Enzymatic Hydrolysis Temperature Research Articles

A novel ultrasound-assisted enzyme extraction method of total flavonoids from Viticis Fructus and processed Viticis Fructus: Comparison of in vitro antioxidant activity

In this study, it is the first that the Viticis Fructus (VF) was used as the raw material for extracting total flavonoids using the ultrasound-assisted enzyme extraction (UAE) method. Response surface methodology was employed to determine the optimal extraction parameters. The optimal conditions were as follows: 60 % ethanol solution as the extract solvent, material–liquid ratio of 1:25, pH value of 4, enzyme addition amount of 1.5 %, enzymatic hydrolysis time of 30 min, enzymatic hydrolysis temperature of 40 ℃, and ultrasonic time of 50 min. Comparing the total flavonoid yield of VF and processed VF (PVF) extracted using different methods, it was observed that UAE resulted in a higher total flavonoid yield compared to traditional ultrasound extraction and enzyme extraction. Additionally, the total flavonoid yield of PVF extracted by all three methods was generally higher than that of VF. The PVF solution extracted by UAE also demonstrated better in vitro antioxidant activity compared to VF. These results suggest that UAE is an effective method to enhance the activity of natural total flavonoids. The study of the physicochemical properties and in vitro antioxidant activity of VF and PVF showed that the total flavonoid yield and antioxidant activity significantly increased after VF stir-frying, indicating that their efficacy can also be enhanced.

A novel tyrosinase inhibitory peptide obtained from Sipunculus nudus gelatin hydrolysate: Preparation, identification, and action mechanism

Tyrosinase inhibitory peptides (TIPs) derived from food resources have attracted immense attention in the food, cosmetic, and pharmaceutical industries owing to their excellent biological safety and ease of absorption. In this study, single-factor experiment and response surface optimization were used to prepare the Sipunculus nudus gelatin enzymatic hydrolysis (SNGH) product. Subsequently, a novel tyrosinase inhibitory peptide was screened out through ultrafiltration classification, biological activity prediction, molecular docking, and molecular dynamics simulation, and its inhibitory mechanism was analyzed. The results revealed that the optimal enzymatic hydrolysis process was: the enzyme addition was 4768.87 U/g, the enzymatic hydrolysis time was 4.72 h, and the enzymatic hydrolysis temperature was 42.76 °C. A total of 287 peptides were detected in the ultrafiltration component Ⅰ (SNGH-Ⅰ) with a high tyrosinase inhibitory activity, and 161 peptides with binding energies < −7.0 kcal/mol were screened out through molecular docking. Subsequently, 91 peptides with a biological activity score >0.3 were obtained after biological activity screening. Finally, 5 possible TIPs were screened out according to their hydrophobicity, peptide score, and molecular weight. Among them, IIAPPERKY, VWDESFKVF, and FAGDDAPRAVFPS exhibited a superior tyrosinase inhibitory activity. Molecular dynamics simulations revealed that IIAPPERKY had better conformational stability and belonged to the group of reversible competitive inhibitors.

Optimization of extraction of crude polysaccharide from Cassia seed via ultrasonic assisted cellulase and study on its antioxidant activity

In order to improve the current low extraction rate of cassia polysaccharide and improve its extraction efficiency, the ultrasonic wave assisted cellulase extraction method of cassia polysaccharide is first proposed and optimized. Orthogonal test is conducted to analyze the influence of six factors related to cellulase and ultrasonic wave on the extraction of cassia polysaccharide, and at the same time to explore the antioxidant activity of the extracted polysaccharide in vitro. The results show that the main and secondary factors affecting the extraction rate of cassia polysaccharide are pH &gt; ultrasonic power &gt; enzyme dosage &gt; enzymatic hydrolysis temperature &gt; enzymatic hydrolysis time &gt; ultrasonic time. The optimal conditions are: enzyme dosage: 1.6%, enzymatic hydrolysis time: 80 min, enzymatic hydrolysis temperature: 52 ℃, enzymatic hydrolysis pH: 6.4, ultrasonic time: 30 min, ultrasonic power: 276 W, the polysaccharide yield under the optimal conditions is 12.29%, which is 5.31% and 14.43% higher than the single enzyme extraction method and ultrasonic extraction method reported in the literature, respectively, indicating that the composite extraction method is superior to the single extraction method. The antioxidant activity analysis shows that the half inhibitory concentrations of cassia polysaccharide against DPPH·, ABTS·+, ·OH, ·O2- free radicals are 0.894 mg/mL, 1.175 mg/mL, 1.908 mg/mL and 7.601 mg/mL, respectively, which shows good antioxidant activity in vitro. Therefore, ultrasonic assisted cellulase extraction of crude polysaccharide from cassia seed is a new efficient and rapid extraction method.

