Wheat Gluten Hydrolysates Research Articles

In-situ and real-time monitoring of enzymatic process of wheat gluten by miniature fiber NIR spectrometer

The degree of hydrolysis (DH), peptide content and angiotensin I-converting enzyme (ACE) inhibitory activity of hydrolysates are the three most important indexes for process control in proteins enzymatic hydrolysis and ACE inhibitory peptides preparation. In this study, a miniature fiber optical spectrometer in conjunction with multivariate analysis for in-situ and real-time monitoring of the parameters (DH, peptide content and ACE inhibitory activity) in wheat gluten (WG) hydrolysis process. Synergy interval partial least square (Si-PLS) algorithm was performed to variables selection and the regression model was calibrated. The performance of a model was evaluated by the correlation coefficient (Rp) and the root mean square error (RMSEP) in the prediction set. The results showed that Rp=0.9270, RMSEP=1.73% for DH; Rp=0.9673, RMSEP=0.79mg/ml for peptide content; Rp=0.9507, RMSEP=5.12% for ACE inhibitory. This work demonstrated that miniature fiber optical spectrometer combined Si-PLS model can be used as an alternative method for in-situ and real-time monitoring the enzymatic process of WG.

Effect of deamidation-induced modification on umami and bitter taste of wheat gluten hydrolysates.

Bitter taste is the main limiting factor for various applications of protein hydrolysates. Frequently used physicochemical methods for debittering protein hydrolysates come with some undesired side effects. Deamidation-induced modification would be a very promising technique to improve the flavor of wheat gluten hydrolysates (WGHs). This study was designed to determine the effect of deamidation with certain enzymes or acid treatment on the chemical composition, bitterness and umami properties of WGHs. The difference between umami peptide and free glutamic acid on the suppression of bitterness is emphatically discussed. The optimal scheme is proposed based on the flavor of WGHs and the yield of peptides. The generation of umami substances suppressed bitter signal transduction. When the content of umami substances was relatively high, the umami-enhancing properties of umami peptides were obviously effective. The intensity of umami taste was high enough to further suppress bitter taste in the course of neurocognitive functioning. When WGHs were treated with Glutaminase for 180 min, the umami taste score increased from 1.62 to 4.27 and the bitter taste score decreased from 1.33 to 0.65. © 2016 Society of Chemical Industry.

The synergistic effect of heat treatment on alcalase-assisted hydrolysis of wheat gluten proteins: Functional and antioxidant properties

In order to confirm the gluten potential for inclusion into functional foods, the synergistic effect of the heat treatment and controlled enzymatic hydrolysis on the functional and the antioxidant properties of alcalase-assisted wheat gluten hydrolysates (AWGHs) will be discussed. For this purpose, wheat gluten was heat-treated during 30 min at 75 °C and intensively hydrolyzed with alcalase at degree of hydrolysis (DH) 16.1%, 22.9%, and 30.2%. All the hydrolysates had excellent solubility over a pH range of 2–12. Emulsifying activity and stability were also improved, while proteolysis was deleterious to foam capacity and stability, water-holding capacity, fat-binding capacity and did not show improvement at higher DH (22.9% and 30.2%). As well, controlled hydrolysis of heat-treated gluten resulted in a remarkable improvement in antioxidant activities. The results show that the heat-treated AWGHs were superior to the untreated hydrolysate in the functional and antioxidant properties tested. Practical applications This report examines existing evidence regarding the wheat gluten proteins (WGP), which is a byproduct from wheat starch processing. It is known that enzymatic hydrolysis is frequently used to improve functional properties of protein hydrolysates and largely dependent on the degree of hydrolysis (DH), which needs to be controlled to elude redundant proteolysis that can deteriorate functionality and cause unfavorable effects. The DH is a substantial factor which affect the hydrolysates' performances and an appropriate selection of protease for WGP hydrolysis will result in maximum biological activity and improved functionalities. Heat treatment is often used to facilitate the proteolysis of proteins. Thus, functional and antioxidant properties of WGP hydrolysates, as a function of heat treatment and the DH were adequately examined in this study and results showed that by combining heat prehydrolysis treatment under controlled conditions, hydrolysates with improved properties can be produced enhancing utilization of WGP in food products.

