Evaluation of the binding affinity and antioxidant activity of phlorizin to pepsin and trypsin

Phlorizin(PHL)is a natural compound with strong antioxidant properties mainly found in apples.In this paper,the interaction mechanism of PHL with pepsin and trypsin was comparatively evaluated by computer simulation,fluorescence spectra,circular dichroism(CD),and Fourier transform infrared(FT-IR)spectra at a molecular level.Fluorescence spectra showed that PHL quenches the pepsin/trypsin by static quenching.Thermodynamic parameters indicated that PHL binds to pepsin mainly through hydrogen bonds and van der Waals forces,and that of trypsin was electrostatic forces.The ground state complexes PHL and protease have a moderate affinity of 105 L/mol PHL binds more strongly to trypsin than to pepsin.CD and FT-IR spectra results showed that pepsin/trypsin decreased theβ-sheet content and slightly changed its secondary structure upon PHL.These experimental results are mutually verified with the predicted computer-aid simulation results.Upon PHL and trypsin binding,the antioxidant capacity of PHL was elevated.Nevertheless,the antioxidant capacity of PHL was decreased after binding to pepsin.This work elucidates the binding of PHL binding mechanisms to pepsin/trypsin and provides useful information for the digestion of PHL to improve the application of PHL in food processing.

State of Pepsin and Acidic Protease in Solid Phase System and the Factors Increasing Their Destabilization

The adsorption state and catalytic properties of pepsin and acidic protease from microorganisms Asp. awamori and Asp. oryzae were studied in solid phase system （in presence of sorsilen, DEAE- and CM-cellulose）. According to the results, adsorption capacity and catalytic activity of enzymes depend on the physical nature of surface groups of the solid phase. Changing the stability of enzymes in the system with solid phase is observed even the adsorption bond is less stable （in the case of DEAE- and CM-cellulose in acidic media）. Injection to the medium ethanol, surfactants, sodium chloride and changing the temperature of the incubation medium could prevent the negative effects of the solid phases. When sorsilen is used as solid phase, pepsin and acidic protease from Asp. awamori suffer from high surface inactivation. Various surfactants influence adsorption state of enzymes differently. Non-ionic surfactants （Triton X-100） prevent adsorption and restore catalytic properties of enzymes.

Interaction of hispidin with pepsin:Multi-spectroscopic analyses and docking simulation

Hispidin is a pyranone compound found in edible and medicinal mushrooms of the Phellinus and Inonotus genera.This investigation used fluorescence spectroscopy,UV absorption spectroscopy,and molecular docking to examine the interaction of hispidin with pepsin.The Stern-Volmer method was used to perform the fluorescence quenching measurements at different temperatures(298 K,303 K,and 310 K).According to the findings,hispidin induced a static quenching mechanism in pepsin that resulted in the creation of a hispidin-pepsin complex with binding constants(Ka)ranging from 9.56×10^(4) to 3.45×10^(5) L mol^(-1).The positive values ofΔH(84.6 kJ mol-1)andΔS(337.9 J mol^(-1) K^(-1))demonstrated that hydrophobic forces contributed to forming the hispidin-pepsin complex.The findings of UV-vis absorption,synchronous fluorescence,and 3D fluorescence spectraspectra demonstrated that hispidin altered the conformation and microenvironment of pepsin.According to the analysis of molecular docking,hispidin got into the pepsin's active cavity.The research clarifies the molecular mechanisms by which hispidin binds to pepsin and helps understand its possible biological activity in vivo.

Studies on Digestive Enzymes in Alimentary Tract of Southern Sheatfish (Silurus meridionalis Chen) Larvae Ⅰ-Trypsin and Pepsin

The pepsin and trypsin activities and some of the properties of the two enzymes of southern sheatfish larvae were studied. The results were as follows: the highest level of trypsin activity is in the foregut in all measured tissues; from foregut to hindgut, trypsin activities decrease; the pH optimum of trypsin activity is pH9.0; the strongest pepsin activity is in the stomach; the proper density of haemoglobin for detecting pepsin activity is 1.0%. These data are useful in solving applied nutritional problems, such as the adequacy of artificial food to the digestive abilities of the fish.

