Gluten Hydrolysate Research Articles

Enzymatic Hydrolysis as an Effective Method for Obtaining Wheat Gluten Hydrolysates Combining Beneficial Functional Properties with Health-Promoting Potential.

The byproduct from wheat starch production contains approximately 70% gluten (WG) and is an inexpensive but demanding protein raw material for the food industry. This study attempted to determine the optimal hydrolysis conditions for such raw material to obtain peptides combining beneficial functional characteristics with health-promoting activity. The proteases Bromelain, Alcalase, Flavourzyme, and a protease from A. saitoi were used for hydrolysis. It was shown that the tested proteases differ both in terms of the effective hydrolysis conditions of gluten and the profile of the released hydrolysates. Bromelain was particularly effective in converting gluten into peptides, combining beneficial health and functional properties. It achieved maximum activity (189 U/g) against WG at pH 6 and 60 °C, and the best-balanced peptides in terms of desired properties were released at a dose of 2.5 U/g. These peptides were free from most allergenic epitopes, effectively inhibited ACE, and, at 0.34 g, were equivalent to the approved dose of BHT. Their emulsifying activity was higher than that of gluten, and the foaming formation and stabilization potential exceeded that of ovalbumin by 10% and 19%, respectively. It seems that Bromelain-released WG hydrolysates are a promising candidate for a safe fat stabilizer and egg white substitute.

Screening and characterization of umami peptides from enzymatic and fermented products of wheat gluten using machine learning

To explore the umami peptides from wheat gluten hydrolysates (WGHs) with different degrees of hydrolysis (DH) after Corynebacterium glutamicum (C. glutamicum) fermentation, a high-throughput screening method for umami peptides was established based on machine learning. The results showed fermented WGHs with high DHs exhibited more favourable taste profile after 4 days of fermentation and produced more small molecule peptides. Fermentation increased the number of characteristic peptides in WGHs with DH10, DH15, and DH20. Among, fermented WGHs with DH15 were the important source of potential umami peptides. Six potential umami peptides were screened (QQLPQFEE, LSFE, EELR, YTCE, YTTD, EEDQ) and were demonstrated to form stable complex with the T1R1/T1R3 receptor through hydrogen bonding interactions, electrostatic interactions, and hydrophobic interactions forces. The affinity of peptides and receptor were in the range of −6.3 to −8.1 kcal/mol. GLU735, GLY755, SER734, SER757 and THR227 were the critical binding sites. Sensory evaluation and electronic tongue showed that six peptides exhibited umami profile and had the capacity to enhance umami and saltiness taste. EEDQ presented the strongest intensity of umami taste and QQLPQFEE had the lowest umami thresholds. This study provides reliable methods for effectively screening new microbial-derived umami peptides.

Enzymatic hydrolysis of wheat gluten employing cysteine proteases from noni fruits (Morinda citrifolia L.)

Previous reports have shown that noni (Morinda citrifolia L.) puree extract (NPE) is a promising source of cysteine peptidases with potential applications in the food industry. This study expanded the applications of cysteine peptidases from noni fruit by analyzing their potential for wheat gluten hydrolysis and assessing their in vitro toxicity as a new food ingredient. NPE cysteine proteases hydrolyze both gliadins and glutenins, reaching a degree of hydrolysis of 24.40% and 31.78%, respectively. In addition, they reduced gliadin content by 96.80% after 4 h of incubation, as displayed by the R5 ELISA test kit for detecting gliadin based on the monoclonal antibody R5. The chromatographic fractionation of NPE cysteine proteases on a cation exchange column (Source 30S) resulted in an unretained peak (P1) and a retained peak (P2), which showed proteolytic activity with different electrophoretic and zymographic profiles. P1 and P2 had similar (p < 0.05) degrees of hydrolysis for gliadins (approximately 9%). P1 performed better (10.68%) than P2 (8.55%) for glutenins. NPE cysteine proteases did not exhibit genotoxicity or cytotoxicity against mouse fibroblasts (L929 cell line). These findings suggest that NPE cysteine proteases have potential for use as novel ingredients to produce gluten-free foods.