Exploring the Relationship between Structural and Air-Water Interfacial Properties of Wheat (Triticum aestivum L.) Gluten Hydrolysates in a Food System Relevant pH Range.

The relationship between structural and foaming properties of two tryptic and two peptic wheat gluten hydrolysates was studied at different pH conditions. The impact of pH on foam stability (FS) of the samples heavily depended on the peptidase used and the degree of hydrolysis reached. Surface dilatational moduli were in most, but not all, instances related to FS, implying that, although the formation of a viscoelastic protein hydrolysate film is certainly important, this is not the only phenomenon that determines FS. In contrast to what might be expected, surface charge was not a major factor contributing to FS, except when close to the point-of-zero-charge. Surface hydrophobicity and intrinsic fluorescence measurements suggested that changes in protein conformation take place when the pH is varied, which can in turn influence foaming. Finally, hydrolyzed gluten proteins formed relatively large particles, suggesting that protein hydrolysate aggregation probably influences its foaming properties.

Long form of thymic stromal lymphopoietin of keratinocytes is induced by protein allergens

A growing body of evidence suggests that epicutaneous sensitization of protein allergens induces immediate-type hypersensitivity (IHS) following induction of Type 2 immune responses in animals and humans. Thymic stromal lymphopoietin (TSLP) derived from keratinocytes is a cytokine that can activate dendritic cells and has been implicated in development of inflammatory Type 2 helper T-cells. However, there is no direct evidence that allergens directly regulate TSLP expression in keratinocytes. This study aimed to evaluate the response of TSLP to protein allergens in cultured human keratinocytes and to identify appropriate endpoints for IHS. The transcription of long-form TSLP (loTSLP) was strongly induced by ovalbumin, wheat gluten (WG), acid-hydrolyzed WG (acid-HWG), and extracts from feces of Dermatophagoides pteronyssinus and D. farina, and trypsin, but not by rare allergens, human serum albumin (HSA), or extracts of mite bodies. In acid-HWG, loTSLP mRNA was significantly augmented by acid hydrolysis of WG for 0.5 h compared to WG. However, prolonged acid hydrolysis attenuated this induction similarly to that reported in previous animal studies. These results suggested that intense loTSLP transcriptional induction was a characteristic of a high-allergenic protein. Additionally, TSLP production was induced by exposure to ovalbumin, WG, and acid-HWG in combination with a trio of cytokines, i.e. interleukin (IL)-4, IL-13, and tumor necrosis factor (TNF)-α. However, no TSLP protein was detected following exposure to HSA, even in the presence of these cytokines. With acid-HWG, TSLP protein release was consistent with loTSLP transcription. Thus, intense loTSLP transcriptional induction and TSLP protein expression are each effective indicators that can be used for in vitro screening of IHS.

Generation of wheat gluten hydrolysates with dipeptidyl peptidase IV (DPP-IV) inhibitory properties.

Wheat gluten, a Pro-rich dietary protein, was investigated for its potential to produce dipeptidyl peptidase IV (DPP-IV) inhibitory peptides during enzymatic hydrolysis with Debitrase HYW20. Nine gluten hydrolysates (H1-H9) were generated using a 2 factor × 3 level design of experiments (DOE) including the incubation temperature (40, 50 and 60 °C) and the enzyme: substrate ratio (E : S, 0.5, 1.0 and 1.5% (w/w)). Their DPP-IV half maximal inhibitory concentration (IC50) ranged from 0.24 ± 0.02 (H9) to 0.66 ± 0.06 mg mL-1 (H2A and H7) and their degree of hydrolysis (DH) from 31.7 ± 0.9 (H7) to 62.2 ± 3.0% (H6). Gluten and H9, the most potent DPP-IV inhibitory hydrolysate, were subjected to simulated gastrointestinal digestion (SGID), yielding Gluten_CorPP and H9_CorPP, respectively. H9_CorPP had a higher DPP-IV inhibitory potency than Gluten_CorPP (i.e., DPP-IV IC50 values of 0.33 ± 0.03 vs. 1.45 ± 0.26 mg mL-1, respectively). H9 and H9_CorPP both contained relatively potent DPP-IV inhibitory peptides such as Val-Pro-Leu, Trp-Leu and Trp-Pro which were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, several sequences possessing features of DPP-IV inhibitory peptides, mostly consisting of a penultimate or C-terminal Pro, were identified within H9. The presence of Pro-containing peptides within H9 may contribute to its stability to digestive enzymes. Gluten hydrolysates may have antidiabetic potential for humans.