Pepsin nanofilm self—assembly on the positively charged poly （ethylene terephthalate）substrate

Pepsin was assembled on the surface of prepared poly(ethylene terephthalate)(PET-NH3^+) substrates.The composition and structure of the pepsin/PET-NH3^+ assembling films in different condition were characterized by X-ray photoelectron spectroscopy(XPS) and atomic force microscopy(AFM).

Characterization and antioxidant activities of marine pepsin soluble collagen from the skin of yellow goosefish Lophius litulon

Characteristics and antioxidant activities of pepsin-soluble collagen （PSC） from yellow goosefish （Lophius litulon） skins were investigated. PSC was characterized as a type I collagen, and its imino acid content was 193 residues/1 000 residues. PSC＇s denaturation temperature was -17.56℃ and Fourier transform infrared spectra confirmed the presence of triple helices. Solubility analysis showed good solubility at acidic pH （1-6） or low NaCl concentrations （≤2%）. PSC showed scavenging activity against hydroxyl radicals and superoxide anions in a concentration-dependent manner. Furthermore, PSC could protect D-galactose-induced skin aging by significantly controlling malondialdehyde formation and improving the activity of superoxide dismutase, glutathione peroxidase, catalase, glutathione, and hydroxyproline. PSC may be a promising antioxidant in appropriate applications.

Comparative Properties of Pepsin Hydrolyzed Jellyfish Protein from Salted Jellyfish

The aim of this study was to compare chemical and biochemical properties of crude jellyfish protein extracted from salted sand jellyfish （Rhopilema hispidum） and white jellyfish （Lobonema smithii）. Sodium dodecyl sulfate polyacylamide gel electrophoresis （SDS-PAGE） showed that the principal proteins of collagen standard types I and II were found among the protein constituents of desalted jellyfish collagen prepared in this study. The amino acid compositions of extracted collagen were typical collagen. The denaturation temperature （Td） of extracted collagen samples was 28-29 ℃. The solubility of all extracted collagens was found up to 4% NaCl. The results indicated that the commercial salting process could partially denature native jellyfish protein functionalities. However, the collagen could be extracted from desalted jellyfish for future use as a food ingredient.

Preparation and Characterisation of Collagen from Freshwater Fish Scales

Acid-soluble collagen (ASC) and pepsin-solubilized collagen (PSC) were prepared from the waste freshwater carp fish scales. The results of SDS-PAGE showed that purified collagens were composed of at least two different chains which were in accordance with the type I collagen with α chain composition of (α1)2α2. Compared with the carp fish ordinary muscle type I collagen , porcine dermis type I collagen and other seawater fish collagens, freshwater carp fish scales collagen contained relative high half-cystine (Cys-s), but lower denaturation temperature(Td) than the porcine dermis type I collagen. These collagens had evident absorption at 230 nm by UV-Vis spectra. The spectrum X-ray diffraction showed that the collagen remained single-helix and tri-helix configuration with the minimum values of the repeat spacings (d) of about 4.48 ? and 11.87 ?. Therefore, to make more effective use of limited-resources, carp fish scales can be a potential resource for the extraction of type I collagen or gelatin.

Separation of growth-stimulating peptides for Bifidobacterium from soybean conglycinin

AIM： To isolate and identify the soybean conglycinin peptides that selectively stimulates the growth of bifidobacteria in vitro, and to investigate the effect of soybean conglycinin peptides on intestinal ecosystem in vivo. METHODS： Soybean conglycinin was purified from soybean seeds by gel filtration （Sepharose-CL-6B）. These proteins were submitted to hydrolysis by pepsin. Several growth-stimulating peptides for bifidobacteria were isolated chromatographically from pepsin hydrolysis of soybean conglycinin and identified by means of matrixassisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS）. Parallel to in vitro study, in vivo experiments with soybean conglycinin peptides were performed in mice. Ninety male KM mice were randomly assigned into five groups of 16 mice each, and each group was administered for 21d intragastrically with physiological saline （control）, conglycinin, pepsin-treated conglycinin （PTC）, the most active fraction which isolated from pepsin-treated conglycinin （P2-PTC） and HCl-full hydrolysis of conglycinin （HCl-FHC）, respectively. Intestinal microflora were evaluated by standard microbiologic methods and biochemical assays of cecal content samples after treatment. RESULTS： The results showed that the peptides which were isolated from soybean conglycinin could stimulate the growth of bifidobacteria in vitro, and the molecular mass of purified peptides with MALDI-TOF-MS ranged from 693.32 to 1829.55. Compared with control group, in vivo experiments showed that P2-PTC group decreased cecal pH （7.08±0.08 vs7.21±0.09, P〈0.05） and enterococcicounts （5.38±0.26 log10CFU/g vs 5.78±0.19 log10CFU/g, P〈0.05）, significantly increased sIgA level （172.08±35.40 ng/g vs 118.27±33.93 ng/g, P〈0.01） and β-galactosidase activity （1.28±0.23 U/g vs 1.82±0.58 U/g, P〈0.05） CONCLUSION： The results have shown that conglycinin is good source for enzyme-mediated production of peptides which stimulate the growth of bifidobacteria. These peptides are inactive within the sequence of the parent protein but can be released during enzymatic hydrolysis, and in vivo experiments demonstrate that conglycinin peptides may be beneficial for improving gastrointestinal health.

