinal processes [52]. However, the present study demonstrates that native whey can be directly converted into antioxidant-rich peptides in a single enzymatic step, bypassing the need for extensive downstream purification or hydrolysis optimization. For example, one study identified the peptide WYSLAMAASDI from β-lactoglobulin A using Corolase PP, which was found to have potent antioxidant activity (ORAC-FL value of 2.621 µmol TE/µmol peptide) that was attributed to the presence of tryptophan and tyrosine in its sequence [53]. Similarly, the authors of [54] isolated the tetrapeptide Trp–Tyr–Ser–Leu (WYSL) from Alcalase-treated whey protein hydrolysates, which exhibited high DPPH- and superoxide-radical-scavenging activities (IC50 values of 273.6 and 558.4 µM, respectively).

These examples highlight the potential of one-pot systems to yield functional peptide fractions comparable in structure and activity to those obtained using more complex or segmented processes. Furthermore, the coexistence of GOSs in the matrix in this study may enhance the synergistic health-promoting effects of the final product.

3.5.3. Effect of Reaction Conditions on Technological Properties

The effects of time and reaction conditions on the emulsifying and foaming properties of the bioconverted native whey obtained using the one-pot system were assessed (Figure 6).

The pH 5.3 and 51 °C conditions are close to the isoelectric point of the main whey serum proteins, namely, β-lactoglobulin (pI ≈ 5.2), α-lactalbumin (pI 4.8–5.1), and bovine serum albumin (pI 4.8–5.3) [55]. Under such conditions, low solubility and, consequently, reduced technological performance would be expected. However, as described by [56], β-lactoglobulin and BSA retain relatively high solubility at their pI due to their surfaces being rich in hydrophilic residues and the coexistence of positive and negative charges that sustain protein hydration. For instance, although emulsifying activity was moderate during the final reaction stages (200–250 m²/g), emulsion stability reached high values (90.3 ± 3.9%) (Figure 6A). This behavior could be attributed to progressive hydrolysis, which generates peptides capable of diffusing toward the interface, as well as the presence of sugars in the medium following β-galactosidase action on lactose, thereby increasing the viscosity of the continuous phase and strengthening the interfacial film. Likewise, a high foaming capacity was observed at extended reaction times (198.3 ± 2.9%), although with lower foam stability (70.6 ± 4.2%) (Figure 6D). This decline in stability may be associated with reduced electrostatic repulsion between bubbles under isoelectric conditions, which promotes coalescence and drainage.

At pH 6 and 60 °C, the emulsifying activity index (EAI) was initially high, reaching a maximum at 30 min (365.6 ± 9.2 m²/g), which subsequently markedly decreased over time while emulsion stability peaked at 90 min and then declined (Figure 6B). This behavior could be explained by the degree of hydrolysis achieved at this pH and temperature, which generates peptides of intermediate size. During the early stages, native or partially hydrolyzed proteins were still present and able to exert effective surface-active properties (Figure 5C). However, as the reaction progressed, further fragmentation did not yield a peptide profile with an adequate balance between hydrophilic and lipophilic groups, thereby limiting the ability to maintain stable oil–water interfacial films. For contextualization, EAI values for commercial WPC typically range between 150 and 300 m²/g depending on the protein concentration, degree of denaturation, and processing conditions [35]. Enzymatic hydrolysis has been shown to modulate interfacial performance, where moderate degrees of hydrolysis may enhance emulsifying properties due to improved molecular flexibility, while excessive hydrolysis can reduce interfacial film cohesion [22,45]. Therefore, the maximum EAI values shown here are above the upper range commonly reported for conventional whey-based ingredients.

In contrast, the foaming capacity index (FCI) remained relatively consistent throughout the reaction, with moderate variations between 170 and 187%. Partially hydrolyzed whey proteins typically exhibit foaming capacity values between 140 and 200% depending on the extent of hydrolysis and environmental conditions [22,56]. The FCI values observed here fall within this reported range, suggesting that the integrated one-pot treatment maintains a functional aeration capacity while enabling concurrent carbohydrate valorization. These results support the potential applicability of the developed ingredient in emulsified beverages, nutritional emulsions, and aerated dairy systems, where ingredients with both prebiotic and techno-functional properties may be advantageous. In addition, foam stability showed an initial increase, reaching a maximum at 60 min (80.4 ± 1.8%), followed by a gradual decrease to 74.3 ± 2.1% at the end of the reaction (Figure 6E). This sustained trend, without any abrupt changes, suggests that moderate hydrolysis generated peptides with sufficient flexibility to maintain stable foaming properties over time. Conversely, emulsification exhibited a different pattern. This divergence between foaming and emulsifying behaviors is consistent with the observations in [56], which noted that proteins can be effective foaming agents without necessarily being good emulsifiers, and vice versa. Although emulsions and foams share common formation and stabilization principles, the energetic characteristics of their interfaces differ, and, therefore, the structural requirements of proteins are not identical for both properties.