Preparation of chickpea peptide-calcium chelates with antioxidant activity

Chelation of antioxidant chickpea peptides with calcium can improve the absorption rate of calcium in human intestinal tract. In the present work, the optimum enzymatic hydrolysis scheme was determined by a single-factor test with an orthogonal experimental design with the following conditions: material-liquid ratio, 1:15; enzymatic hydrolysis time, 10 min; enzyme-substrate ratio, 1:20 (papain protease); and enzymatic hydrolysis temperature, 60°C. The protein hydrolysate obtained under these conditions had high antioxidant activity. The free radical scavenging rates of •OH, O2-•, DPPH•, and H2O2 were 49.44, 63.64, 66.57, and 57.64%, respectively. The reducing power was 0.75. The optimum conditions for the preparation of calcium chelate from chickpea peptides were determined by an orthogonal optimisation test: the peptide-calcium ratio, 7:1; the chelation temperature, 50°C; the initial pH of the reaction solution, 7.0; the chelation time, 40 min; and the chelation rate, 39.95%. Antioxidant chickpea peptide-calcium chelates could have the potential to induce synergistic physiological effects.

Development and Identification of Novel α-Glucosidase Inhibitory Peptides from Mulberry Leaves.

The mulberry leaf is a botanical resource that possesses a substantial quantity of protein. In this study, alcalase hydrolysis conditions of mulberry leaf protein were optimized using the response surface method. The results showed that the optimum conditions were as follows: substrate protein concentration was 0.5% (w/v), enzymatic hydrolysis temperature was 53.0 °C, enzymatic hydrolysis time was 4.7 h, enzyme amount was 17,800 U/g, and pH was 10.5. Then mulberry leaf peptides were separated by ultrafiltration according to molecular weight. Peptides (<3 kDa) were screened and subsequently identified using LC-MS/MS after the evaluation of α-glucosidase inhibition across various fractions. Three novel potential bioactive peptides RWPFFAFM (1101.32 Da), AAGRLPGY (803.91 Da), and VVRDFHNA (957.04 Da) with the lowest average docking energy were screened for molecular dynamics simulation to examine their binding stability with enzymes in a 37 °C simulated human environment. Finally, they were prepared by solid phase synthesis for in vitro verification. The former two peptides exhibited better IC50 values (1.299 mM and 1.319 mM, respectively). These results suggest that the α-glucosidase inhibitory peptides from mulberry leaf protein are potential functional foods or drugs for diabetes treatment, but further in vivo studies are needed to identify the bioavailability and toxicity.

Effect of Peanut Protein Treated with Alkaline Protease and Flavorzyme on BALB/c Mice.

This article aims to analyze the effects of enzyme treatment concentration, temperature, and time on peanut protein so as to obtain an optimal enzymatic hydrolysis condition for flavorzyme (Fla) and alkaline protease (Alk). The results were as follows: enzymatic hydrolysis temperature 60 °C and 55 °C, enzyme concentration 10% and 4%, enzymatic hydrolysis time 80 min and 60 min, and double enzyme hydrolysis ratio 2% Fla + 5% Alk, respectively. The BALB/c mice were sensitized with gavage of peanut protein before and after enzyme treatment to evaluate the effects of different enzyme treatments on peanut allergenicity. Compared with the mice sensitized with raw peanuts, the weight growth rate of the mice sensitized with enzyme treatment peanut increased but not as much as the control, the degranulation degree of mast cell and basophils decreased, the inflammatory infiltration and congestion in jejunum and lung tissue decreased, the expression of proinflammatory factors and thymic stromal lymphopoietin (TSLP) gene decreased, and the secretion of specific antibodies (IgE, and IgG) decreased, and the binding ability of peanut protein with peanut-specific IgE antibodies decreased as well. The results above indicate that the allergenicity of peanut protein decreases after enzyme treatment and the dual enzyme (Fla + Alk) treatment can be much more efficient.

