Two-step Enzymatic Hydrolysis Research Articles

Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8±1.8mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6±0.6mg calcium/g protein with a high association constant of 420M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

Influence of sonication pretreatment on antiradical and anti-ACE activity of protein hydrolysates from fermented pork loins

The aim of this study was to assess whether ultrasound treatment (sonification time: 5, 15, and 30 min; constants: ∼40 kHz, ∼2.5 W cm2) can be applied prior to hydrolysis to enhance the anti-radical and angiotensin converting enzyme inhibiting (anti-ACE) effect of the hydrolysates from fermented pork loins. Enzymatic hydrolysis was performed using pepsin, followed by pancreatin. The influence of meat matrix on the course of hydrolysis, shaped using a lactic acid bacteria (LAB)-based starter culture, was also analyzed. It was found that proteases caused a systematic increase in the content of peptides, while pancreatin limited the peptide content in the protein hydrolysate from the loins subjected to spontaneous fermentation. Moreover, for these tests, sonication time had a negligible effect on the peptides content of the hydrolysates. On the other hand, for the sample of LAB-fermented products, both sonication time and stage of hydrolysis promoted the biological activity of the hydrolysates. Samples from the LAB-fermented meat had more peptides at the stage of digestion with pepsin and pancreatin, exhibiting much faster antiradical and anti-ACE activity compared to the control sample. The obtained results suggest that the use of LAB promotes the release of antiradical peptides during the two-step enzymatic hydrolysis, the duration of which can be shortened to achieve satisfactory biofunctionalities. Additional application of sonication pretreatment allows controlling the course of the hydrolysis, as the pro-health, biological effect of some protein-derived sequences is associated with the content of peptides.

Preparation and identification of antioxidant peptides from Quasipaa spinosa skin through two-step enzymatic hydrolysis and molecular simulation

Frog skin, a by-product of Quasipaa Spinosa farming, is rich in protein and potentially a valuable raw material for obtaining antioxidant peptides. This study used papain combined with acid protease to digest frog skin in a two-step enzymatic hydrolysis method. Based on a single factor and response surface experiments, experimental conditions were optimized, and the degree of hydrolysis was 30 %. A frog skin hydrolysate (QSPH-Ⅰ-3) was obtained following ultrafiltration and gel filtration chromatography. IC50 for DPPH, ABTS, and hydroxyl radical scavenging capacities were 1.68 ± 0.05, 1.20 ± 0.14 and 1.55 ± 0.11 mg/mL, respectively. Peptide sequences (17) were analyzed and, through molecular docking, peptides with low binding energies for KEAP1 were identified, which might affect the NRF2-KEAP1 pathway. These findings suggest protein hydrolysates and antioxidant peptide derivatives might be used in functional foods.

Construction of hypoallergenic microgel by soy major allergen β-conglycinin through enzymatic hydrolysis and lactic acid bacteria fermentation

Soy allergenicity is a public concern, and the combination of multiple processing methods may be a promising strategy for reducing soy allergenicity. In this study, a novel two-step enzymatic hydrolysis followed by lactic acid bacteria fermentation was proposed for the construction of hypoallergenic soybean protein microgel. β-Conglycinin was used as the main soy allergen. The effects of different enzymatic hydrolysis (Alcalase, Neutrase, and Protamex) and LAB fermentation on β-conglycinin microgel formation and its immunoreactivity were investigated. Results showed that the use of different enzymes and the attainment of different degrees of hydrolysis affected the particle distribution and zeta potential in the microgels and leads to differences in microstructure and immunoreactivity. All hydrolysates compared with intact protein accelerated the formation of gel during LAB fermentation. Among the three assayed enzymes, fermented Protamex hydrolysates at 60 min (PF-60) demonstrated a microgel with an overall reduced average particle size (741.20±7.18 nm), lower absolute values of zeta potential (10.43±0.65 mV), and regular gel network. The antigenicity and IgE-binding capacity decreased to the lowest value of 0.30 % and 6.93 %, respectively. Peptidomics and immunoinformatic analysis suggested that PF-60 disrupted 17/30, 16/44, and 23/75 epitopes in the α, α′, and β subunits, respectively. Unlike the LAB-fermented unhydrolyzed β-conglycinin disrupted epitopes mostly located at the loop domain, PF-60 primarily promoted the exposure and disruption of allergen epitopes with β-sheet structure located at the core barrel domain. These findings can provide new perspectives on the preparation of hypoallergenic soybean-gel products on edible particulate systems.

Structural identification and combination mechanism of iron (II)-chelating Atlantic salmon (Salmo salar L.) skin active peptides.