Effect of limited hydrolysis with endopeptidase on bitterness properties of wheat gluten hydrolysates

The application of limited hydrolysis on wheat gluten (WG) has been found to be a highly effective method for improving a wide range of functional properties associated with plant protein. However, the bitter peptides formed during this process have an unpleasant bitterness. To provide a theoretical framework for de-bittering techniques, this study examined the modulation of bitterness intensity in wheat gluten hydrolysates (WGHs) by manipulating enzyme cleavage sites and degree of hydrolysis (DH). It was found that the bitter peptide's release content increased significantly, and the bitterness threshold decreased markedly with increasing DH. Furthermore, the structure-bitterness threshold association of bitter peptides was investigated. The results indicated that peptide segments in molecular weights (MW) from 500 to 1000 exhibited the greatest influence on bitterness threshold compared to peptides with other MWs. The percentage of total hydrophobic amino acid content and position of some hydrophobic amino acids in the amino acid sequence had a significant effect on the bitterness threshold. Finally, 47 peptides were identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. These peptides were mainly small peptides with less than 13 amino acid residues and MW below 1500 Da, and most of them had Q values higher than 5855 J/mol.

Enzymatic Preparation and Processing Properties of DPP-IV Inhibitory Peptides Derived from Wheat Gluten: Effects of Pretreatment Methods and Protease Types.

The choice of appropriate proteases and pretreatment methods significantly influences the preparation of bioactive peptides. This study aimed to investigate the effects of different pretreatment methods on the hydrolytic performance of diverse proteases during the production of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides derived from wheat and their foaming and emulsion properties. Dry heating, aqueous heating, and ultrasound treatment were employed as pretreatments for the protein prior to the enzymatic hydrolysis of wheat gluten. FTIR analysis results indicated that all pretreatment methods altered the secondary structure of the protein; however, the effects of dry heating treatment on the secondary structure content were opposite to those of aqueous heating and ultrasound treatment. Nevertheless, all three methods enhanced the protein solubility and surface hydrophobicity. By using pretreated proteins as substrates, five different types of proteases were employed for DPP-IV inhibitory peptide production. The analysis of the DPP-IV inhibitory activity, degree of hydrolysis, and TCA-soluble peptide content revealed that the specific pretreatments had a promoting or inhibiting effect on DPP-IV inhibitory peptide production depending on the protease used. Furthermore, the pretreatment method and the selected type of protease collectively influenced the foaming and emulsifying properties of the prepared peptides.

Optimization of Enzymatic Hydrolysis by Protease Produced from Bacillus subtilis MTCC 2423 to Improve the Functional Properties of Wheat Gluten Hydrolysates

This study is aimed at investigating the reutilizing of gluten protein from the wheat processing industry by Bacillus subtilis MTCC 2423 protease to obtain gluten hydrolysates with high added value. Gluten protein hydrolysis using protease achieved a 34.07% degree of hydrolysis with 5% gluten protein, at a hydrolysis time of 2 h for 1000 U/mL at pH 8.0 and temperature of 40°C. Compared to the wheat gluten, the obtained hydrolysates exhibited enhanced functional attributes, including heightened solubility (43%), increased emulsifying activity (93.08 m2/g), and improved radical scavenging properties. Furthermore, these hydrolysates demonstrated enhanced antioxidant potential, as evidenced by elevated ABTS (2,2 ′‐azino‐bis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) of 81.25% and DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) of 56.46% radical scavenging activities and also exhibited a higher α‐amylase inhibitory effect of 33.98%. The enhancement in functional characteristics of wheat gluten hydrolysates was observed by Fourier transform infrared spectroscopy. The percentage of free amino acids obtained by protease‐mediated hydrolysates increased significantly compared to the unhydrolyzed wheat, which was observed by high‐performance liquid chromatography. These findings suggest that wheat gluten hydrolysates hold promising potential as functional and nutritional food ingredients in the food industry, owing to their enhanced functionalities and potential antioxidant and antidiabetic properties.