Screening of Ultraviolet-Induced Thermotolerant Yeast Mutants and Their Performance

The simultaneous saccharification and fermentation (SSF) technique holds promise for the conversion of lignocellulose to ethanol. However, the optimal fermentation temperature of yeast is lower than the enzymatic hydrolysis temperature of the saccharification process, which leads to the temperature of the actual production process of SSF usually being lower than 38 °C. In this work, two ultraviolet (UV)-induced mutations were performed step by step using Saccharomyces cerevisiae BY4742 as the original strain to enable the yeast to perform well at higher temperatures. Thermotolerant strains obtained through mutagenesis and screening, YUV1-1 and YUV2-2, were utilized for fermentation and SSF at a targeted temperature of 40 °C. They obtained ethanol yields comparable to those at 38 °C in SSF, whereas the ethanol yields of the original strain at 40 °C decreased by about 10% compared to those at 38 °C. This study proves that thermotolerant strains adapted to elevated fermentation and SSF temperatures can be obtained through UV mutagenesis and screening, thereby increasing the stability of the fermentation and SSF processes and lowering the subsequent distillation costs.

Milk Thistle Oil Extracted by Enzyme-Mediated Assisted Solvent Extraction Compared with n-Hexane and Cold-Pressed Extraction.

Silymarin and milk thistle oil have unique biological benefits; however, applying silymarin to milk thistle oil remains a challenge. In this research, the content of silymarin in milk thistle oil conditions using enzyme-mediated solvent extraction was investigated and optimized by response surface methodology. The optimal extraction conditions using enzyme-mediated solvent extraction were as follows: the enzyme-added content was 3.06 mg/mL, the enzymatic hydrolysis temperature was 55.09 °C, and the enzymatic hydrolysis time was 66.28 min. Oil extracted by the enzyme-mediated assisted solvent was further compared with those extracted with n-hexane and cold pressing. Results indicated that the oil extraction using the enzyme-mediated assisted solvent had a lower acid value (2.20 ± 0.01 mg/g) and the highest α-tocopherol content (0.62 ± 0.00 mg/g), total phenols (7.67 ± 0.01 mg/g), and flavonoids (1.06 ± 0.13 mg/g). Furthermore, the antioxidant capacity of milk thistle oils was further investigated. The results showed that the enzyme-mediated assisted solvent-extracted oil had the strongest antioxidant capacity with lower lipid oxide content. Therefore, enzyme-mediated solvent extraction is an excellent method for extracting milk thistle oil.

Synthesis of activated carbon nanofibers by bio-enzymatic method as electrode material for supercapacitors

In this work, the activated carbon nanofibers were prepared by hydrolysis of nanofibers mat using bio-enzymatic method. The nanofiber mat was hydrolyzed by medium temperature α-amylase, and carbonized to obtain activated carbon nanofibers. The results indicated that CNFs had good fiber structure, small electrolyte diffusion resistance, rich pore structure, as well as large specific surface area (268.09 m2/g). Furthermore, the effects of enzymatic hydrolysis temperature, enzyme dosage and enzymatic hydrolysis time on the mass loss rate of CNFs were investigated. The CNFs achieved the best electrochemical performance when the amount of β-cyclodextrin was 20 wt%. The specific capacitance of CNFs could reach 213 F/g under current density of 0.5 A/g. Also, the capacitance retention rate of CNFs could reach 94.31 % after 5000 cycling times under current density of 2 A/g.

Study on the Effect of Different Compound Proportion of Oyster and Mantis Shrimp on the Quality of Flavor Condiments

Oyster and mantis shrimp were used as raw materials to investigate the effects of their compound ratio on the degree of hydrolysis, sensory and color difference, and the volatile flavor components of the best enzymatic hydrolysis products were analyzed. The results showed that: according to the sensory evaluation score, the best compound ratio of oyster and mantis shrimp was 5:1.Under the conditions of enzymatic hydrolysis time of 2h, enzymatic hydrolysis temperature of 55℃, ratio of trypsin to flavourzyme of 3:1, addition amount of 0.7% and pH of 8.0, the umami taste score and degree of hydrolysis of enzymatic hydrolysis products were 5.71 and 28.22% respectively. In the determination of volatile flavor components, 16 volatile flavor components were detected, mainly including 2 alcohols (34.41%), 6 aldehydes (14.64%), 1 ketone (0.31%), 2 esters (0.69%) and 5 alkanes (2.53%).