Activity of Brucea javanica oil emulsion against gastric ulcers in rodents

The present study aims to investigate the gastroprotective effect of Brucea javanica oil emulsion(BJOE) in animals. Gastroprotective potential of BJOE was studied on absolute ethanol,aspirin, reserpine and restraint plus water immersion-induced gastric ulcers in mice as well as glacial acetic acid(GAA) and pyloric ligation(PL)-induced gastric ulcers in rats. Except for ulcer scores, total acidity as well as pepsin activity as for the PL-induced gastric ulcer model and ulcer incidence as for the GAA-induced gastric ulcer model were also determined. Histopathological evaluation as for aspirin, reserpine, PL-induced models was conducted. Results showed that BJOE significantly(P < 0.05) reduced ulcer index in the mouse and rat models in a dose-dependent manner. It had significant(P < 0.05) suppressive effect on total activity of gastric juice as well in PL-induced model. Histopathological examination for the stomach samples confirmed the findings in the aspirin, reserpine or PLinduced gastric lesion models, which showed relatively complete mucosa structure and less inflammation. It is concluded that BJOE could be effective on gastric ulcer in rodents and its gastroprotective activity might be related to antioxidant, anti-inflammatory ability and promote gastric mucus secreted. The results may provide beneficial basis for increasing BJOE's clinical indication in future.

COMPARISON OF FOUR METHODS TO GENERATE IMMUNOREACTIVE FRAGMENTS OF A MURINE MONOCLONAL ANTIBODY OC859 AGAINST HUMAN OVARIAN EPITHELIAL CANCER ANTIGEN

In the present study, four different proteases (pepsin, papain, bromelain and ficin) were screened with a murine monoclonal antibody OC859 , in order to verify whether different digestion procedures could improve yield and stability of the F(ab’)2 or Fab fragments. The yields of F(ab’)2 or Fab fragments from digestion with pepsin, papain , bromelain and ficin were respectively 20. 3+/-2. 0%, 50. 5+/-5. 0%,74. 4+/-2. 7% and 82. 8 +/-10. 2% of the theoretical maximum. Immunoreactivity in a noncompetitive solid-phase radioimmunoassay (SPRIA) of the fragments generated by the four proteases were respectively 10+/-5%, 36+/-5%, 60+/-6% and 75+/-6%of the intact OC859 IgG. These results suggested that the fragmentation of OC859 with ficin gave a higher yield of superior immunoreactive fragments.

Effects of enzyme treatments on the functionality of commercial pea and pea blended protein ingredients

The effects of enzyme treatments on the functional properties of commercial pea protein,pea:rice protein blend(3:2),pea:oat protein blend(1:1),and pea:hemp protein blend(1:1)were investigated.Treatments were applied through hydrolysis(pepsin or papain),crosslinking(transglutaminase,TG),and their combination(pepsin+TG or papain+TG).The protein content of each protein blend increased slightly or remained unchanged under treatments involving papain but decreased after treating with pepsin or pepsin+TG.Surface hydrophobicity increased for pea,pea:rice,and pea:oat blends after all enzyme treatments.Water holding capacity decreased for all samples compared to the untreated materials.For oil holding capacity,there was only improvement in the pea:rice blend with TG and pea:hemp with papain and papain+TG.Emulsion stability of pea:rice and pea:oat blends increased after all treatments;pepsin treatment specifically improved the emulsion stability to over 90%.Foaming capacity of each protein blend increased under all enzyme treatments whereas the foaming stability improved for the pea:rice blend but decreased for the pea:hemp blend.All treatments increased protein solu-bility.Specific enzyme treatment can be used to tailor the functionality of commercial plant protein blends.

