Enzymatic hydrolysis of silkworm pupa and its allergenicity evaluation by animal model with different immunization routes

Silkworm pupa is a nourishing food with high nutritional value,but its consumption has been greatly limited given its allergenicity.Enzyme hydrolytic technique is recognized as an effective method to reduce the allergenicity of protein.In this study,we aimed to investigate the effect of enzymolysis on the allergenicity of silkworm pupa.Crude silkworm pupa protein was extracted through alkali extraction and acid precipitation,which included 5 proteins with the molecular weights ranging from 34 kDa to 76 kDa,and silkworm pupa were then hydrolyzed by alkaline protease.The allergenicity of silkworm pupa protein and its enzymatic hydrolysates was evaluated by establishing BALB/c mice model,and the mice were immunized via intragastric gavage and intraperitoneal injection,respectively.The results indicated that the intraperitoneal inj ection immunization route induced more by detecting with antibodies,histamine and Th2-related cytokines.Moreover,mice treated with silkworm pupa protein peptide displayed no obvious allergic symptoms,indicating that enzyme hydrolytic technique could significantly reduce the allergenicity of silkworm pupa.

Structural and antioxidative properties of royal jelly protein by partial enzymatic hydrolysis

The objective of this study was to investigate the structural and antioxidative properties of royal jelly protein(RJP)at different degrees of hydrolysis(DH)by partial enzymatic hydrolysis. RJP was hydrolyzed by alcalase for 0 min, 15 min, 1 h, 5 h and 8 h to obtain hydrolysates at DH of 5.34%, 11.65%, 15.19%, 21.38% and 23.91%, respectively. With the increased DH, the RJP hydrolysates showed elevated antioxidative activities. The molecular weight of RJP hydrolysates was significantly decreased but their primary backbone kept unchanged. Analysis of circular dichroism spectra revealed that the enzymolysis reduced the content of α-helix but increased the contents of β-sheet, β-turn and random coil. Meanwhile, the surface hydrophobicity and fluorescence intensity of RJP hydrolysates were decreased and a red shift occurred. As the enzymolysis continued, the surface morphology of RJP was gradually changed from a sheet-like structure into microparticles. Changes in antioxidative activities and structures generally followed a DH-dependent manner, however these changes became insignificant for samples at DH beyond 20%. Taking into consideration of both effectiveness and productivity, the optimum enzymatic duration was determined at 5 h.

Structure, composition and enzymatic hydrolysis of steam-exploded lespedeza stalks

Pretreatment of lespedeza stalks by steam explosion has been studied. The results indicate that steam-exploded pretreatment has strong effects on physical features, morphology, crystallinity, and composition of lespedeza stalks as shown by scanning electron microscopy （SEM）, infrared （IR）, and X-ray diffraction spectrometry methods. After steam explosion, the cellulose and lignin contents of lespedeza stalks varied only slightly, but the hemicellulose content had decreased from 29.34% to 7.48%. The cellulose obtained by steam-exploded pretreatment had a higher degree of crystallinity than that of the raw material. At the explosion condition of 2.25 MPa and 4 min, lignocellulose is easier to hydrolyze by enzyme than the original lignocellulose. The concentration of reduced sugar in the hydrolyzate liquid increased from 71.77 to 162.84 g·L^-1.

Pretreatment of agricultural residues by supercritical CO_2 at 50–80 °C to enhance enzymatic hydrolysis

Various agricultural crop residues including corn stover,corn cob,and sorghum stalk with a moisture content of 75 wt%were subjected to a long pretreatment(12–60 h)with supercritical CO_2(scCO_2),at low temperature(50–80°C)and a pressure of 17.5–25.0 MPa.The sugar yields from the enzymatic hydrolysis(EH)of the pretreated samples were as much as three-to fourfold greater than those afforded by the raw materials.However,when pretreatment was conducted within a short time(e.g.0.5 h),as previously reported in the literature,only a slight increase in the EH sugar yields was observed.The proposed sc CO_2pretreatment mechanism demonstrated the role of moisture in the system.Wetting,softening,and swelling were observed to mainly affect the lignocellulose when a suitable amount of water was added.Finally,the samples were analysed by X-ray diffraction and scanning electron microscopy,before and after pretreatment,to investigate the changes in the microscopic structure of the biomass.