A two-stage heating process with low energy consumption for treatment of preliminary desalinated impregnation effluent to produce xylose

Production of xylose from preliminary desalinated impregnation effluent (PDSIE) containing hemicellulose is an economical way of the effluent treatment. However, due to the high alkali and salt contents in PDSIE, its resource utilization is limited in the developed treatment process for pulp-impregnated effluent that has been deeply desalted. In this study, based on analysis of reaction rate and kinetic characteristics of pre-hydrolysis, a two-stage pre-hydrolysis heating process including heating at a high temperature for a short time followed by slow cooling to enzymatic hydrolysis temperature (HCP) with low energy consumption was developed for PDSIE. The heating time was shortened from 2 h to 0.5 h, the inhibition of conversion from hemicellulose to xylose by the high alkali and salt contents in PDSIE was alleviated, and the loss of xylose was reduced by HCP, compared to those under the constant temperature heating conditions. The heating cost based on coal consumption was reduced by 75.54% in HCP according to the preliminary economic analysis. Based on xylose yield and energy-saving results, the temperature in the first stage of HCP was determined to be 120 °C, and the cooling time in the second stage was determined to be 6 h.

Optimization of Enzymatic Preparation Process of Hypoglycemic Peptide from Moringa oleifera Seeds by Response Surface Methodology and Its Hypoglycemic Activities Evaluation in Vitro

In order to improve the resource utilization of Moringa oleifera seeds powder, enzymatic method was used to prepare hypoglycemic peptide from Moringa oleifera seeds. Taking the the degree of proteolysis and α-glucosidase inhibition rate as the response value, single-factor experimental screening and response surface method was used to optimize the optimum enzymatic hydrolysis time, liquid-to-material ratio, hydrolysis pH, enzyme addition, and enzymatic hydrolysis temperature. The hypoglycemic peptide with a molecular weight of <3 kDa were prepared by coarse separation by ultrafiltration, and their inhibitory effects on human liver cancer cells (HepG2 cells) were analyzed by MTT method. The results showed that the optimal conditions of enzymatic hydrolysis were as follows: Time 4.6 h, ratio of liquid to material 40.5:1, pH8.3, enzyme dosage 5.5%, temperature 55 ℃, and the inhibition rate of α-glucosidase was 23.62%±0.14%. The α-glucosidase inhibition rate of the ultrafiltration fraction with a molecular weight of less than 3 kDa had an IC50 value of 5.56 mg/mL. When the concentration of ultrafiltration fraction was 300 μg/mL, the proliferation of HepG2 cells was significantly inhibited after 48 h(P<0.05). The research can lay the foundation for the further separation and purification of hypoglycemic peptide from Moringa oleifera seeds.

A temperature-induced chitosanase bacterial cell-surface display system for the efficient production of chitooligosaccharides.

To establish a temperature-induced chitosanase bacterial cell-surface display system to produce chitooligosaccharides (COSs) efficiently for industrial applications. Temperature-inducible chitosanase CSN46A bacterial surface display systems containing one or two copies of ice nucleation protein (InaQ-N) as anchoring motifs were successfully constructed on the basis of Escherichia coli and named as InaQ-N-CSN46A (1 copy) and 2InaQ-N-CSN46A (2 copies). The specific enzyme activity of 2InaQ-N-CSN46A reached 761.34 ± 0.78 U/g cell dry weight, which was 45.6% higher than that of InaQ-N-CSN46A. However, few proteins were detected in the 2InaQ-N-CSN46A hydrolysis system. Therefore, 2InaQ-N-CSN46A had higher hydrolysis efficiency and stability than InaQ-N-CSN46A. Gel permeation chromatography revealed that under the optimum enzymatic hydrolysis temperature, the final products were mainly chitobiose and chitotriose. Chitopentaose accumulated (77.62%) when the hydrolysis temperature reached 60 °C. FTIR and NMR analysis demonstrated that the structures of the two hydrolysis products were consistent with those of COSs. In this study, chitosanase was expressed on the surfaces of E. coli by increasing the induction temperature, and chitosan was hydrolysed directly without enzyme purification steps. This study provides a novel strategy for industrial COS production.