Foam fractionation as a tool to study the air-water interface structure-function relationship of wheat gluten hydrolysates

Enzymatic hydrolysis of wheat gluten protein improves its solubility and produces hydrolysates with foaming properties which may find applications in food products. First, we here investigated whether foam-liquid fractionation can concentrate wheat gluten peptides with foaming properties. Foam and liquid fractions had high and very low foam stability (FS), respectively. In addition, foam fractions were able to decrease surface tension more pronouncedly than un-fractionated samples and liquid fractions, suggesting they are able to arrange themselves more efficiently at an interface. As a second objective, foam fractionation served as a tool to study the structural properties of the peptides, causing these differences in air-water interfacial behavior. Zeta potential and surface hydrophobicity measurements did not fully explain these differences but suggested that hydrophobic interactions at the air-water interface are more important than electrostatic interactions. RP-HPLC showed a large overlap between foam and liquid fractions. However, a small fraction of very hydrophobic peptides with relatively high average molecular mass was clearly enriched in the foam fraction. These peptides were also more concentrated in un-fractionated DH 2 hydrolysates, which had high FS, than in DH 6 hydrolysates, which had low FS. These peptides most likely play a key role in stabilizing the air-water interface.

Sensory and antigenic properties of enzymatic wheat gluten hydrolysates produced in an enzyme membrane reactor in comparison with batch

The aim of this study was to compare the sensory and antigenic properties of enzymatic wheat gluten hydrolysates produced with Flavourzyme and glutaminase in batch and continuous process, respectively. The hydrolysate samples were obtained by a standard batch process with microfiltration (16 µm) and ultrafiltration (10 kDa) and an optimized continuous enzyme membrane reactor (EMR) process with an ultrafiltration membrane (10 kDa). Two drying methods were applied: oven and freeze drying. Sensory evaluation showed that the EMR hydrolysates were significantly different and had an improved taste impact for the attributes “overall intensity,” “salty,” “umami” and “mushroom.” The antigenic potential of the EMR hydrolysates was reduced (222 ± 4 ppm gliadin) compared to batch microfiltered hydrolysate (9030 ± 89 ppm) and batch ultrafiltered hydrolysate (843 ± 49 ppm). The superior quality of wheat gluten hydrolysate from the EMR process compared to batch process was proven since sensory properties of wheat gluten hydrolysate were improved and the antigenic potential was reduced.

Improvement of Functional Properties of Wheat Gluten Using Acid Protease from Aspergillus usamii.

Hydrolysis parameters (temperature, E/S ratio, pH, and time) for acid protease (from Aspergillus usamii) hydrolysis of wheat gluten were optimized by response surface methodology (RSM) using emulsifying activity index (EAI) as the response factor. A temperature of 48.9°C, E/S ratio of 1.60%, pH 3.0, hydrolysis time of 2.5 h was found to be the optimum condition to obtain wheat gluten hydrolysate with higher EAI. The solubility of wheat gluten was greatly improved by hydrolysis and became independent of pH over the studied range. Enzymatic hydrolysis resulted in dramatically increase in EAI, water and oil holding capacity. Molecular weight distribution results showed that most of the peptides above 10 kDa have been hydrolyzed into smaller peptides. The results of FTIR spectra and disulfide bond (SS) and sulfhydryl (SH) content suggested that a more extensional conformation was formed after hydrolysis, which could account for the improved functional properties.

Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins.

Protein hydrolysis enables production of peptides and free amino acids that are suitable for usage in food and feed or can be used as precursors for bulk chemicals. Several essential amino acids for food and feed have hydrophobic side chains; this property may also be exploited for subsequent separation. Here, we present methods for selective production of hydrophobic amino acids from proteins. Selectivity can be achieved by selection of starting material, selection of hydrolysis conditions, and separation of achieved hydrolysate. Several protease combinations were applied for hydrolysis of rubber seed protein concentrate, wheat gluten, and bovine serum albumin (BSA). High degree of hydrolysis (>50 %) could be achieved. Hydrophobic selectivity was influenced by the combination of proteases and by the extent of hydrolysis. Combination of Pronase and Peptidase R showed the highest selectivity towards hydrophobic amino acids, roughly doubling the content of hydrophobic amino acids in the products compared to the original substrates. Hydrophobic selectivity of 0.6 mol-hydrophobic/mol-total free amino acids was observed after 6 h hydrolysis of wheat gluten and 24 h hydrolysis of rubber seed proteins and BSA. The results of experiments with rubber seed proteins and wheat gluten suggest that this process can be applied to agro-industrial residues.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7441-8) contains supplementary material, which is available to authorized users.