A Study of the Enzymatic Hydrolysis of Fish Frames Using Model Systems

A model system was employed to study the operating conditions and primary parameters of enzymic hydrolysis of cod proteins. Pancreatin, papain, and bromelain were used to hydrolyse minced cod fillets under controlled conditions and with the rate of hydrolysis being continually monitored via both the pH-stat and TNBS method. The two methods were compared and evaluated. The rate of protein solubilisation was plotted against the degree of hydrolysis (DH). Dry fish protein hydrolysate (FPH) powders having short, medium and high degrees of hydrolysis (DH of approximately 8%, 11% and 16% respectively) were produced and analysed for their molecular weight distribution, using size exclusion chromatography. Almost complete protein solubilisation (75 g soluble protein per kg hydrolysis solution) could be achieved within an hour, at 40oC, at 1% enzyme/substrate ratio (w/w) with papain and bromelain. The pH-stat was found capable of continuously following the rate of hydrolysis but only at low DH. The TNBS could be accurately used even at high DH to estimate the percentage of the peptide bonds cleaved, but required chemical analysis of withdrawn samples.

Dynamic changes of peptidome and release of polysaccharide in sea cucumber(Apostichopus japonicus) hydrolysates depending on enzymatic hydrolysis approaches

Enzymatic hydrolysis has been widely used to produce bioactive hydrolysates from sea cucumber body wall.Here,inspired by the clarification of Apostichopus japonicus genome,we investigated the enzymatic hydrolysis of sea cucumber body wall by using the omics strategy.Shared proteins,including major yolk proteins,collagens,extracellular matrix glycoproteins and muscle proteins,were released from the body wall by different hydrolysis condition.A portfolio of 216 shared peptides were detected in the peptidome by papain with different hydrolysis time,while 32 shared peptides were detected in the peptidome by differing proteases.Unshared peptides and the relative abundance distribution profiles of shared peptides changed depending on hydrolysis approaches,indicating dynamic changes of peptidome during hydrolysis.Moreover,release of sulfated fucan and fucosylated chondroitin sulfate changed with the hydrolysis condition.The monitoring of dynamic enzymatic hydrolysis process at a molecular scale would contribute to production and quality control of sea cucumber hydrolysates.

Wet Oxidation Pretreatment of Poplar Waste for Enhancing Enzymatic Hydrolysis Efficiency

In this paper, we described the optimization of the wet oxidation pretreatment conditions to enhance enzymatic hydrolysis efficiency, using poplar waste from the stock section of a paper mill as the raw material. We showed that the optimal conditions of the pretreatment for poplar waste were an initial p H value of 10, a temperature of 195℃, a holding time of 15 min, and an oxygen pressure of 1.2 MPa. In this case, the yield of the obtained solid material produced by the process was 51.7% and the reducing sugar yield was 46.8%. The solid part obtained from the pretreatment process was hydrolyzed by cellulase L-10. The optimal enzymatic conditions were a temperature of 49℃, a duration time of 56 h, an enzyme dosage of 38 FPU/g at a p H value of 4.8, and a solid-to-liquor ratio of 1∶50. The resulting cellulose conversion rate reached 96.4% in terms of the pretreated substances. In addition, a chemical composition analysis of the poplar waste and pretreated material indicated that about 92% of the hemicelluloses and 43% of the lignin in the raw material were degraded and dissolved. In addition, the crystallization decreased from 57.5% to 54.8%. An obvious fibrillation of the fiber pretreated by the wet oxidization process was observed by SEM. Moreover, high-performance liquid chromatography(HPLC) results showed a high xylose content and monosaccharide degradation products in the pretreatment solution. In conclusion, the wet oxidation pretreatment process could efficiently degrade or remove the lignin and hemicellulose, as well as reduce the crystallinity of the lignocellulosic material, which resulted in animprovement of the enzymatic ability and an increase in the cellulose conversion rate.