Optimization and Quality Analysis of Different Extraction Methods of Palm Seed Oil

The extraction process of palm seed oil was optimized. Using palm seed as raw material, oil extraction rate was used as an index. The effects of flash extraction, ultrasonic-assisted extraction, supercritical extraction, and aqueous enzymatic extraction on the yield of palm seed oil were investigated. The extraction methods and technological conditions of palm seed oil were optimized by the orthogonal method on the basis of single factor. The seed oil was analyzed and detected. The results showed that the water enzymatic extraction method was the best, and the optimal extraction conditions were as follows: enzymatic hydrolysis time 16 h, enzymatic hydrolysis temperature 50°C, and enzymatic content 2.0%. The oil yield of palm seed was 16.48%. Conclusion. Water enzymatic extraction process of palm seed oil is reasonable, the active ingredients are rich, and the quality of seed oil is better, providing reference for the development and research of palm seed oil.

Extraction, purification, and biological activities of flavonoids from branches and leaves of Taxus cuspidata S. et Z.

The optimal operational and process parameters were determined for the enzymatic hydrolysis and ultrasound-assisted extraction (EHUE) method of flavonoids extracted from Taxus cuspidata branches and leaves (TCBL), and the biological activity of obtained flavonoids was evaluated. According to single factor and central composite design experiments, the optimum key experimental parameters for EHUE were that pectinase enzyme concentration was 0.10 mg·mL-1, enzymatic hydrolysis temperature was 48 °C, and enzymolysis time was 39 min. The yield of flavonoids from TCBL under the optimized conditions was 5.23% ± 0.18%. Four purified flavonoid compounds from TCBL extract were identified as 1) (E)-1-methoxy-2-O-(p-coumaroyl)-myo-inositol, 2) catechin, 3) epicatechin, and 4) quercetin-3-O-glucoside. Among the 4 compounds, compounds 2 and 3 showed higher antioxidant capacities, α-amylase, and α-glucosidase inhibitory activities. The statistical analysis showed that epicatechin and catechin were potent antioxidants and active agents for inhibiting type II diabetes. In addition, all 4 compounds exerted clear antitumor activity against MCF-7, Hela, and HepG2 cells. Especially, compound 4 had highest antitumor capacity against MCF-7 and Hela, while compound 1 was best at suppressing the proliferation of HepG2 cells.

Study on the stability of intermediates in the process of enzymatic hydrolysis of phosphatidic acid by phospholipase A1

In the degumming process of crude soybean oil by phospholipase A1 (PLA1), the content of free fatty acids (FFA) in the product increases excessively. In this study, PLA1 was used to hydrolyse simulated crude soybean oil. It was found that the acyl group at the sn-2 position migrated to the sn-1 position, the content of FFA in the oil was as high as 1.26 ± 0.03 g/100 g, and the acyl migration rate was up to 38.9 ± 0.10% after 2.8 h. Under the conditions of H3BO3 addition of 2 g/100 g, pH of 5, enzymatic hydrolysis temperature of 50 °C and enzymatic hydrolysis time of 2 h, the acyl migration rate of sn-2-HPA was 16.8 ± 0.50%, the content of FFA was 1.14 ± 0.02 g/100 g, and the enzymatic hydrolysis efficiency was increased by 28.57 ± 0.07%.

Optimization of the Temperature and Time for the Autolysis and Enzymolysis of Perna Viridis Protein

