Enzymatic Hydrolysis Research Articles

Antioxidant Activity and DPP-IV Inhibitory Effect of Fish Protein Hydrolysates Obtained from High-Pressure Pretreated Mixture of Rainbow Trout (Oncorhynchus mykiss) and Atlantic Salmon (Salmo salar) Rest Raw Material

The use of fish rest raw material for the production of fish protein hydrolysates (FPH) through enzymatic hydrolysis has received significant interest in recent decades. Peptides derived from fish proteins are known for their enhanced bioactivity which is mainly influenced by their molecular weight. Studies have shown that novel technologies, such as high-pressure processing (HPP), can effectively modify protein structures leading to increased biological activity. This study investigated the effect of various HPP conditions on the molecular weight distribution, antioxidant activity, and dipeptidyl-peptidase IV (DPP-IV) inhibitory effect of FPH derived from a mixture of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) rest raw material. Six different treatments were applied to the samples before enzymatic hydrolysis; 200 MPa × 4 min, 200 MPa × 8 min, 400 MPa × 4 min, 400 MPa × 8 min, 600 MPa × 4 min, and 600 MPa × 8 min. The antioxidant and DPP-IV inhibitory effects of the extracted FPH were measured both in vitro and at cellular level utilizing human intestinal Caco-2 cells. The results indicated that low and moderate pressures (200 and 400 MPa) increased the proportion of larger peptides (2–5 kDa) in the obtained FPH, while treatment at 600 MPa × 4 min resulted in a higher proportion of smaller peptides (1–2 kDa). Furthermore, HPP led to the formation of peptides that demonstrated increased antioxidant activity in Caco-2 cells compared to the control, whereas their potential antidiabetic activity remained unaffected.

Urinary excretion profiles of the orexin receptor antagonist suvorexant and its metabolites.

Suvorexant is an orexin receptor antagonist used in the treatment of insomnia. In this study, we investigated the urinary excretion profiles of suvorexant and its major metabolites, including conjugates, to obtain fundamental information for proving exposure to suvorexant in criminal cases. Urine specimens were collected from three subjects for maximum 168h after a single oral ingestion of suvorexant (10mg), and suvorexant and its metabolites in urine were determined using liquid chromatography-tandem mass spectrometry with a C18 semi-micro column. The carboxylic and hydroxy metabolites (M4 and M9) were identified with authentic standards synthesized in our laboratory, and their glucuronides and other hydroxy metabolites (M8 and M10) were tentatively detected based on measured exact masses and product ion spectra of them. Suvorexant, M4 and M9 would be detectable for 20-34h, 6-7days and 42-61h after intake, respectively. The quantitative results demonstrated that the molar ratios of accumulated amounts of M4 and M9 including their glucuronides excreted in urine to dose ranged about 2.6-6.2% and 0.37-0.51%, respectively, while that of the unchanged parent was much lower (0.011-0.013%). The ratios of the amount of glucuronide to the total amount of M4 and M9 excreted in urine was less than 10% and approximately 90%, respectively. The urinary excretion profiles indicated that M4 and M9 would be effective indicators for proving suvorexant intake, and M4 could be detected until one week after intake even without enzymatic hydrolysis (limit of detection: 0.05ng/mL).

Valine potentiates cefoperazone-sulbactam to kill methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is possibly the most infamous example of antibiotic resistance and new antibiotics are urgently needed to control it. The present study used metabolic state-reprogramming approach to identify an ideal biomarker as an antibiotic adjuvant for reversing the metabolic state of MRSA. The most repressed valine was identified as the adjuvant. Exogenous valine most effectively potentiated cefoperazone-sulbactam (SCF) to kill MRSA in vitro and in vivo. Viability of 18 clinical MRSA isolates was reduced by the top 276.64-fold in the presence of valine and SCF. In mouse models, lower bacterial load in liver, spleen, kidney, thigh, and higher survival were determined in the SCF + valine than valine or SCF alone. Valine promoted MRSA to increase SCF uptake that overcomes the efflux and enzymatic hydrolysis. It also extended the PAE of SCF. These occur because valine activates the pyruvate cycle to elevate proton motive force by NADH and increases membrane permeability by lauric acid. Therefore, the combination of valine and SCF is a new drug candidate effective against MRSA.