Cascade Enzymatic Hydrolysis Coupling with Ultrafine Grinding Pretreatment for Sugarcane Bagasse Saccharification

The biorefinery process for sugarcane bagasse saccharification generally requires signifcant accessibility of cellulose. We reported a novel method of cascade cellulase enzymatic hydrol- ysis coupling with ultrafine grinding pretreatment for sugarcane bagasse saccharification. Three enzymatic hydrolysis modes including single cellulase enzymatic hydrolysis, mixed cellulase enzymatic hydrolysis, and cascade cellulase enzymatic hydrolysis were compared. The changes on the functional group and surface morphology of bagasse during cascade cellulase enzymatic hydrolysis were also examined by FT-IR and SEM respectively. The results showed that cascade enzymatic hydrolysis was the most efficient way to enhance the sugarcane bagasse saccharification. More than 65% of reducing sugar yield with 90.1% of glucose selectivity was achieved at 50 ℃, pH=4.8 for 72 h （1200 r/min） with cellulase I of 7.5 FPU/g substrate and cellulase II of 5 FPU/g substrate.

Conversion of Sugarcane Shoots and Leaves into Reducing Sugars by Pretreatment and Enzymatic Hydrolysis

Sugarcane shoots and leaves consist of 38% cellulose, 30.6% hemicellulose and 12.8% lignin on dry solid （DS） basis and have the potential to serve as low cost feedstocks for ethanol production. The pretreatment and enzymatic hydrolysis conditions include particle size, alkali （NaOH）/dilute acid （H2SO4） pretreatment, chemical and substrate concentrations, temperature, autoclaving time for pretreatment, enzyme concentration, pH and temperature for hydrolysis varied were evaluated for conversion of sugarcane shoots and leaves cellulose and hemicellulose to reducing sugar. The optimum conditions were accomplished by using 14% w/v DS of 0-10 mm sugarcane shoots and leaves in particle size, pretreated with 1.5% w/v of dilute sulfuric acid at 121℃, 15 lbs/in2 for 15 min and enzymatic saccharification using 40 FPU/g DS cellulose at 50℃ and pH 5, After incubating at 160 rpm for 12 hrs, 59 g/L or 386,38 mg/g DS of reducing sugar and 50.69% saccharification were obtained.

Enzymatic Hydrolysis of Fish Frames Using Pilot Plant Scale Systems

Papain was used to hydrolyse fish frames under controlled conditions at a batch-pilot plant scale- process, for the production of fish protein hydrolysates (FPH). Mass balance calculations were carried out so that the rate of hydrolysis, rate of protein solubilisation and yields could be estimated. Almost complete hydrolysis could be achieved in 1 hour, at 40oC, with no pH adjustment, at 0.5% (5 g.kg-1) enzyme to substrate ratio (E/S, were S is Kjeldahl protein) using whole fish frames (including heads and flaps). This was achieved both with the addition of water (1/1 to 2/1 frames/water) but more importantly from commercial considerations without the initial addition of water (after mincing of the fish material). The degree of protein solubilisation ranged between 71% - 86% w/w. Four different processes are described, namely: 1) a soluble spray-dried FPH powder 2) a liquid FPH 3) a partly soluble, spray dried FPH powder and 4) a crude, drum-dried protein for animal consumption. The amino acid profile of the FPH was identical to that of the parent substrate (fish frames).