In order to utilize salmon skin for high value, and investigate the structural identification and combination mechanism of iron (II)-chelating peptides systemically, Atlantic salmon (Salmo salar L.) skin, a by-product of Atlantic salmon processing, was treated by two-step enzymatic hydrolysis to obtain salmon skin active peptides (SSAP). Then they reacted with iron (II) to obtain iron (II)-chelating salmon skin active peptides (SSAP-Fe) with a high iron (II) chelating ability of 98.84%. The results of Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalenesulfonic acid ammonium salt hydrate (ANS) combined fluorescence measurement, isothermal titration calorimetry (ITC) and full wavelength ultraviolet (UV) scanning showed that the structural characteristics of SSAP changed before and after chelating iron (II). Reverse phase high performance liquid chromatography (RP-HPLC) and mass spectrometry were used to identify and quantify the peptides in SSAP-Fe. Four peptide sequences (STEGGG, GIIKYGDDFMH, PGQPGIGYDGPAGPPGPPGPPGAP and QNQRESWTTCRSQSSLPDG) were identified. The content of PGQPGIGYDGPAGPPGPPGPPGAP was the highest, at 25.17μg/mg. The pharmacokinetic and pharmacodynamic properties of these four peptides were also investigated, and the results indicated that they have satisfactory predicted ADMET properties. Molecular docking technology was used to analyze the binding sites between iron (II) and SSAP, and it was found that PGQPGIGYDGPAGPPGPPGPPGAP had the lowest predicted binding energy with iron (II) and the most stable predicted binding energy with iron (II). This results showed that the stability of SSAP-Fe were closely related to the number of covalent bonds and the types of amino acids. This study revealed the structure and combination mechanism of SSAP-Fe, and indicated that SSAP-Fe prepared by chelation may be used as a Fe supplement that can be applied in functional foods or ingredients.

Efficient co-production of xylo-oligosaccharides and probiotics from corncob by combined lactic acid pretreatment and two-step enzymatic hydrolysis

Lactic acid (LA) is efficient in xylo-oligosaccharides (XOS) production from poplar. However, the role of LA in XOS production from corncob has not been carefully elucidated, and the co-production of probiotics of Bacillus subtilis from corncob residue has not been reported. In this study, LA pretreatment was combined with enzymatic hydrolysis to produce XOS and monosaccharides from corncob. An XOS yield of 69.9% was obtained from corncob by combining 2% LA pretreatment and xylanase hydrolysis. Yields of 95.6% glucose and 54.0% xylose were obtained from corncob residue via cellulase, and the resulting cellulase hydrolysate was used to culture Bacillus subtilis YS01. The resulting viable count of the strain was 6.4×108 CFU/mL, and the glucose and xylose utilization were 99.0% and 89.8%, respectively. This study demonstrated a green, efficient, and mild process for producing XOS and probiotics from corncob by combining LA pretreatment and enzymatic hydrolysis.

Selective removal effect of subcritical fluid extraction on egg yolk lipids and characterization and enzymatic improvement of defatted egg yolk powder

This study investigated the effects of subcritical fluid extraction on lipid removal and the physicochemical properties of egg yolk powder. In addition, two-step enzymatic hydrolysis was used to improve the physicochemical properties. The content of total lipids and cholesterol decreased to 30.2% and 10.5 mg/g after subcritical propane-butane extraction, respectively, while phospholipids were retained. Protein solubility, surface hydrophobicity and total sulfhydryl results showed that this partial defatting avoided further quality deterioration of egg yolk powder. Scanning electron microscopy and confocal microscopy showed that the surface morphology and distribution of proteins and lipids changed with defatting degree. After two-step enzymatic hydrolysis, protein solubility of subcritical propane-butane extraction egg yolk powder significantly increased to 31.4% and soluble peptides with an average molecular weight lower than 1000 Da accounted for 70.0%. The improved defatted egg yolk powder is rich in protein, peptides and phospholipids and has great potential as a nutritional supplement.