Application of AprX from Pseudomonas paralactis for the improvement of the emulsifying properties of milk, plant and insect protein and estimation of their hydrolysate’s bitter potential

AbstractProtein properties can be modified by selective enzymatic hydrolysis. In this study, the alkaline metalloendopeptidase AprX (Serralysin; EC 3.4.24.40) from Pseudomonas paralactis was used for the tailored hydrolysis of different food proteins resulting in the production of protein hydrolysates with improved emulsifying properties. Sodium caseinate, wheat gluten and buffalo worm protein were used for AprX hydrolysis at 40 °C and pH 8 to cover a spectrum of different protein sources. A maximum degree of hydrolysis (DH) of 13.1 ± 0.2%, 14.2 ± 0.1% and 20.7 ± 0.1% was reached for sodium caseinate, wheat gluten and the worm protein, respectively. The corresponding hydrolysate properties were analyzed regarding their particle size, peptide composition, solubility, viscosity, surface hydrophobicity and interfacial tension. The emulsifying properties were investigated by the oil-droplet size, ζ-potential and stability of emulsions prepared from the hydrolysates. Using partially hydrolyzed sodium caseinate (DH = 10.6%) as an emulsifier lead to an eightfold increase of the emulsion stability (t1/2 = 180 ± 0 min) compared to unhydrolyzed sodium caseinate. The emulsion stability using wheat gluten hydrolysates (DH = 11.9%) was increased 30-fold (t1/2 = 45 ± 5 min). Simultaneously, the solubility of gluten was increased by 60%. Buffalo worm hydrolysates (DH = 14.6%) had a twofold (t1/2 = 85 ± 5 min) increased emulsion stability. In conclusion, AprX can be used to improve the solubility and emulsifying properties of food proteins at a relatively high DH.

Genomic Diversity and Virulence Factors of Clostridium perfringens Isolated from Healthy and Necrotic Enteritis-Affected Broiler Chicken Farms in Quebec Province

Avian necrotic enteritis due to the Gram-positive bacterium Clostridium perfringens has re-emerged following the ban on antibiotic growth promoters in many poultry producing countries. The limited number of previous studies has left important gaps in our understanding of the genetic diversity and virulence traits of the pathogen. To address these knowledge gaps, in this study, we sequenced the genomes of 41 Clostridium perfringens isolates recovered from commercial broiler chicken flocks in Quebec, Canada, including isolates from healthy birds and those affected by necrotic enteritis. We sought to understand the pangenome diversity and interrogated the genomes for key virulence factors involved in necrotic enteritis pathogenesis. On average, the genomes had a GC content of 28% and contained 3206 coding sequences. A variable presence of toxins, degradative hydrolytic enzymes, and collagen-binding proteins was also found. Through pangenome analysis, we revealed a total of 10,223 genes, 652 (6.4%) of which formed the core genome. Additionally, we identified 17 different plasmids, 12 antibiotic resistance genes, and nine prophage regions. Overall, our results demonstrated a relatively high genetic diversity among chicken Clostridium perfringens isolates collected from the same geographical location, offering new insights into potential virulence mechanisms and adaptation of the pathogen within poultry populations.

ENZYMATIC HYDROLYSIS OF STEAM EXPLODED STRAW WITH THE ADDITION OF ACETIC ACID

The effect of steam explosion on the enzymatic hydrolysis of straw was investigated in the presence of 5, 10, 15 and 20% wt. addition of acetic acid. Analysis was performed at temperatures of 160, 170, 180, 190, 200 and 210°C. The concentration of monosaccharides obtained after enzymatic hydrolysis was considered the main indicator of the increased availability of cellulose due to their release into the solution. The results indicate that the addition of acetic acid increases the concentration of monosaccharides, but only at lower temperatures. The temperature of 180°C corresponded to the most effective pretreatment by steam explosion in the presence of acetic acid with the highest concentration of 10%, which corresponds to the conversion of polysaccharides to monosaccharides of 74.78%. At high temperatures above 200°C, the addition of acetic acid results in a decrease in the concentration of monosaccharides due to the high severity factor in the range of 3.94 – 4.24.