Antioxidant Activities of Okra Protein Concentrate and Isolate after Enzymatic Hydrolysis

Three different proteolytic enzymes (pepsin, trypsin and papain) were used to prepare okra seed protein concentrate and okra seed protein isolate hydrolysates. The hydrolysates were assayed for antioxidant properties using radical scavenging, reducing power and metal chelating assays. The highest degree of hydrolysis (after 360 min) for okra protein isolates was 35.20%, 35.21% and 10.53% for pepsin, papain and trypsin respectively. The highest degree of hydrolysis (after 360 min) for okra protein concentrates was 26.8%, 28.59% and 6.47% for pepsin, papain and trypsin respectively. Pepsin hydrolysates showed higher metal chelating activity and radical scavenging activity than trypsin and papain hydrolysates. Trypsin hydrolysates showed the lowest antioxidant activities, which may be due to the low degree of hydrolysis. In general, for antioxidant activity, there was an increase in activity with an increase in the degree of hydrolysis. Similar antioxidant activity was found in both the okra protein isolate and concentrate hydrolysates except for metal chelating activity which was higher in okra protein isolate hydrolysates. This may be due to the higher ash concentration in the concentrates (9.4% in concentrates vs. 2.6% in isolates). In this study, pepsin hydrolysates with a final DH of 35.2% showed higher reducing power and metal chelating activity than trypsin and papain hydrolysates. Okra protein hydrolysates were found to have varying levels of antioxidant activity, which was dependent on the specificity of the protease and proportional to the degree of hydrolysis achieved.

Enzymatic Hydrolysis of Hairtail Surimi in an Ultra-High Pressure Bioreactor

Amino acids have been extracted from Hairtail surimi using enzymes in an ultra-high pressure bioreactor. The extraction efficiency of different enzymes including papain, trypsin, and proteases (acid, neutral, alkaline) also has been evaluated, and it has been discovered that neutral protease behaved the best. The amino acids were analyzed using automatic amino acid analyzer, and the enzymatic digestion conditions were optimized. For neutral protease, the optimal condition was 50℃, 250 MPa, pH 7.0. Material to liquid ratio of enzyme is 6%. More than 29 amino acids were detected after 24 hours of hydrolysis;the enzymatic hydrolysis rate can reach 83.29%. The results show that enzymatic digestion under ultra-high-pressure provides a very promising approach to extract amino acids from Hairtail surimi.

DETERMINATION OF AMINO ACIDS IN TWO POLYSIPHONIA SPECIES AND STUDY OF ENZYMATIC HYDROLYSIS METHOD

The total content of the rich amino acids in two common red algae, Polysiphonia urceolata and Polysiphonia japonica growing in the Qingdao seashore were determined. The algae powder was hydrolyzed by 6 mol/L HCl at 110℃ for 48 h and determined by amino acid analyzer. The content was 25.35% and 24.16%, respectively, much higher than that of some other species. In addition, a nutritive liquid with abundant amino acids was prepared (by the enzymatic hydrolysis method using Polysiphonia urceolata ) as raw material for a kind of health beverage. The dried seaweed was decolored by 0.25% KMnO 4 and 0.5% active carbon, then enzymalized. In the selection of enzymalizing condition, the orthogonal experimental design was used. Four factors including kinds of enzyme, quantity of enzyme, temperature and time were studied at 3 levels. According to the orthogonal design results, we can choose an optimal condition: hydrolyzing at 45℃ by neutral proteinase (0.25%, w/w) for 2 h, adjusting pH to 8.5, then adding trypsin (0.25%, w/w) and hydrolyzing for 2 h. Finally the above solution was alkalized by NaOH and neutralized by casein. After the hydrolyzed liquid was filtered and concentrated, suitable additives were added. The final products contain rich amino acids.

Study on Enzymatic Hydrolysis Technology and Antioxidant Activity of Glutinous Rice Glutinous Enzymatic Processes and Antioxidant Activity