To determine the optimal conditions for autolysis, the autolysis of mussel (Perna viridis) protein was studied. Using P. viridis meat as the raw material and the degree of hydrolysis as the index, the effects of hydrolysis temperature and hydrolysis time, were researched. Single-factor experiments of autolysis of P. viridis were conducted, taking the degree of hydrolysis as the index. Keeping the pH, time, and solid/liquid ratio unchanged, the temperature was controlled at 30, 40, 50, 60, and 70 °C to study the effects of temperature on the autolysis of P. viridis protein. When the enzymatic hydrolysis temperature was 30 °C, the degree of protein hydrolysis was very low. The results showed that: with increasing temperature up to 50 °C, the degree of hydrolysis increased, reaching a maximum of 15.84%. Keeping the pH, temperature, and solid/liquid ratio unchanged, hydrolysis times of 2, 3, 4, 5, and 6 h were tested to determine the effect of time on the autolysis of P. viridis protein. The results showed that: hydrolysis occurred between 2 and 5 h, when the degree of hydrolysis increased from 13.62% to 15.31%, exhibiting a marked change in the extent of hydrolysis. The continuous increase in hydrolysis could be explained by the fact that P. viridis increased the amino nitrogen content in the hydrolysate via enzymatic hydrolysis and microbial degradation of endogenous enzymes. Therefore, the optimum conditions obtained in these experiments were a temperature of 50 °C, time of 5 h.

Effect of Papain on the Demulsification of Peanut Oil Body Emulsion and the Corresponding Mechanism.

This study investigated the effect of papain on the demulsification of peanut oil body emulsion extracted using an aqueous enzymatic method and the associated mechanism. The highest free oil yield using papain (92.39%) was obtained under the following conditions: an enzymatic hydrolysis temperature of 55°C, sample-to-water ratio of 1:3, enzyme concentration of 1400 U/g, and an enzymatic hydrolysis time of 3 h. Papain degraded the peanut oil body protein to small-molecular-weight peptides (≤ 14.4 kDa). Compared to the emulsion before enzymatic hydrolysis, the amino acid content in the aqueous phase was higher after enzymatic hydrolysis, the viscosity of the oil body emulsion was lower, and the particle diameter of the emulsion was significantly larger. The following demulsification mechanism was derived. Papain degrades the protein on the peanut oil body and dissolves it in water. The outer side of the oil body loses the protection of electrostatic repulsion and steric hindrance provided by the membrane protein. This causes the viscosity of the emulsion system and the molecular steric hindrance to decrease. As a result, the oil droplets gather and eventually demulsify. The results of this study provide the theoretical basis for the instability in oil body emulsions and are expected to promote the application of enzymatic demulsification in industry.

Pretreatment of lignocellulosic feedstock to produce fermentable sugars for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production using activated sludge

The goal of this research was to release fermentable reducing sugar from lignocellulose (poplar) by hot water pretreatment, and then employed to synthesize poly(3-hydroxybutyrate-co-3-hydroxy-valerate) (PHBV) using activated sludge to replace traditional petroleum-based polymers. The orthogonal experiment was used to optimize the conditions of different pretreatment temperature, pretreatment time, enzymatic hydrolysis temperature and enzymatic hydrolysis time and the optimal condition for producing sugars was pretreated at 200 °C for 30 min and enzymatic hydrolysis at 45 °C for 3d. A maximum yield was 530.3 mg/g of reduced sugar, while the furfural and 5-HMF produced in the optimum conditions were 512.61 mg/L and 239.34 mg/L. Moreover, the effects of increasing concentration of hot water pretreated poplar hydrolysates (500–1700 mg/L) on PHBV production were investigated. Poplar hydrolysate (1700 mg/L) exhibited maximum PHBV concentration of 637.556 mg/L.

Novel fusants of two and three clostridia for enhanced green production of biobutanol

The objective of the present study is to improve biobutanol production by utilizing renewable resources of agriculture residues (i.e. wheat straws) in simultaneous saccharification and fermentation (SSF). Three strains of anaerobic gram-positive clostridia (Clostridium beijerinckii, Clostridium thermocellum, and Thermoanaerobacterium saccharolyticum) were fused through a protoplast fusion technique. Results show that protoplast fusion of thermophilic and mesophilic clostridia led to improved thermostability in a fermentation medium at 45 °C. This represents the optimum temperature for enzymatic hydrolysis. Results also show that the fused strain produced essential hydrolysis enzymes, which eliminated the need to add any enzymes during the hydrolysis step. Furthermore, results in the present study demonstrate that the fused culture of bacteria was able to tolerate the elevated concentration of acetone, butanol, and ethanol during production, which resulted in higher biobutanol production of 13.8 g/L. This study includes a comparison with the co-culture as a benchmark to account for the effects of protoplast fusion.