Finally, at pH 6.5 and 60 °C, a clear relationship between the degree of hydrolysis and technological properties was observed (Figure 6C). The highest DH recorded under this condition translated into enhanced emulsifying capacity, which reached its maximum at 180 min (379.5 ± 8.1 m²/g), together with stability values exceeding 99% at the end of the reaction. This effect can be explained by the generation of low-molecular-weight peptides with high interfacial mobility, which are able to rapidly diffuse to the oil –water interface, reduce interfacial tension, and uniformly cover oil droplets. Moreover, since these conditions are further from the pI of whey proteins, the higher net charge of the molecules increases electrostatic repulsion, thereby reducing coalescence and improving emulsion stability. Similar results were reported in [22], which employed Alcalase at pH 8 and 50 °C and achieved a DH close to 15%, which led to a reduction in the >18 kDa fraction and an increase in peptides in the 3–18 kDa and <0.6 kDa ranges, which are partially consistent with the size profiles shown in Figure 5C. Such a molecular weight redistribution would favor emulsification, as small and intermediate peptides can effectively reduce interfacial tension and stabilize oil droplets. In contrast, the foaming properties did not benefit to the same extent under these conditions (Figure 6F). The foam formation capacity decreased markedly toward the end of the reaction (148.3 ± 2.9%), although foam stability increased slightly (75.3 ± 1.7%). This behavior could be attributed to the fact that very short peptides, despite their rapid diffusion to the air–water interface, lack sufficient chain length to form the cohesive and elastic films required for a high foaming capacity. Nevertheless, the increase in continuous-phase viscosity, attributable to the accumulation of longer sugars (GOS) and protein fragmentation, may have contributed to the delayed drainage.

3.6. Result Synthesis and Practical Implications

In summary, Y_GOS and DH could not be simultaneously optimized, revealing intrinsic trade-offs in one-pot multi-enzyme systems. Maximal GOS production was favored under mildly acidic pH and moderate temperatures, whereas higher pH and temperature enhanced proteolytic activity and peptide release, which is consistent with the optimal activity of each enzyme. However, antioxidant capacity did increase under all tested conditions, but its magnitude was strongly dependent on pH, temperature, and reaction time. A higher pH and temperature promoted faster generation of antioxidant-active compounds, likely due to extensive protein hydrolysis and low-molecular-weight peptide formation, while milder conditions supported a slower yet sustained increase. The antioxidant functionality was dependent on the combined effects of the peptide profile, reaction intensity, and reaction time rather than from GOS yield or hydrolysis extent alone, with Maillard reactions potentially also playing an important role. Hence, LC-MS/MS-based peptide identification represents a logical next step to further elucidate structure–function relationships. Analytical assessments (e.g., browning index, carbonyl compounds, and furosine quantification) could also be conducted in future studies to determine the effect of non-enzymatic transformations and how they modulate enzyme activity.

On the other hand, technological properties also exhibited similar differences. Emulsifying and foaming behaviors were affected by the reaction conditions, with no single condition producing the highest values for all properties. Conditions near the isoelectric point favored high emulsion stability despite moderate emulsifying activity, whereas a higher pH and temperature enhanced emulsifying capacity through increased peptide mobility and electrostatic repulsion. Conversely, extensive hydrolysis improved emulsion stability but compromised foaming capacity, reflecting distinct structural requirements for oil–water and air–water interfaces.

Reaction time was a critical variable for all properties. Short to intermediate times preserved partially hydrolyzed proteins and intermediate-sized peptides that support balanced interfacial functionality, while extended reactions favored smaller peptides with higher antioxidant potential but reduced foaming performance. Overall, none of the tested conditions simultaneously maximized Y_GOS, DH, antioxidant activity, and technological properties. Instead, the one-pot system provides a flexible processing method in which pH, temperature, and time can be tuned to tailor ingredients for specific applications, such as foam beverage-based formulations with prebiotic effects or antioxidant-rich nutritional emulsions, highlighting its potential for sustainable dairy ingredient innovation.

4. Conclusions

This study demonstrated the technical feasibility of simultaneously converting lactose and whey proteins in a single enzymatic reactor using native whey as the sole substrate. The proposed one-pot system successfully integrates β-galactosidase from Aspergillus oryzae and Alcalase under compatible operational conditions, enabling concurrent GOS synthesis and controlled protein hydrolysis without significant enzyme incompatibility.

The results confirmed the existence of intrinsic trade-offs between optimal conditions for GOS production and protein hydrolysis, highlighting the complexity of multi-enzyme systems. Multi-response optimization identified intermediate pH and temperature conditions that achieved a balanced production of both components. The antioxidant capacity and technological properties were strongly influenced by reaction intensity and time, indicating that functionality arises from the dynamic evolution of peptide profiles and lactose conversion rather than from endpoint yields alone.

From an application perspective, the system generated a multifunctional whey-based ingredient that exhibits antioxidant potential and tunable emulsifying and foaming properties. These findings support the concept of application-driven process design, where reaction conditions can be adjusted according to the specific industrial application. Overall, the one-pot strategy represents a viable and flexible approach for native whey valorization, contributing to process intensification in the dairy sector and providing a platform for the development of integrated whey-derived ingredients. Future work should further elucidate the molecular mechanisms underlying the observed functional modifications and test combinations of proteases and β-galactosidases from different sources.