Protective effect of wheat gluten peptides against ethanol-stress damage in yeast cell and identification of anti-ethanol peptides

In the study, the protective effects of wheat gluten hydrolysates (WGH) on the ethanol-induced injury in yeast cells were investigated, and the peptides that enhanced the resistance to 10% (v/v) ethanol-stress were identified. Results showed that the growth and viability of yeast under ethanol-stress were significantly enhanced by WGH, and it indicated that there were high activity fractions in WGH. Interestingly, eight wheat gluten peptides (WGP) were obtained and identified as EP, LL, LW, LPL, LML, LLL, LLW, and EFPL, respectively, and five (LL, LLL, LW, LML and EP) of them worked best. Furthermore, WGP could enhance the yeast resistance to ethanol-stress, and the WGP apparently maintained the morphology and structure (especially the cell walls and membranes) of yeast under ethanol-stress. Further studied found that WGP (especially LL and LW) contributed to increasing cell wall integrity and cell membrane fluidity while also reducing cell membrane permeability. TEM analysis found that due to the existence of LL and LW, both the thickness of cell wall and the integrity of cell membrane were enhanced, on the contrary, the cell membrane degradation and cytoplasmic efflux were reduced. These results revealed that WGP could be considered as an effective protector of yeast against ethanol-stress damage.

Potential use of gluten hydrolysate as a plasticizer in high-moisture soy protein–gluten extrudates

Potential use of gluten hydrolysate as a plasticizer in high-moisture soy protein–gluten extrudates

Enhancement of in vitro cholesterol-lowering and anti-diabetic activities of gluten hydrolysate prepared using kiwifruit protease as affected by ultrasound and atmospheric cold plasma post-treatments

Enhancement of in vitro cholesterol-lowering and anti-diabetic activities of gluten hydrolysate prepared using kiwifruit protease as affected by ultrasound and atmospheric cold plasma post-treatments

Применение нового комплексного ферментного препарата на основе штамма Aspergillus oryzae при гидролизе пшеничного глютена

Пшеничный глютен является источником незаменимых аминокислот и биоактивных пептидов, а его ферментативные гидролизаты широко используются в качестве пищевых добавок и усилителей вкуса. В то же время высокое содержание пролина препятствует гидролизу глютена пищеварительными протеазами, что приводит к возникновению аллергических реакций и нарушениям пищеварительной системы. Глубокий ферментативный гидролиз способствует снижению аллергенности глютена, повышает его пищевую ценность и расширяет область применения в пищевой промышленности. Некоторые комплексные ферментные препараты (ФП), содержащие эндо- и экзопептидазы, за счет их синергетического действия обеспечивают эффективный гидролиз глютена. На основе штамма Aspergillus oryzae 21-154 LAP – продуцента комплекса эндо- и экзопептидаз с увеличенной активностью лейцинаминопептидазы (LAP) – получен комплексный ФП Протооризин LAP, по активности LAP сравнимый с широко используемым в пищевой промышленности ФП Flavourzyme® 1000L, при высоком уровне общей протеолитической активности, обусловленном действием эндопептидаз. По увеличению содержания растворимого белка и свободных аминокислот при гидролизе пшеничного глютена Протооризин LAP превосходил импортные ФП Flavourzyme® 1000L и Alcalase 2.4L как при индивидуальном, так и при совместном применении. Электрофоретический анализ полученных гидролизатов показал, что Протооризин LAP интенсивно расщеплял все основные белки глютена. Через 24 ч гидролиза оставалась только слабая полоса белка с молекулярной массой ~37 кДа. ФП Alcalase 2.4L расщеплял пшеничный белок с меньшей эффективностью, чем Протооризин LAP, оставляя через 24 ч, помимо полосы на уровне ~37 кДа, некоторое количество пептидов с молекулярной массой менее 14,4 кДа. Наименьшей способностью к гидролизу белков пшеницы обладал Flavourzyme® 1000L, однако при совместном использовании с Alcalase 2.4L препарат эффективно гидролизовал низкомолекулярные белковые фракции. Протооризин LAP успешно заменял совместное действие ФП Flavourzyme 1000L и Alcalase 2.4L, что показывает перспективность его использования при получении гидролизатов пшеничного глютена. Wheat gluten is a good source of essential amino acids and bioactive peptides. Its hydrolysates are widely used as food additives and flavor enhancers. At the same time, the high proline content interferes with the gluten hydrolysis by digestive proteases, which leads to allergic reactions and digestive system disorders. Deep enzymatic hydrolysis contributes to the reduction of gluten allergenicity, increases its nutritional value and expands its scope in the food industry. Some complex enzyme preparations (EP) containing endo- and exopeptidases, due to their synergistic action, provide effective hydrolysis of gluten. Using Aspergillus oryzae 21-154 LAP strain, a producer of endo- and exopeptidases complex with increased leucine aminopeptidase (LAP) activity, the EP Protoorizin LAP was obtained, in terms of LAP activity comparable to Flavourzyme® 1000L widely used in the food industry, and possessing a high level of total proteolytic activity due to endopeptidases presence. During wheat gluten hydrolysis Protoorizin LAP was superior to Flavourzyme® 1000L and Alcalase 2.4L, both when used individually and when combined, by the increase in the soluble protein and free amino acids content. Electrophoretic analysis of the obtained hydrolysates showed that Protoorizin LAP intensively cleaves all major gluten proteins. After 24 h of hydrolysis, the ~14 kDa faint band remained at the level of ~37 kDa. Alcalase 2.4L cleaved wheat protein with less efficiency than Protoorizin LAP, leaving after 24 h, in addition to the ~37 kDa band, some peptides with a molecular weight of less than 14.4 kDa. Flavourzyme® 1000L had the least ability to hydrolyze gluten, however, when used together with Alcalase 2.4L, the EP contributed to a more complete hydrolysis of low molecular weight protein fractions. Protoorizin LAP successfully replaced the combined action of Flavourzyme 1000L and Alcalase 2.4L, which shows the promise of its use in the wheat gluten hydrolysates production.