Enzymatic Hydrolysis of Residual Soybean Oil with Simultaneous Separation Using Membrane

AbstractThis study investigates the simultaneous separation process using a batch reactor coupled to a cellulose acetate membrane for the enzymatic hydrolysis of residual soybean oil. The membrane was manufactured with 30 s pre‐evaporation time and 1‐mm thickness. Their morphologies and wettability were analyzed. Pichia pastoris lipase was characterized based on temperature and pH and was applied to the hydrolysis of residual soybean oil at 45 °C and pH 7 in batches, with and without simultaneous separation. The results show that the process efficiency with the cellulose acetate membrane resulted in 55.58 % ± 2.78 % free fatty acids in enzymatic hydrolysis, compared with 39.72 % ± 1.99 % without the membrane. The membrane also achieved 100 % oil rejection.

Exopolygalacturonase Production from the Novel Strain Lichtheimia sp. UV-16 and Enzyme Hydrolysis Properties.

A pectinase-producing strain, Lichtheimia sp. X-8, was isolated from the soil for the first time. Subsequently, Lichtheimia sp. UV-16, with a 1.23-fold increase in pectinase activity, was obtained via UV mutagenesis, and optimization of its liquid fermentation process boosted pectinase activity from 455.6 ± 12.7 to 3202.0 ± 82.1 U/mL. The crude enzyme was purified by salting out and anion exchange resin, with a purification ratio of 2.28-fold and a yield of 36.5%. The optimal reaction temperature for the pure enzyme was 60 °C with an optimal pH of 5.5. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) confirmed that the enzyme was an exopolygalacturonase, achieving over 99% efficiency in pectin hydrolysis. Furthermore, incorporating pure enzymes into juice pulps can substantially enhance the juice yield, which makes this polygalacturonase a promising application in the beverage industry.

Discovery and Characterization of Mannan-Specialized GH5 Endo-1,4-β-mannanases: a Strategy for Açaí (Euterpe oleracea Mart.) Seeds Upgrading.

The pulp of açaí palm fruits (Euterpe oleracea Mart.) is a valuable export commodity in Brazil. Its production generates 1.6 million tons/year of açaí seeds, a resource largely wasted. The seeds consist mainly of linear β-mannan, offering potential for prebiotic β-mannan-derived oligomers and mannose production. However, the crystalline structures of β-mannan hinder enzymatic hydrolysis. This study aimed to discover and characterize fungal enzymes targeting açaí seed β-mannan using a palm β-mannanase (EgMan5A) as a guide. Recombinant expression, enzyme optimization, kinetics, substrate specificity, and structural modeling were performed. The two fungal enzymes, JaMan5A and SlMan5A, were found to be specific for unsubstituted mannan, showing no activity toward galacto- and glucomannan. Among them, SlMan5A showed the highest activity on açaí seed β-mannan (∼24 U/mg) and other unsubstituted mannan substrates, likely due to its greater thermal robustness. These results provide valuable insights into β-mannan specificity contributing to the sustainable valorization of açaí seeds.

Study on the Effects of Ginger Extract on the Degree of Hydrolysis, Sensory Evaluation, Color Difference, and Volatile Flavor Components of Oyster Enzymatic Hydrolysate

Study on the Effects of Ginger Extract on the Degree of Hydrolysis, Sensory Evaluation, Color Difference, and Volatile Flavor Components of Oyster Enzymatic Hydrolysate

Optimization of Hydrolysis Conditions and Bio-functional Properties of Whey Protein Hydrolysate Obtained from Ash Gourd (Benincasa hispida) Protease