Optimization of an enzymatic wheat gluten hydrolysis process in an enzyme membrane reactor using a design of experiment approach

The aim of this study was to determine the optimal conditions for operating an enzyme membrane reactor system to obtain a high yield of amino acids and peptides released from wheat gluten with Flavourzyme™. The optimal operating conditions were determined using multivariate analysis. A fractional factorial design made up of 27 runs amended by eight axial points was followed in a bench-scale enzyme membrane reactor system (V = 2 L), and responses were recorded over 8 h. Optimal conditions determined were a temperature of 50 ± 1 °C and a Flavourzyme™ activity of 116 ± 4 nkatLeu-pNA/mL. Modeling resulted in an optimal substrate feed rate of 19.2 g/L/h and a permeate flux of 57 L/m2/h. The enzymatic wheat gluten hydrolysis in an enzyme membrane reactor was conducted for 72 h with an average total product space–time yield of 12.6 ± 1.3 g/L/h (n = 4) and a free amino groups space–time yield of 4.4 ± 0.4 g/L/h (n = 4), and the substrate utilization was 0.82 ± 0.01 (g/g) (n = 4). The resulting product–enzyme ratios were 7.7 ± 0.6 mgTP/nkatLeu-pNA (n = 4) and 3.0 ± 0.2 mgFAA/nkatLeu-pNA (n = 4). The productivity of the enzyme membrane reactor improved compared to the reference batch process and doubled compared to previously proposed enzyme membrane reactor processes for the hydrolysis of wheat gluten.

Changes in the enzyme-induced release of bitter peptides from wheat gluten hydrolysates

Extraction of wheat gluten hydrolysates prepared using Proteax with isobutyl alcohol has opened a new avenue for exploring the release characteristics of bitter peptides.

Effect of sequential hydrolysis with endo- and exo-peptidase on bitterness properties of wheat gluten hydrolysates

After the bitterness of wheat gluten hydrolysates using single-enzyme treatment was assessed by taste dilution analysis, schemes of sequential hydrolysis with endo- and exo- peptidases were designed to prepare low-bitterness small-peptide powders.

Batch-to-batch variation and storage stability of the commercial peptidase preparation Flavourzyme in respect of key enzyme activities and its influence on process reproducibility

The synergy of endopeptidases and exopeptidases is the key for an efficient hydrolysis of proteins. Flavourzyme is sold as a commercial peptidase preparation from Aspergillus oryzae that exhibits various endo- and exopeptidase activities and, therefore, generates protein hydrolysates with high degrees of hydrolysis. The manufacturer (Novozymes) standardizes the enzyme preparation for one peptidase activity, determined with the marker substrate H-Leu-pNA. However, seven peptidases of Flavourzyme were recently identified and purified, and the significant contribution of six of them to wheat gluten hydrolysis was demonstrated. The knowledge about the batch-to-batch variation and storage stability of the Flavourzyme preparation regarding the other peptidase activities are still unclear, and this is important information for the usage of the enzyme preparation to gain reproducible protein hydrolysis processes. In the present study, we tested 12 Flavourzyme batches for the activity of the seven peptidases. The impact of the storage time on the peptidase activities and the magnitude of the batch-to-batch variation were investigated. In contrast to the activity determined with H-Leu-pNA as a substrate, the variations of the other peptidase activities were noticeable. The variation of the endopeptidase activity was most distinct and the activity decreased during the storage time of the preparation. The variation of the Flavourzyme composition also affected the reproducibility of a casein batch hydrolysis process, which should be taken into account for any future research and industrial application.

Production of wheat gluten hydrolysates with reduced antigenicity employing enzymatic hydrolysis combined with downstream unit operations.