Hydrolase Activities of Sourdough Microorganisms

Sourdough is renowned for improving bakery products’ nutritional and quality characteristics through the enzymes produced by its microbiota. Among the enzymatic framework present in sourdough fermentation, amylase, cellulase, and peptidase are responsible for many of the properties valued in sourdough products. Furthermore, there is an increasing concern regarding the allergenic potential of gluten, which motivates the investigation of enzymatic gluten hydrolysis. This study aimed to select probiotics, isolate and identify microorganisms from sourdough, and assess their amylase, cellulase, and peptidase profiles. Additionally, a rapid screening method was developed for gluten and wheat flour hydrolysis, and gluten zymography and enzymography were performed. As a result, 18 microorganisms were isolated from sourdough and identified. The probiotic Bacillus licheniformis LMG-S 28935, and three microorganisms isolated from sourdough, the Limosilactobacillus fermentum, Pediococcus pentosaceus, and Saccharomyces cerevisiae, completed the profile of analyzed hydrolases and presented the capacity to hydrolyze gluten. These findings contribute to a better understanding of sourdough microorganisms’ hydrolase activities in the bakery science and technology field. In addition, an efficient, fast, and economical method for screening extracellular glutenase, produced by microorganisms, was applied. To our knowledge, it was the first time that amylase, cellulase, and peptidase activities were assessed from sourdough microorganisms.

Evaluation of antioxidant, α-amylase-inhibitory and antimicrobial activities of wheat gluten hydrolysates produced by ficin protease

Evaluation of antioxidant, α-amylase-inhibitory and antimicrobial activities of wheat gluten hydrolysates produced by ficin protease

Taste characteristic and the mechanism of light-colored Maillard reaction products derived from gluten hydrolysate

Light color and savory flavor are difficult to be integrated in ‘Thermal Process Flavoring’ through traditional Maillard reaction. A light-colored seasoning from gluten peptide-glucose system was prepared through gradient temperature-elevating Maillard reaction with extra-added l-cysteine. Greater saltiness and umami enhancement effect of Maillard reaction products (MRPs) were observed from weaker reaction, while lower temperature and longer heating time were conducive to yield kokumi-taste compounds. With the addition of l-cysteine, intensities of fluorescence and UV-VIS absorption of MRPs were weakened. An inhibition of browning and a decrease of α-dicarbonyls concentration caused by l-cysteine were observed. The addition of l-cysteine also led to an increase of peptides below 500 Da (from 53.27 mg/mL to 56.50 mg/mL) and a decrease of cross-linking products in 1–3 kDa, which accounted for superior umami and gentle kokumi attribute of the light-colored products compared with the MRPs without l-cysteine addition.