Background: Enzymatic hydrolysis alters the bio-functionality of whey proteins by generating bioactive peptides. Plant serine proteases as an alternative to animal and microbial proteases, recently considered useful for whey protein hydrolysate preparation to enhance nutritional and bio-functional properties. Objectives: The present study thus aims at optimization of whey protein hydrolyzing conditions employing ash gourd protease (AGP) and evaluation of 2, 2 diphenyl -1-picrylhydrazyl (DPPH) radical scavenging (antioxidant) and angiotensin I- converting enzyme (ACE) inhibition activities and potency of the generated hydrolysate. Methods: Optimization was accomplished using response surface methodology and generating treatment combinations applying central composite design in two steps: first temperature (50-90°C) and pH (6-9) was optimized adding same concentration of AGP (0.3% v/v) at reconstituted WPC (0.6% protein) and then E/S ratio (2-6 %v/v) and hydrolysis time (1-6h) was optimized at reconstituted WPC (4% protein). The effect of hydrolysis time at optimized condition was also evaluated with response to degree of hydrolysis, antioxidant and ACE inhibition activities of the generated hydrolysate and its ultra-centrifuged fractions ≤3kDa and >3kDa. The AGP (Adjusted to activity 2U/mL) obtained after partially purification by three phase partitioning method was used for hydrolysis experiments. Results: The optimum conditions for enzymatic hydrolysis of whey protein to achieve maximum degree of hydrolysis (13.99%) and DPPH radical scavenging activity (23.05%) were observed at 7.48 and 66.40°C. Similarly, the enzyme performed optimally at enzyme by substrate ratio of 5.85% (v/v) for 6 hours of hydrolysis, providing 19.31 % of degree of hydrolysis and 47.71 % of DPPH radical scavenging activity. AGP hydrolyzed whey protein compared to un-hydrolysed whey protein had significantly higher degree of hydrolysis, DPPH radical scavenging and ACE inhibition activity. The lower molecular weight peptides fractions ≤3kDa was found to be more effective (1.45 times) than the peptide fractions >3kDa however was less effective (1.19 times) than the whole hydrolysate regarding antioxidant activity. Similarly, ≤3kDa fraction of hydrolysate had 1.9 times higher ACE inhibition potential as compared to its whole hydrolysate. Conclusion: Overall, the study showed that the AGP employed whey protein hydrolysate has potential for use as a natural antioxidant and ACE inhibition agent.

Biocontrol potential of natamycin-producing Streptomyces lydicus JCK-6019 against soil-borne fungal diseases of cucumber and characterization of its biocontrol mechanism.

Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG-4 are the two most important fungal pathogens causing soil-borne fungal diseases of cucumber; they are difficult to control and cause serious economic losses. Given the detrimental effects of the indiscriminate use of chemical fungicides, biocontrol emerges as an efficient and ecofriendly alternative for managing soil-borne fungal diseases. Streptomyces lydicus JCK-6019 (hereafter, JCK-6019) was isolated from rhizosphere soil. Its fermentation filtrate and volatile organic compounds exhibited broad-spectrum antifungal activity against various phytopathogenic fungi and oomycetes. JCK-6019 produced natamycin as an agar-diffusible antifungal metabolite. It also produced indole-3-acetic acid and various hydrolytic enzymes. In vivo experiments revealed that a ten-fold-diluted optimized JCK-6019 fermentation broth exhibited 100% control efficiency against cucumber damping-off disease and 62.5% control efficiency against cucumber Fusarium wilt disease. Pretreatment of cucumber seedlings with 1000-fold-diluted optimized JCK-6019 fermentation broth resulted in 68.18% and 23.91% disease control values against cucumber damping-off and Fusarium wilt disease, respectively. Moreover, peroxidase activity in cucumbers after 1 day of treatment was 1.5-fold higher than that in the control. Similarly, polyphenol oxidase activity in cucumbers after 3 days of treatment was 2.34-fold higher than that in the control, indicating that JCK-6019 can induce plant resistance. The natamycin-producing strain JCK-6019 could effectively suppress the development of cucumber Fusarium wilt and damping-off disease by inducing plant resistance and producing antifungal metabolites, including natamycin and volatile organic compounds. Thus, JCK-6019 possesses high potential for application in the development of biocontrol agents against soil-borne fungal diseases of cucumber. © 2024 Society of Chemical Industry.