Due to allergies or other health disorders a certain segment of the population is not able to safely consume some plant proteins, which are the main protein support in human nutrition. Coeliac disease is a prominent autoimmune disorder and requires a strict adherence to a gluten-free diet. The aim of this study was to identify suitable combinations of enzymatic hydrolysis and common unit operations in food processing (centrifugation, ultra-filtration) to produce gluten-free wheat gluten hydrolysates for food application. To analyse the hydrolysates, a simple and cheap competitive ELISA protocol was designed and validated in this study as well. The competitive ELISA was validated using gliadin spiked skim milk protein hydrolysates, due to the latter application of the assay. The limit of quantification was 4.19 mg kg(-1) , which allowed the identification of gluten-free (<20 mg kg(-1) ) hydrolysates. Enzymatic hydrolysis, including the type of peptidase, and the downstream processing greatly affected the antigenicity of the hydrolysates. Enzymatic hydrolysis and downstream processing operations, such as centrifugation and ultra-filtration, reduced the antigenicity of wheat gluten hydrolysates. Gluten-free hydrolysates were obtained with Flavourzyme after centrifugation (25 g L(-1) substrate) and after 1 kDa ultra-filtration (100 g L(-1) substrate). A multiple peptidase complex, such as Flavourzyme, seems to be required for the production of gluten-free hydrolysates. © 2015 Society of Chemical Industry.

Surface topography, nano-mechanics and secondary structure of wheat gluten pretreated by alternate dual-frequency ultrasound and the correlation to enzymolysis

The effects of alternate dual-frequency ultrasound (ADFU) pretreatment on the degree of hydrolysis (DH) of wheat gluten (WG) and angiotensin I-converting enzyme (ACE) inhibitory activity were investigated in this research. The surface topography, nano-mechanics and secondary structure of WG were also determined using atomic force microscope (AFM) and circular dichroism (CD). The correlations of ACE inhibitory activity and DH with surface topography, nano-mechanics and secondary structure of WG were determined using Pearson’s correlation analysis. The results showed that with an increase in either pretreatment duration or power, the ACE inhibitory activity of the hydrolysate also increases, reaching maximum at 10min and 150W/L, respectively, and then decreases thereafter. Similarly, AFM analysis showed that as the pretreatment duration or power increases, the surface roughness also increase and again a decrease occurs thereafter. As the pretreatment duration or power increased, the Young’s modulus and adhesion of WG also increased and then declined. Young’s modulus and adhesions average values were compared with ACE inhibitory activity reversely. The result of the CD spectra analysis exhibited losses in the relative percentage of α-helix of WG. Pearson’s correlation analysis showed that the average values of Young’s modulus and the relative percentage of α-helix correlated with ACE inhibitory activity of the hydrolysates linearly and significantly (P<0.05); the relative percentage of β-sheet correlated linearly with DH of WG significantly (P<0.05). In conclusion, ADFU pretreatment is an efficient method in proteolysis due to its physical and chemical effect on the Young’s modulus, α-helix and β-sheet of WG.

Wheat gluten hydrolysis using isolated Flavourzyme peptidases: Product inhibition and determination of synergistic effects using response surface methodology

The commercial peptidase preparation Flavourzyme is derived from the Aspergillus oryzae strain ATCC 42149/RIB 40 (Yellow koji mold) and is widely used for protein hydrolysis in various industrial and research applications. However, a biochemical characterization of the Flavourzyme peptidases is difficult, because obtaining purified proteins is essential when functional and structural characterization studies are targeted. Key enzyme activities (three endopeptidases, two aminopeptidases, two dipeptidyl peptidases) have recently been identified and isolated from this commercially available enzyme preparation. The impact and the synergism of theses peptidases on the complex wheat gluten hydrolysis are yet unclear. However, the knowledge about the latter is crucial for an efficient protein hydrolysis. In the present study, we determined the product inhibition for the seven isolated peptidases and analyzed the impact of each peptidase on the wheat gluten hydrolysis using response surface methodology. In general, both aminopeptidases and the three endopeptidases were of major importance. One of the endopeptidases (alkaline protease 1) was least affected by product inhibition and showed the highest effect on the wheat gluten hydrolysis. In the case of the aminopeptidases, the leucine aminopeptidase 2 showed a higher impact on the hydrolysis compared to the leucine aminopeptidase A, but exhibited the highest product inhibition sensitivity. The dipeptidyl peptidases were of only minor impact on the wheat gluten hydrolysis. To conclude, six out of the seven peptidases contributed significantly (p<0.05) to the wheat gluten hydrolysis and should be considered when designing such a process.