Sourdough yeast-bacteria interactions results in reduced immunogenicity by increasing depolymerization and hydrolysis of gluten

Sourdough is claimed to increase organoleptic and nutritional quality of baked products, to improve digestion and decrease immunogenicity of dough through enzymatic degradation of proteins. The specific role of sourdough strains in protein degradation and immunogenicity reduction has not been clarified, limiting further commercial applications. The current study comparatively investigated the effects of strains (Saccharomyces cerevisiae JM4, Torulaspora delbrueckii JM1 and Pediococcus acidilactici XZ31) selected from sourdough on gluten hydrolysis, depolymerization and immunogenicity during dough fermentation. The results showed that fermentation treatment degraded the polymerization and immunogenicity of gluten mainly by weakening the polymerization ability of glutenin and hydrolyzing glutenin peptides. Gluten in dough fermented with Pediococcus acidilactici XZ31 were more prone to depolymerize than with Saccharomyces cerevisiae JM4 and Torulaspora delbrueckii JM1. The application of Pediococcus acidilactici XZ31 significantly degraded gluten peptides and reduced gluten immunogenicity, however, Saccharomyces cerevisiae JM4 and Torulaspora delbrueckii JM1 had no effect on this. The mixed inocula of Pediococcus acidilactici XZ31 and yeast showed superiority over monoculture in degrading toxic/immunogenic peptides.

Wheat gluten hydrolysates with embedded Ag-nanoparticles; a structure-function assessment for potential applications as wound sorbents with antimicrobial properties

Numerous approaches have been used to prevent bacterial infection from injured skin, such as bandages and topical creams. However, the higher level of reactive oxygen species, bacterial infections, and excess wound exudates remain the major challenges for wound healing. In this study, we have tailored the structure of wheat gluten hydrolysates (WGH) as a continuous matrix by compositing it with a minimal amount of PVA, PVP, and PEG as polymer crosslinkers (0.5 wt%) to provide film structure integrity. Silver nanoparticles (AgNPs) were impregnated into the WGH to develop a control release matrix of the AgNPs. Scanning electron microscopy, X-ray diffractogram, and functional group patterns of WG and AgNPs indicate a successful integration of AgNPs into the wheat gluten matrix. The swelling capacity of the films was tested at acidic, neutral, and basic pH and was found to be highest in WG/PEG/Ag at pH 9 with 389%. The gradual release of Ag+/AgNPs from the films significantly scavenged free radicals and increased the antibacterial activity with up to a 12 mm inhibition zone against Pseudomonas aeruginosa. According to these findings, WGH with AgNPs has been successfully cast in films with increased absorption capacity, free radicals scavenging, oxidant quenching, and antibacterial capabilities, along with the sustained release of silver ions. The results, therefore, show the potential of the developed films in biomedical applications such as wound dressing.

Screening and Identification of Novel Soluble Epoxide Hydrolase Inhibitors from Corn Gluten Peptides.

The objective of this study was to investigate the soluble epoxide hydrolase (sEH) inhibitory properties of corn gluten peptides. In total, 400 dipeptides and 8000 tripeptides were first virtually screened by molecular docking and 30 potential sEH inhibitory peptides were selected. Among them, WEY, WWY, WYW, YFW, and YFY showed the highest sEH inhibitory activities with IC50 values of 55.41 ± 1.55, 68.80 ± 7.72, 70.66 ± 9.90, 96.00 ± 7.5, and 94.06 ± 12.86 μM, respectively. These five peptides all behaved as mixed-type inhibitors and were predicted to form hydrogen bond interactions mainly with Asp333, a key residue located in the catalytic active site of sEH. Moreover, it was found that the corn gluten hydrolysates of Alcalase, Flavourzyme, pepsin and pancreatin all exhibited high sEH inhibitory activities, with IC50 values of 1.07 ± 0.08, 1.19 ± 0.24, and 1.46 ± 0.31 mg/mL, respectively. In addition, the sEH inhibitory peptides WYW, YFW, and YFY were successfully identified from the corn gluten hydrolysates by Alcalase using nano-LC-MS/MS. This study demonstrated the sEH inhibitory capacity of peptides for the first time and corn gluten might be a promising food protein source for discovering novel natural sEH inhibitory peptides.