Enhanced Saccharification Efficiency of Corn Straw by Laccase‐Cu2O Under Light/Dark Cycles

To strengthen the clean utilization of biomass waste, laccase modified cuprous oxide (Cu2O) composite catalyst is synthesized. The effects of catalyst type, catalyst concentration, pH, sunlight exposure time, and lighting method on the reducing sugar yield are investigated. The composite catalyst in combination with sunlight irradiation can effectively enhance the reducing sugar yield of corn straw. The optimum conditions were pretreatment time of 55 min, catalyst concentration of 58 mg L−1, pH of 5.5, enzyme hydrolysis process using a 30 DL (namely 20 min of dark/20 min of light alternating cycle for 30 h) scheme, and enzyme loading of 25 FPU g−1. Validation experiments show that the lignin removal percentage can reach 95.63%, and the reducing sugar yield can reach 124.79 mg g−1 under the optimum condition. The pretreatment and saccharification of straw executed twice are most advantageous for sugar production. Laccase‐Cu2O can be reused three times.

Semi-Continuous Microwave-Assisted Oil Extraction and Enzymatic Hydrolysis of Butia Odorata Seeds: Efficient Recovery of Oil and Fermentable Sugars in a Sequential Method

Semi-Continuous Microwave-Assisted Oil Extraction and Enzymatic Hydrolysis of Butia Odorata Seeds: Efficient Recovery of Oil and Fermentable Sugars in a Sequential Method

Enhanced enzymatic hydrolysis of Bermuda grass to improve monosaccharide yield for biofuel production using onsite-produced cellulase cocktails

Enhanced enzymatic hydrolysis of Bermuda grass to improve monosaccharide yield for biofuel production using onsite-produced cellulase cocktails

Discovery and Optimization of Dihydroquinolin-2(1H)-ones as Novel Highly Selective and Orally Bioavailable Phosphodiesterase 5 Inhibitors for the Treatment of Pulmonary Arterial Hypertension.

Phosphodiesterase 5 (PDE5) is a cGMP-specific hydrolytic enzyme and widely distributed in versatile tissues. PDE5 has been identified as a valid therapeutic target for treating erectile dysfunction and pulmonary arterial hypertension (PAH). Herein, a hit-to-lead structural optimizations were performed on the PDE1 inhibitor 10c, leading to compound 14b possessing great potency against PDE5A (IC50 = 3 nM) with high selectivity over PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, PDE9, PDE10, and PDE11 by more than 1125-fold, and remarkable safety properties. Furthermore, oral administration of 14b (5.0 mg/kg) exerted much better pharmacodynamics effects on both mPAP (mean pulmonary artery pressure) and RVHI (index of right ventricle hypertrophy) than sildenafil citrate (10.0 mg/kg) in a monocrotaline-induced PAH rat model. Overall, these results proposed a novel highly selective PDE5 inhibitor 14b which could serve as a potential candidate for treatment of PAH.

Sulfite Pretreatment Enhances Tobacco Stalk Deconstruction for Cellulose Saccharification and Lignin Pyrolysis

Sulfite-catalyzed acid pretreatment to overcome the inherent recalcitrance of biomass offers a significant advantage in terms of obtaining high glucose conversion. However, the residual lignin after enzymatic hydrolysis has not been fully exploited. Herein, this study introduced a joint approach using sulfite-catalyzed acid pretreatment (SPROL) and pyrolysis to upgrade tobacco stalk to produce fermentable sugar, and the resulting lignin is used to produce bio-oil and bio-char. The results suggest that SPROL pretreated tobacco stalk yields a high cellulose-based glucose selectivity of 75.9% with 15 FPU/g substrate enzyme dosage at 50 °C after 72 h of enzymolysis. Lignin characterization reveals that sulfonation occurred during SPROL pretreatment, and as the dosage of sulfonating agent increased, the thermal stability of the residue lignin decreased. After sample pyrolysis at 600 °C for 30 min, approximately 22%, 33%, and 45% of the lignin undergoes conversion into bio-oil, bio-char, and gas products, respectively. The bio-oil analysis results demonstrated that acetic acid is the most abundant identified GC-MS component at around 69.91% at the optimal condition, which implied that it could be of high value when utilized for pyroligneous acid. This research provides a synthetic approach using the SPORL technique to process tobacco stalk into fermentable sugar, bio-oil, and bio-char, which is significant for the commercial utilization of agricultural waste into value-added products.