Application of efficient pre‐treatment by physical fields for improving the taste and flavor of processed chicken enzymatic hydrolysate

In the condiment industry, people usually use enzymatic hydrolyzate to improve the umami of the product, and there are few reports on the umami intensity of the enzymatic hydrolysate. To explore the differences in taste and flavor of the samples pretreated with different physical fields, the chicken was pretreated by ultrasound (US, 600 W, 30 min), infrared radiation (IR, 90°C, 30 min), and radio frequency (RF, 120 mm, 30 min), respectively, and followed by a two-step enzymatic hydrolysis. Results revealed that the chicken enzymatic hydrolyzate after RF pretreatment (120 mm, 30 min) had the best overall taste and stronger umami. The sample treated with 90°C IR has a richer flavor, while there were no significant changes in taste of the 600 W ultrasound treatment group compared with the “control.” Therefore, RF pretreatment (120 mm, 30 min) is more suitable for industrial to produce low cost and high umami chicken enzymatic hydrolyzates. Practical applications In the condiment food industry, people usually use umami-rich enzymatic hydrolysates to improve the umami taste of products. The 120 mm plate spacing radio frequency pretreatment proposed in this study for 30 min before enzymolysis can significantly improve the taste characteristics of the chicken enzymolysate, significantly enhance its saltness and umami, and make the overall taste more harmonious. It provides a certain theoretical basis for the industrial production of low-cost umami-enhancing enzymatic hydrolysis products.

Ultrasonication for production of nanoliposomes with encapsulated soy protein concentrate hydrolysate: Process optimization, vesicle characteristics and in vitro digestion.

This study presents the state-of-art research about the assembly of soy proteins in nanocarriers, liposomes, and its design includes different physicochemical strategies and approaches: two-step enzymatic hydrolysis of soy concentrate, hydrolysate encapsulation by using phospholipids and cholesterol, and application of ultrasonication. Achieved results revealed that ultrasonication, together with cholesterol addition into phospholipid layers, improved the stability of nanoliposomes, and a maximum EE value of 60.5% was obtained. Average size of peptide-loaded nanoliposomes was found to be from 191.1 to 286.7nm, with a ζ potential of -25.5 to -34.6mV, and a polydispersity index of 0.250-0.390. Ultrasound-assisted encapsulation process did not lead to a decrease in the antioxidant activity of the trapped peptides. FTIR has indicated an effective hydrophobic interaction between phosphatidylcholine and hydrolysate peptides. TEM and SEM have confirmed the spherical nanocarrier structure and unilamelarity. Prolonged gastrointestinal release and stability of peptides have been enabled by liposome nanocarriers.

Purification and Identification of an ACE-Inhibitory Peptide from Gracilaria tenuistipitata Protein Hydrolysates

Edible marine species are valuable sources of bioactive peptides. This study investigated the ACE-inhibitory activity of protein hydrolysates from the red algae Gracilaria tenuistipitata. Fifteen groups of protein hydrolysates were prepared by a two-step enzymatic hydrolysis of G. tenuistipitata: initial hydrolysis with several glycolytic enzymes, followed by three separate proteolytic reactions (Alcalase, Neutrase and Flavourzyme) for 2–10 h. Results showed that the hydrolysate GTN4H had the highest ACE-inhibitory activity in vitro. Furthermore, oral administration of GTN4H significantly reduced systolic blood pressure in spontaneously hypertensive rats. Fraction A derived from GTN4H displayed the highest ACE-inhibitory activity among fractions. Further purification of fraction A by RP-HPLC obtained a purified peptide (MW: 1776 Da) with 17 amino acids and 95.4% ACE-inhibitory activity.

Free Radical Scavenging, Redox Balance and Wound Healing Activity of Bioactive Peptides Derived from Proteinase K-Assisted Hydrolysis of Hypophthalmichthys molitrix Skin Collagen.

Various protocols for enzymatic hydrolysis of fish by-products are increasingly tested to ensure value-added products with functional and biological properties important for food, cosmetic and medical applications. In addition, they attempt to minimize waste from industrial processing and environmental requirements. This study aims to establish an efficient protocol based on two-step enzymatic hydrolysis of freshwater fish skin and to evaluate the effect of resulting bioactive peptides on free radical scavenging, redox balance and regulation of fibroblast proliferation and migration. Pepsin-soluble collagen extracted from silver carp (Hypophthalmichthys molitrix) skin was hydrolyzed by proteinase K at specific sites under controlled conditions. The molecular mass of ultrafiltration permeate was determined by gradient electrophoresis and gel filtration chromatography. The biological activity of intermediate and small size bioactive peptides was evaluated in experimental models in vitro mimicking oxidative stress and skin wound conditions. Extracted fish collagen was hydrolysed using proteinase K, the most efficient enzyme for the cleavage of the primary structure of the molecule, as previously found in silico. Established optimal conditions increased the enzyme specificity and the process yield. Bioactive peptides exerted significantly higher scavenging activity on free stable radicals and hydroxyl radicals often found in vivo, compared to fish collagen. They stimulated fibroblast metabolism in a dose-dependent manner and up-regulated cell migration in a scratch wound model. Pretreatment of fibroblasts with induced oxidative stress using optimal concentrations of fish peptides prevented the increase of reactive oxygen species production. In conclusion, bioactive peptides from carp skin demonstrated valuable properties of maintaining redox balance and skin wound healing process improvement, which indicated further potential applications in the development of pharmaceutical and nutraceutical formulations. In this study the enzymatic hydrolysis was applied to isolated protein, in contrast to previous studies using waste tissue with variable composition. Recovered bioactive peptides acted not only as antioxidant agents, but also as regulators of oxidative stress and wound healing processes in skin cell models. Their nutritional and cosmetic application is recommended in novel formulations fighting skin ageing phenomena.