Characteristics of the enzyme-induced release of bitter peptides from wheat gluten hydrolysates.

Bitter peptides in the enzymatic hydrolysates were prepared and purified from wheat gluten using aqueous ethanol solutions and macroporous resin, which has opened a new road for the extraction and separation of bitter peptides. This report contains the release regularity of bitter peptides and the factors affecting the change of bitter intensity during enzymatic hydrolysis, providing a scientific basis for the research on debitterizing method. In this study, the effects of different degrees of hydrolysis (DH) and enzyme active sites on the bitter peptide content and bitter taste thresholds were discussed. The relationship between amino acid composition, molecular weight distribution, surface hydrophobicity and bitter taste thresholds was extensively researched. The results showed the exposure of hydrophobic amino acids and the bitterness intensity of the hydrolysates increased as the DH increased, and the bitterness of wheat gluten hydrolysates (WGHs) hydrolyzed by Alcalase was stronger than that of Trypsin. According to correlation analysis, the proportion of total hydrophobic amino acid is the first factor that affects the sensory properties of bitter peptide, and the release content of bitter peptides and the content of total bitter amino acids are the second, following by the content of peptide in the molecular weight range of 500–1,000 Da and the surface hydrophobicity. The amino acid sequence of bitter peptides from WGHs were identified and predicted using high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) and bioinformatics. It was found that the molecular weight of most of the peptides was below 1,500 Da, and the Q value was higher than 5.86 kJ/mol.

Improved functional properties of wheat gluten hydrolysate by covalent conjugation with chlorogenic acid

SummaryAppropriate modification of wheat gluten with polyphenols or other natural compounds can greatly increase its functional properties and provide a high‐quality protein source at a relative lower price. In this paper, covalent modification of wheat gluten hydrolysate (WGH) was performed with chlorogenic acid (CGA) by free‐radical grafting. The structural changes in WGH by conjugating with CGA were characterised by ultraviolet spectrum, FTIR spectroscopy, differential scanning calorimeter (DSC) and fluorescence scanning, as well as physicochemical properties and biological functions. Results showed that WGH‐CGA conjugates had higher thermal stability, better emulsifying and foaming capabilities, stronger antioxidant activity and lower allergenicity than the non‐conjugated WGH. These findings indicated that the WGH‐CGA conjugates had potential use value as functional food materials and additives in the food industry.

Wheat gluten hydrolysates prepared by sequential treatment with different combinations of commercial proteases

Wheat gluten hydrolysates prepared by sequential treatment with different combinations of commercial proteases

Biomarkers to Monitor Adherence to Gluten-Free Diet by Celiac Disease Patients: Gluten Immunogenic Peptides and Urinary miRNAs.

Celiac disease (CD) is a multifactorial autoimmune enteropathy with a prevalence greater than 1% in the pediatric population. The only therapy for CD patients is a strict gluten-free diet (GFD). Gluten-free food contamination by other cereals during packaging and cooking or accidental ingestion of gluten may cause several intestinal and extraintestinal symptoms in CD patients. Therefore, the monitoring of gluten contamination in food and assessing the level of ingested gluten by analytical biomarkers has been of great interest in recent years. To this aim, small gluten immunogenic peptides (GIPs) obtained by the hydrolysis of gluten and present in urine and feces have been studied as biomarkers of gluten intake and to monitor adherence to GFD by CD patients. More recently, the use of circulating, fecal and urinary miRNAs has emerged as a novel diagnostic tool that can be potentially applied to assess adherence to GFD. Moreover, the presence of GIPs and miRNAs in both feces and urine suggests a similar excretion modality and the possibility of using urinary miRNAs, similarly to GIPs, as potential biomarkers of GFD in CD patients.