Separation and purification of proteinases from Gastric Mucosa of Northern Sheatfish,Silurus soldatovi

The separation and purification method for pepsin of Northern Sheatfish (Silurus soldatovi) was established by using the combination technology of salting-out,gel chromatography and gel electrophoresis,and the enzymic properties were also analyzed.The experimental results indicated that 28% and 56% (NH4)2SO4 saturation could separate the activated protease from the pepsin extract of gastric mucosa of Sheatfish (Silurus soldatovi) ;compared with the homogenate extraction,the pepsin specific activity of purified extraction by Sephadex G-75 gel chromatography system increased 598 fold,was 5 times higher than that of activated liquid,and the total production rate was 10.1%.The purified pepsin liquor at the conditions of pH3.3 0.01 M alanine-formic acid buffering solution,60 cm chromatography column,and the flowing rate of 0.8 ML/min was analyzed by SDS-PAGE,which indicated that there were two bands and the molecular weight was 34.0 kDa and 40.4 kDa,respectively.There were two peaks in the enzyme activity determination of the separated collecting liquor in gel chromatography,and the SDS-PAGE showed the concentrations of the two proteins was different,which indicated that it existed at least two pepsins in the gastric mucosa of Sheatfish (Silurus soldatovi).

Laryngopharyngeal reflux disease management for recurrent laryngeal contact granuloma:A case report

BACKGROUND Laryngeal contact granuloma(LCG)is difficult to treat and frequently associated with high persistence and recurrence,despite the availability of both surgical and pharmacological treatment options.An appropriate strategy is therefore needed to help patients with multiple recurrences of LCG to potentially avoid unnecessary surgery.CASE SUMMARY We describe the case of a 34-year-old male patient with recurrent LCG in which a good response was achieved through successful management of laryngopharyngeal reflux disease using a combination pharmacotherapeutic regimen consisting of anti-reflux therapy,pepsin secretion inhibition,bile acid neutralization,and lifestyle modifications.This patient underwent surgery to excise the granuloma,then relapsed,underwent a second surgery,which was followed by a second recurrence.The granuloma then disappeared after 9 mo of combined treatment with ilaprazole enteric-coated capsules(10 mg qd),mosapride tablets(5 mg tid)and compound digestive enzyme capsules(2 tablets).The drug regimen was discontinued after one year,and no recurrence of the lesion has been reported during the one-year follow-up period.CONCLUSION We report a combination of pharmacotherapeutics and lifestyle modifications for the management of laryngopharyngeal reflux disease to address recurring LCG.

Preparation of lignin derivatives and their application as protease adsorbents

Synthesis of two lignin derivatives, lignophenol and lignin-aminophenol, were presented in this article. The chemical structure and the func-tional groups of lignin derivatives were charac-terized through FT-IR analysis. The immobiliza-tion of three proteases (papain, trypsin and pepsin) on lignin and lignin derivatives was carried out using adsorption technique. The influence of contact time and pH on the enzyme adsorption by different adsorbents was inves-tigated. Furthermore, enzyme activity recovery was also evaluated. Results showed that lignin and lignin derivatives could adsorb proteases effectively and the adsorption capacity of lingo- phenol and lignin-aminophenol was higher than that of pure lignin. Meanwhile, the activity re-covery of papain and pepsin immobilized on lignin derivatives was very high. This pheno- menon suggested that there is a supramole- cular interaction between enzymes and lignin derivatives which do not inhibit enzyme activity. Therefore, lignophenol and lignin-aminophenol are both promising adsorbents for enzyme im-mobilization under acid and neutral conditions.

Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa

An unprecedented enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction of 2-aminobenzaldehyde and a,b-unsaturated aldehydes is achieved. Pepsin from porcine gastric mucosa provided mild and efficient access to diverse substituted 1,2-dihydroquinolines in yields of 38%–97% with 6%–24%enantiomeric excess（ee）. This work not only provides a novel method for the synthesis of dihydroquinoline derivatives, but also promotes the development of enzyme catalytic promiscuity.