Effect of pretreatments on production of xylooligosaccharides and monosaccharides from corncob by a two-step hydrolysis

Xylan extraction by alkali is usually a prerequisite for traditional xylooligosaccharide (XOS) production from corncob. In this study, to avoid xylan isolation before XOS production, corncob was delignified by hydrogen peroxide-acetic acid (HPAA), followed by a two-step enzymatic hydrolysis for XOS and monosaccharide production. High lignin removal (55.5%–82.6%) was observed upon pretreatment with 75%–100% HPAA and using 50–100 mM H2SO4 as a catalyst. A high XOS yield of 27.8% was obtained from 75% HPAA-pretreated corncob with 75 mM H2SO4, and the xylose/XOS ratio was only 0.09, indicating that the purity of XOS was relatively high. The highest glucose yield (79.1%) was obtained from 100% HPAA-pretreated corncob with 50 mM H2SO4. Finally, 58.3 g of XOS, 186.9 g of glucose, and 56.4 g of xylose were obtained from 1 kg of corncob. This study provides a promising approach for XOS and monosaccharide production from corncob without xylan isolation.

Study on the Extracting Technology for Antioxidant Oligopeptides from Donkey Meat by Two-Step Enzymatic Hydrolysis

Study on the Extracting Technology for Antioxidant Oligopeptides from Donkey Meat by Two-Step Enzymatic Hydrolysis

Preparation of angiotensin I-converting enzyme (ACE) inhibitory peptides from Tie Guanyin tea residue protein using two-step enzymatic hydrolysis

In this study, we attempted to prepare novel angiotensin I-converting enzyme inhibitory (ACEI) peptides from Tie Guanyin tea residue protein (TTRP). Different proteases were used to hydrolyze TTRP to prepare ACEI peptides, neutral protease and alkaline protease hydrolysates exhibited higher ACEI activity and was chosen in further hydrolysis treatment. Response surface methodology (RSM) was used to optimize the enzymatic condition, and the results indicated that the optimal conditions of enzymatic hydrolysis were: 4% (w/v) of TTRP in water was hydrolyzed with 3500 U/g neutral protease at 50 °C and pH 6.5 for 2.5 h in the first step and then with 5000 U/g alkaline protease at 50 °C and pH 10.0 for 3 h in the second step. Under the optimum condition, the ACE inhibitory rate of the final products was 88.45% ± 0.45% at the concentration of 1.0 mg/mL. Moreover, ACEI peptides derived from TTRP showed good stability under different temperatures, pHs, metal ions and gastrointestinal digestion. These results indicated that the TTRP has potential to be used for preparing ACEI peptides.

Flavor Formation of a Novel Cream-Soy Protein Isolate Mixed Flavor by Fermentation in Combination with Two-Step Enzymatic Hydrolysis

Flavor Formation of a Novel Cream-Soy Protein Isolate Mixed Flavor by Fermentation in Combination with Two-Step Enzymatic Hydrolysis

Effect of two-step enzymatic hydrolysis on the antioxidant properties and proteomics of hydrolysates of milk protein concentrate

Food protein and peptides are generally considered a source of dietary antioxidants. The antioxidant activity and peptide profiles of four extensive hydrolysates of milk protein concentrate (MPC) were examined using the two-step enzymatic method. The hydrolysis combinations were Alcalase-Flavourzyme (AE), Alcalase-ProteAXH (AH), Alcalase-Protamex (AX) and Alcalase-Protease A 2SD (AD). The results showed that highest degree of hydrolysis corresponded to the AE sample (20.41%). High-efficiency gel-filtration chromatography results indicated that the relative proportions of extensive hydrolysates with molecular weights < 3 kDa were 99.89%, 99.57%, 99.93%, and 99.89% for AX, AE, AD and AH, respectively. The hydrolysates of the MPC exhibited increased radical-scavenging capacity, as evidenced through an analysis with 1,1-diphenyl-2-pycryl-hydrazyl (DPPH), 2,2-azinobis (3-ethylbenzothiazo-line-6-sulfonic acid) diammonium salt (ABTS), reducing power and hydroxyl-radical scavenging activity testing. The main bioactive peptides were identified through EASY-nLC-orbitrap MS/MS and bioinformatics. The study may provide useful information regarding the antioxidant properties of extensive hydrolysates of MPC.