[ Objective] This study aimed to investigate enzymatic hydrolysis technology of glutinous rice and the oxidation resistance activity of the enzymatic hydrolysis solution. [ Method ] White glutinous rice was hydrolyzed using four kinds of proteases including neutral protease, alkaline protease, papain and trypsin. Using the scavenging rate of hydroxyl radical （ ·OH） as an indicator and appropriate protease as hydrolytic enzyme, the effects of protein substrate concentration, enzyme dosage, enzymatic hydrolysis temperature and initial pH on the abilities of proteases to scavenge hydroxyl radical from enzymatic hydrolysis solution of glutinous rice were investigated. Based on single-factor test, L9 （34） orthogonal experimental design was adopted, to determine the optimal enzymatic hydrolysis condi- tions leading to the highest oxidation resistance activity of enzymatic hydrolysis solution. [ Result] The optimized process parameters for enzymatic hydrolysis of glu- tinous rice protein with neutral protease were： protein substrate concentration of 2%, enzyme dosage of 24 000 U/g protein （protein meter）, enzymatic hydrolysis temperature of 55 ℃, initial pH of 8.0, and enzymatic hydrolysis duration of 0.5 h; under these conditions, the hydroxyl radical scavenging rate could reach 56. 05% ; protein substrate concentration, enzyme dosage, enzymatic hydrolysis temperature and initial pH had extremely significant effects on the hydroxyl radical scavenging rate. In addition, the activities of antioxidant peptides in glutinous rice hydrolysates were well maintained within a temperature range of 60 - 100℃. [Condusion] The study produced theoretical feasibility reference for the production of functional base powder by spray drying.

Effects of different enzymatic hydrolysis methods on the bioactivity of peptidoglycan in Litopenaeus vannamei

The effects of different hydrolysis methods on peptidoglycan （PG） were assessed in terms of their impact on the innate immunity and disease resistance of Pacific white shrimp, Litopenaeus vannamei. PG derived from Bifidobacteriurn thermophilum was prepared in the laboratory and processed with lysozyme and protease under varying conditions to produce several different PG preparations. A standard shrimp feed was mixed with 0.05% PG preparations to produce a number of experimental diets for shrimp. The composition, concentration, and molecular weight ranges of the soluble PG were analyzed. Serum phenoloxidase and acid phosphatase activity in the shrimp were determined on Days 6-31 of the experiment. The protective activity of the PG preparations was evaluated by exposing shrimp to white spot syndrome virus （WSSV）. Data on the composition of the PG preparations indicated that preparations hydrolyzed with lysozyme for 72 h had more low-molecular-weight PG than those treated for 24 h, and hydrolysis by protease enhanced efficiency of hydrolysis compared to lysozyme. SDS-PAGE showed changes in the molecular weight of the soluble PG produced by the different hydrolysis methods. Measurements of serum phenoloxidase and acid phosphatase activity levels in the shrimp indicated that the PG preparations processed with enzymes were superior to the preparation which had not undergone hydrolysis in enhancing the activity of the two serum enzymes. In addition, the preparation containing more low-molecular-weight PG enhanced the resistance of the shrimp to WSSV, whereas no increased resistance was observed for preparations containing less low-molecular-weight PG. These findings suggest that the immunity-enhancing activity of PG is related to its molecular weight and that increasing the quantity of low-molecular-weight PG can fortify the effect of immunity enhancement.

Enzymatic Hydrolysis of Sugarcane Biomass and Heat Integration as Enhancers of Ethanol Production

The aim of this study is to assess the possibility of increasing ethanol production by introducing the bagasse hydrolysis process into conventional distilleries.Simulations were performed for mass and energy balances using Aspen Plus?software.It was assumed that sugarcane trash and lignin cake—hydrolysis process residues—are available as supplementary fuel.Several cases were evaluated,including:(a)conventional ethanol distillery,(b)conventional plant combined with a hydrolysis process without heat integration,with different solid contents in the hydrolysis reactor,and(c)conventional plant combined with the hydrolysis process applying heat integration by pinch analysis.The highest ethanol yield was achieved in the case of heat integration and concentration of cellulose hydrolysate by the membrane system with a solid content of 5%in the hydrolysis reactor.This represents an increase of 22%over conventional distilleries currently found in the industry.