Clinical relevance of salivary pepsin detection in diagnosing gastroesophageal reflux disease subtypes

Background:Gastroesophageal reflux disease(GERD)is heterogeneous with a varied symptom spectrum and reflux profiles.Its definite diagnosis often requires invasive tools including endoscopy or reflux monitoring.The aim of this study was to investigate the clinical relevance of salivary pepsin detection as a non-invasive screening tool to diagnose GERD of different subtypes.Methods:A total of 77 patients with suspected GERD symptoms and 12 asymptomatic controls were analysed.All participants performed symptom evaluation,upper endoscopy,esophageal manometry,and 24-hour multichannel intraluminal impedance-dual pH probe monitoring.Saliva was self-collected across three different time points:at early fasting,postprandially,and at symptom occurrence.Salivary pepsin levels were measured via Peptest.The optimal threshold of salivary pepsin for diagnosing distal or proximal reflux was determined according to a receiver-operating characteristic curve.Results:The average salivary pepsin concentration of suspected GERD patients was significantly higher than that of controls(100.63[68.46,141.38]vs 67.90[31.60,115.06]ng/mL,P=0.044),although no difference was found among patients with different symptom spectrums.The distal reflux group had a higher average pepsin concentration than non-reflux patients(170.54[106.31,262.76]vs 91.13[63.35,127.63]ng/mL,P=0.043),while no difference was observed between the distal reflux group and the proximal reflux group.The optimal cut-off value of salivary pepsin concentration for diagnosing pathological distal reflux was 157.10 ng/mL,which was higher than that for diagnosing pathological proximal reflux(122.65 ng/mL).The salivary pepsin concentration was significantly correlated with distal and proximal reflux parameters.Conclusions:Salivary pepsin measurement can help in identifying true GERD with pathological distal reflux or proximal reflux,regardless of different symptom spectrums.A higher threshold should be applied for diagnosing distal reflux than for proximal reflux.

The effect of enzymes on the in vitro degradation behavior of Mg alloy wires in simulated gastric fluid and intestinal fluid

With an upsurge of biodegradable metal implants,the research and application of Mg alloys in the gastrointestinal environment of the digestive tract have been of great interest.Digestive enzymes,mainly pepsin in the stomach and pancreatin in the small intestine,are widespread in the gastrointestinal tract,but their effect on the degradation of Mg alloys has not been well understood.In this study,we investigated the impacts of pepsin and pancreatin on the degradation of Mg-2Zn alloy wires.The results showed that the pepsin and pancreatin had completely different even the opposite effects on the degradation of Mg,although they both affected the degradation product layer.The degradation rate of Mg wire declined with the addition of pepsin in simulated gastric fluid(SGF)but rose with the addition of pancreatin in simulated intestinal fluid(SIF).The opposite trends in degradation rate also resulted in completely different degradation morphologies in wires surface,where the pitting corrosion in SGF was inhibited because of the physical barrier effect of pepsin adsorption.In contrast,the adsorption of pancreatin affected the integrity of magnesium hydrogen phosphate film,causing a relatively uneven degraded surface.These results may help us to understand the role of different digestive enzymes in the degradation of magnesium and facilitate the development and clinical application of magnesium alloy implanted devices for the digestive tract.

Indigestible cowpea proteins reduced plasma cholesterol after long-term oral administration to Sprague-Dawley rats

Cowpea protein isolate(CPI)was subjected to various dry and wet heat pretreatments followed by sequential digestion with pepsin and pancreatin;the undigested residues were isolated as the indigestible cowpea proteins(ICPs).All the ICPs exhibited in vitro bile acid-binding capacity but ICP from the slow cooling-induced gelation had the highest yield(68%)and was used for rat feeding experiments to determine effect on plasma total cholesterol(TC).Groups consisting of 3 male and 3 female Sprague-Dawley rats each were fed hypercholesterolemic diets that contained casein only or casein that was partially substituted with ICP of CPI for 6 weeks.Results showed diet that contained 5%(w/w)ICP was more effective in preventing TC increase(1.8 mmol/L)when compared to increases of 9.34 and 4.15 mmol/L for CPI and casein only diets,respectively.