Removal of phenylalanine from egg white powder: Two-step enzymatic method combined with activated carbon adsorption

Individuals suffering from phenylketonuria require foods containing low levels of phenylalanine (Phe). In this research, we therefore developed a two-step enzymatic hydrolysis method followed by activated carbon treatment to remove Phe from egg white powder (EWP). Nine proteases were tested for their ability to expose or release Phe residues from the egg proteins, while nine kinds of activated carbon were tested as an adsorptive medium for removing Phe residues. Our results showed that trypsin combined with flavourzyme released the most Phe residues, and No.4 activated carbon was most effective at removing Phe residues. The percentage of Phe removal varied from 91.71–97.78% and the final mass yield from 230.08–342.45 g low Phe peptide (LPP) / Kg of EWP. The LPP mixtures obtained mainly containing amino acids and oligopeptides (< 600 Da). The EWP-LPP mixture obtained therefore has the potential to prepare medical foods for patients with phenylketonuria.

Effect of sulfuric acid on production of xylooligosaccharides and monosaccharides from hydrogen peroxide-acetic acid-pretreated poplar

Lignin is one of the main obstacles for enzymatic hydrolysis, which can be selectively removed by hydrogen peroxide-acetic acid pretreatment (HPAC). In this work, the effects of sulfuric acid concentration on chemical composition, structural features, physical properties and enzymatic digestibility of HPAC pretreated poplar were investigated. The increased H2SO4 dosage enhanced the lignin removal of HPAC-pretreated poplar, resulting in the increased accessibility and decreased hydrophobicity. A satisfying glucose yield (91.84%) was obtained from HPAC pretreated poplar (100 mM H2SO4) at 5 FPU/g DM of cellulase loading with the addition of xylanase (30 U/g DM) and Tween 80 (3 g/L). The increment of H2SO4 concentration promoted the yield of xylooligosaccharides from 0.69% to 20.45% and monosaccharides from 5.76% to 92.89% respectively by two-step enzymatic hydrolysis. This work demonstrated that HPAC pretreatment played a critical role in efficient utilization of poplar carbohydrates by enzymatic hydrolysis.

Purification and identification of dual-enzyme hydrolysates obtained from defatted walnut and its antioxidant effects on d-galactose-induced aging mice

The purpose of this study was to explore the antioxidant activity in vivo of dual-enzyme hydrolysates obtained from defatted walnut, and to purify and identify it. The walnut protein hydrolysates (WPH) were obtained through two-step enzymatic hydrolysis of alkaline protease and trypsin. The effects of WPH on the oxidative stress-related biochemical indexes of d-galactose-treated mice were investigated. Next, WPH was passed through a Sephadex G-25 gel chromatography, anion exchange chromatography and Sephadex G-15 gel chromatography. Subsequently, peptides in the high antioxidant activity fraction were identified by RP-nano-HPLC–MS/MS. Finally, 14 potential peptides of length 11 AAs to 21 AAs with antioxidant activity were identified from WPH.

Preparation and characterization of a low-phenylalanine whey hydrolysate using two-step enzymatic hydrolysis and macroporous resin adsorption

A low-phenylalanine (Phe) diet is indispensable for phenylketonuria patients. The objectives of this study were to develop a two-step enzymatic method to effectively release Phe from whey protein, and then to prepare a low-Phe hydrolysate (LPH) by using adsorbent. Thermolysin (T) or Pancreatin (P) combined with Protease A2SD (A2) showed the degree of hydrolysis ranging from 42.62% to 45.43% and released more than 90% Phe from whey protein. The D101 macroporous resin column removed 98.38% Phe from TA2 hydrolysate (TA2H), compared to 64.50% of Phe removal by activated carbon. The protein content of LPH prepared by a magnified D101 resin column was 60.41 g/100 g hydrolysate and its Phe content was 1.38 ± 0.11 mg/g protein equivalent. After adsorption, the percentage of peptides below 600 Da significantly increased from 50.79% to 93.60%, while the intensity of bitterness and astringency measured by electronic-tongue also increased. However, aroma-active compounds such as Millard reaction and lipid oxidation products were reduced after absorption. The LPH prepared in our study could be a suitable protein substitute to make phenylketonuria food.