Development and study of the nutritional value and storage stability of a soft cottage cheese product enriched with collagen and antioxidant-rich plant extracts

This study investigates the development of a novel soft cottage cheese product enriched with collagen concentrate from poultry processing by-products and antioxidant-rich plant extracts for the adaptive nutrition of athletes. Collagen concentrate was obtained from chicken skin, bone tissue, and feet through enzymatic hydrolysis and freeze-drying. Antioxidant-rich extracts were prepared from sea buckthorn and cinnamon rosehip using ethanol extraction. The plant extract demonstrated high antioxidant potential, containing 1.98% phenolic compounds, 29.8 mg/100g vitamin A, 48.9 mg/100g vitamin E, and 756.4 mg/100g vitamin C. The antioxidant extract demonstrated significant immune-boosting effects in experimental rats by enhancing lymphocyte and T-cell counts. Various ratios of collagen concentrate and plant extract were tested in the cottage cheese product. Optimal water-holding capacity and effective viscosity were achieved with a 6:4 or 8:4 collagen-to-extract ratio, balancing collagen’s gelation properties with the antioxidant benefits. The addition of 8% dry collagen concentrate and 4% sea buckthorn and rosehip extract resulted in an enhanced nutritional profile, particularly through increased polyunsaturated fatty acids (Omega-3 and Omega-6), vitamins A, C, E, and essential minerals like calcium, phosphorus, and magnesium. Storage stability studies indicated optimal preservation of product structure at 0-2°C for up to 96 hours, maintaining a viscosity loss coefficient between 15.0-15.8%. This enhanced soft cottage cheese product demonstrates improved nutritional profiles and antioxidant properties while maintaining structural stability, making it a promising functional food for athletes and health-conscious consumers.

Bovine Lung Peptides after Trypsinolysis Reveal Anti-Exudative Activity

Abstract Enzymatic hydrolysates of proteins isolated from cow lungs are used as anti-inflammatory agents for the growth and regeneration of new tissues. Hydrolysates were observed to inhibit the production of free radicals in cells and have a high anti-exudative activity even in low doses. In this study, we established the anti-exudative activity of peptides of protein hydrolysates isolated from bovine lungs. The anti-exudative activity of the peptides sum was compared with acetylsalicylic acid (ASA) and prednisolone, and the dynamics of inflammation were studied by tumour formation using carrageenan and formalin. Between 1 h and 24 h after administration of the drug, the effect of the peptides sum showed a significantly better indicator than the rest of the means. In general, the anti-exudative activity of the peptides sum was 2–3 times higher than the rest of the preparations. Biochemical indicators of prednisolone and peptides sum in two doses of 10−6 μg/kg and 10−4 μg/kg were studied. As a result of the study, it was found that the peptides sum at 10−6 μg/kg dose enhanced alanine aminotransferase (ALT) activity by 17.1% compared to those resulting from prednisolone treatment. In C-reactive protein (CRP) and antistreptolysin O (ASO) indicators, 10−4 μg/kg dose showed high activity. The sum of isolated peptides reduced ALT and ASO levels. This study provides additional support for preparing anti-inflammatory means from the peptides sum isolated from bovine lungs.

Simultaneous Recovery of Vivianite and Humic Acids from Waste Activated Sludge via Ferric Trichloride Flocculation and Enzymatic Hydrolysis Co-Treatment

Synchronously recovering phosphorus as vivianite and humic acids (HAs) from waste activated sludge (WAS) is of great significance for the carbon neutralization of wastewater. In this study, flocculation, enzyme degradation (lysozyme/protease/amylase/cellulase in a 1:1:1:1 ratio), and pH adjustment were used to reclaim vivianite and HAs. After FeCl3 coagulation–precipitation and enzymatic hydrolysis of the sludge for 11 h, the supernatant was enriched with Fe2+ and PO43−, with the molar ratio of Fe2+:PO43− of 2.21. To improve the purity of the vivianite, the crude protein was separated at pI 6.0. The purity of the crystals reached a peak of 97.44 ± 0.04% at pH 7.5. HAs extracted from the residuals had a high affinity for metal adsorption, and the adsorption process was both endothermic and efficient. Overall, this study demonstrates the feasibility and effectiveness of the joint reclaiming of vivianite and HAs, providing new insights into multiple resource recovery from WAS.