Extracellular peptidases from insect- and compost-associated microorganisms: screening and usage for wheat gluten hydrolysis

Screening for microorganisms with an enzyme activity for a specific application is challenging. In this study, we showed a successful way to isolate microorganisms from insects and compost samples, which had a desired enzyme activity, namely the hydrolysis of wheat gluten. This was realized by a two-step screening approach. The first step was an agar plate screening assay for extracellular peptidase activity, and the most promising organisms were further tested in a second screening step: the hydrolysis of a wheat gluten suspension. Here, the extracellular peptidases of two microorganisms showed similar hydrolysis performances compared to the commercial enzyme preparation Alcalase®. The best isolate, which was identified as a Bacillus subtilis species, was cultivated in a bioreactor (35 L scale) to produce a larger amount of the extracellular peptidases. The peptidase activities in the cell-free culture medium were partially purified, as it is common for technical enzyme preparations for the food industry. After centrifugation and ultrafiltration, an ammonium sulfate precipitation was performed. This peptidase preparation was biochemically characterized, and the obtained stability was sufficient for application. Wheat gluten hydrolytic approaches (500 mL scale) were performed under controlled conditions (e.g., pH value). The amount of solubilized gluten protein obtained after 24 h hydrolysis was higher using the enzyme preparation from the isolate compared to Alcalase®. The enzyme stability increased under process conditions, compared to the stability in buffer, due to the stabilizing effect of the substrate. The biochemical characteristics and the hydrolysis performance showed the potential of the novel extracellular peptidases for the hydrolysis of the industrially relevant substrate wheat gluten.

Continuous long-term hydrolysis of wheat gluten using a principally food-grade enzyme membrane reactor system

The potential of the enzyme membrane reactor technology has been shown in several studies. In our study, we designed a principally food-grade continuous long-term hydrolysis process of wheat gluten in an enzyme membrane reactor with the proteolytic enzyme preparation Flavourzyme®. Among others, ethanol was the most suitable food-grade processing aid for avoiding contamination and showed high potential for application. The Flavourzyme®/wheat gluten process was comprehensively characterized in the enzyme membrane reactor. Critical factors for the enzyme stability were the temperature, pump stress and enzyme leakage through the membrane. The hydrolysis and process conditions were optimized to increase the space–time yield. Respective process parameters were chosen to obtain sufficient microbial, enzyme and process stability for the whole process time. The long-term hydrolysis was carried out in the presence of 8% (v/v) ethanol with a substrate concentration of 100gL−1 at 37°C and pH 7.5 for 96h. The continuous process resulted in stable product quality (degree of hydrolysis) and space–time yield (6.33gh−1L−1) over time. However, a discontinuous removal of accumulated dry matter was inevitable for the present enzyme membrane reactor process and was performed for 30min every 24h. Due to the fact that the enzymes were reused, the enzyme productivity could be increased by 450% compared to a reference batch process.

Structure–mechanism relationship of antioxidant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated by pH

The aims of this study were to assess bioactive properties (ACE inhibition and antioxidant capacity) from wheat gluten hydrolysate peptides fractionated by pH (4.0, 6.0 and 9.0), to determine peptide action mechanism, and to relate it to the secondary structure and functional groups of peptides. Gluten hydrolysate extracts (GHE) were enriched in peptides with medium hydrophobicity and molecular weight (≈60% MH and 5.5kDa, respectively). Gluten peptides inhibited ACE I by uncompetitive mechanism and a direct relationship between α-helix structure and IC50% value was obtained (r=0.9127). TEAC and cooper chelating activity from GHE 6.5 were the highest and directly correlated with MH peptides. GHE 9.0 had high carotene bleaching inhibition (47.5±0.3%) and reducing power activity (163.1±2.9mg S2O32− equivalent g−1 protein), which were directly related to disulfide bonds content of peptides (r=0.9982 and 0.9216, respectively). pH was a good alternative to select bioactive peptides from wheat gluten hydrolysate.