Optimization of Liquid Ammonia Treatment for Enzymatic Hydrolysis of Miscanthus sinensis Anderss

Miscanthus biomass yield high, high photosynthetic efficiency, fast growth, easy breeding, widely distributed, is suitable as raw materials for the production of fuel ethanol. In this paper, the method of the pretreatment of liquid ammonia for Miscanthus, using the pretreatment of liquid ammonia to overcome biomass recalcitrance, adding cellulase enzyme, and the monosaccharide content is determined by using high performance liquid chromatography (HPLC). Effect of pretreatment process on the enzyme solution effect of awn, conducts the research to the water processing rate, temperature, residence time, amount of ammonia, the amount of H2O2 parameters. Data shows that, in the moisture content of 80%, temperature 130?C, loading amount of ammonia 2:1, residence time 10 min, the glucan and xylan enzyme conversion rate are 73.23% and 73.28% respectively. After a mass balance, dry base mans per 100 g can get glucose 28.96 g and xylan 17.25 g.

Extraction Hydrolysates from Larimichthys Polyactis Swim Bladder Using Enzymatic Hydrolysis

As a kind of biopolymer,hydrolysates of fish swim bladder,safer than those of land mammals,are widely used in food,cosmetics as well as pharmaceutical and biomedical fields for their biocompatibility,biodegradability,and weak antigenicity.To enhance hydrolysate production,in this paper,the papain and alcalase hydrolysis processes of larimichthys polyactis swim bladder were optimized with orthogonal experiments.With 89.5%hydrolysate yield,the optimal processing conditions for alcalase were solid-liquid ratio of 1:30,enzyme concentration of 0.7%,and extraction time of 6 h.As for papain,under the optimal processing conditions:solid-liquid ratio of 1:20,enzyme concentration of 0.5%,and extraction time of 8 h,the hydrolysate yield was 65.1%.To obtain higher hydrolysate yields,the ultrasonic pretreatments were implemented before the optimal enzyme hydrolysis processes.With ultrasonic waves of 100 W for 50 min,the hydrolysate yields were increased 2.1%(alcalase)and 4.5%(papain),respectively.The Fourier Transform Infrared(FTIR)spectroscopic analysis revealed that the hydrolysates extracted by papain exist in triple-helical forms.The Ultra-Violet(UV)absorption spectra indicated that the aromatic amino acids in the hydrolysates had strong absorptions in the wavelength range of 240 nm–300 nm.The results of this research demonstrate that the alcalase hydrolysates have better solubility in water and the solution is more stable under ambient temperature.However,the hydrolysates extracted by papain have a gel property and are insoluble in weak acid at room temperature,which is more suitable for applications in feedstock of biomedical.

Alleviation of Drought Stress in Wheat Using Exogenous Ulva prolifera Extract Produced by Enzymatic Hydrolysis

Drought is one of the major abiotic stresses that affect plant growth and reduce agricultural productivity.Use of algal extract as a biostimulant is gaining increased attention from researchers.This study aimed to investigate the potential of Ulva prolifera extract(UE)as a biostimulant when enzymatically extracted under conditions of water deficit.UE treatments(0.02%,0.06%,and 0.1%)significantly improved the shoot length,root length,and dry weight of roots after 120 h of drought stress relative to that in treatment with the negative control.An increase in catalase(CAT)and peroxidase(POD)activity was also observed that resulted in improved antioxidant capacity.Application of 0.1%UE reduced the malondialdehyde(MDA)content by 30.06%compared with that in the negative control.In addition,the soluble sugar and protein content in wheat treated with 0.1%UE was increased by 23.10%and 93.51%,respectively,resulting in adjustment of the osmotic pressure.Results suggest that UE could significantly enhance the drought tolerance of wheat.This study provides a basis for increasing the value of UE as a biostimulant.

Conditions for enzymatic hydrolysis of egg white proteins

The study on condition for enzymic hydrolysis of in egg white proteins using a dual quadratic rotary, orthogonal and regressive design shows that the optimum temperature was 68 5 ℃, and the optimum pH is 8 21 at the substrate concentration of 5 5%. Mathematic model has been established to reveal the relationship between enzyme concentration and hydrolytic time with respect to the same NR.