Antioxidant Activity Evaluation of Oviductus Ranae Protein Hydrolyzed by Different Proteases.

As nutrition and a health tonic for both medicine and food, the protein content of Oviductus Ranae is more than 40%, making it an ideal source to produce antioxidant peptides. This work evaluated the effects of six different proteases (pepsin, trypsin, papain, flavourzyme, neutral protease and alcalase) on the antioxidant activity of Oviductus Ranae protein, and analyzed the relationship between the hydrolysis time, the degree of hydrolysis (DH) and the antioxidant activity of the enzymatic hydrolysates. The results showed that the antioxidant activity of Oviductus Ranae protein was significantly improved and the optimal hydrolysis time was maintained between 3-4 h under the action of different proteases. Among them, the protein hydrolysate which was hydrolyzed by pepsin for 180 min had the strongest comprehensive antioxidant activity and was most suitable for the production of antioxidant peptides. At this time, the DH, the DPPH radical scavenging activity, the absorbance value of reducing power determination and the hydroxyl radical scavenging activity corresponding to the enzymatic hydrolysate were 13.32 ± 0.24%, 70.63 ± 1.53%, 0.376 ± 0.009 and 31.96 ± 0.78%, respectively. Correlation analysis showed that there was a significant positive correlation between the hydrolysis time, the DH and the antioxidant activity of the enzymatic hydrolysates, further indicating that the hydrolysates of Oviductus Ranae protein had great antioxidant potential. The traditional anti-aging efficacy of Oviductus Ranae is closely related to the scavenging of reactive oxygen species, and its hydrolysates have better antioxidant capacity, which also provides support for further development of its traditional anti-aging efficacy.

Role of Daucus carota in Enhancing Antiulcer Profile of Pantoprazole in Experimental Animals.

The carrot plant (Daucus carota) and its components are traditionally reported for the management of gastric ulcers. This study was performed to evaluate the role of carrot when administered concurrently with a conventional antiulcer treatment, pantoprazole, in alleviating gastric and duodenal ulcers in female experimental animals. The study involved standard animal models to determine the ulcer preventive effect using pylorus ligation, ethanol, and stress induced acute gastric ulcer models and duodenal ulcer models involving cysteamine. Acetic acid-induced chronic gastric ulcer and indomethacin-induced gastric ulcer models were used to evaluate the ulcer healing effect. Carrot fruit (500 mg/kg) and its co-administration with pantoprazole produced significant protection in an ethanol- and stress-induced acute gastric ulcer and cysteamine-induced duodenal ulcer. The healing of the acetic acid-induced chronic gastric ulcer was also augmented with this combination. Both total proteins and mucin contents were significantly increased in indomethacin-induced gastric ulcers. Similarly, in pylorus ligation, the pepsin content of gastric juice, total acidity, and free acidity were reduced. Overall, both ulcer preventive effects and ulcer healing properties of the pantoprazole were significantly enhanced in animals who received the co-administration of carrot fruit (500 mg/kg).

Thermal stable characteristics of acid- and pepsin-soluble collagens from the carapace tissue of Chinese soft-shelled turtle (Pelodiscus sinensis).

The carapace from the Chinese soft-shelled turtle (Pelodiscus sinensis) is used as a traditional Chinese medicine. Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from turtle carapace were isolated and characterized to screen novel collagen material in this study. Yields of 1.0% and 2.8% were obtained for ASC and PSC which contained glycine as the major amino acid and had high imino acid content. Both collagens had maximum ultraviolet absorption peaks of 220 nm. SDS-PAGE revealed that the structure of both collagens was similar, belonging to type I collagen. Relative viscosities of collagens were decreased as the temperature increased. Collagens showed minimum solubility at pH 8 and maximum solubility at a salt concentration of 3%. The denaturation temperature (Td) of PSC was higher whereas the melting temperature was lower than that of ASC. Both ASC and PSC appeared to be spongy like microstructure with fibrillar pores shown by scanning electron microscopy. The results suggest that collagens isolated from turtle carapace has high thermal stability with potential uses as new substitute for mammalian collagen in medicinal, food or biomaterial fields. However, their biological or pharmacological activities are needed to be further studied.

Multi-spectroscopic studies on the interaction between traditional Chinese herb, helicid with pepsin.

Study on the binding properties of helicid by pepsin systematically using multi-spectroscopic techniques and molecular docking method, and these interactions comprise biological recognition at molecular level and backbone of biological significance in medicine concerned with the uses, effects, and modes of action of drugs. We investigated the mechanism of interaction between helicid and pepsin by using various spectroscopic techniques viz., fluorescence spectra, UV-Vis absorption spectra, circular dichroism (CD), 3D spectra, synchronous fluorescence spectra and molecular docking methods. The quenching mechanism associated with the helicid-pepsin interaction was determined by performing fluorescence measurements at different temperatures. From the experimental results show that helicid quenched the fluorescence intensity of pepsin via a combination of static and dynamic quenching process. The binding constants (Ka) at three temperatures (288, 298, and 308 K) were 7.940 × 107, 2.082 × 105 and 3.199 × 105 L mol-1, respectively, and the number of binding sites (n) were 1.44, 1.14, and 1.18, respectively. The n value is close to unity, which means that there is only one independent class of binding site on pepsin for helicid. Thermodynamic parameters at 298 K were calculated as follows: ΔHo (- 83.85 kJ mol-1), ΔGo (- 33.279 kJ mol-1), and ΔSo (- 169.72 J K-1 mol-1). Based on thermodynamic analysis, the interaction of helicid with pepsin is driven by enthalpy, and Van der Waals' forces and hydrogen bonds are the main forces between helicid and pepsin. A molecular docking study further confirmed the binding mode obtained by the experimental studies. The conformational changes in the structure of pepsin was confirmed by 3D fluorescence spectra and circular dichroism.

Identification of Antimicrobial Peptides of Native and Heated Hydrolysates from Hen Egg White Lysozyme.

Hen eggs are a source of bioactive compounds, of which the hen egg white lysozyme (HEWL) protein. HEWL has a demonstrated antibacterial activity. The aim of this study was to evaluate the antimicrobial activity of native and heated HEWL hydrolysates obtained through hydrolysis with pepsin and to identify their peptides using the reversed phase high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (RP-HPLC-ESI-MS-MS) analysis. Native and heat-treated HEWL was hydrolyzed with pepsin at pH 1.2, and their antibacterial activity was tested against Escherichia coli and Staphylococcus carnosus. Two of the hydrolysates obtained presented high antibacterial activity against Gram-positive and Gram-negative bacteria. Native HEWL hydrolysate was a bactericide at 2.0 mg/mL against E. coli. Fifty-one peptide sequences were identified on the two hydrolysates. Peptides identified are cationic peptides. These peptides are rich in Lys and Arg cationic amino acids and have Trp in their sequences.

Extraction and identification of α-amylase inhibitor peptides from Nephelium lappacheum and Nephelium mutabile seed protein using gastro-digestive enzymes.

The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity.

Improved collagen extraction from jellyfish (Acromitus hardenbergi) with increased physical-induced solubilization processes.

Efficiency and effectiveness of collagen extraction process contribute to huge impacts to the quality, supply and cost of the collagen produced. Jellyfish is a potential sustainable source of collagen where their applications are not limited by religious constraints and threats of transmittable diseases. The present study compared the extraction yield, physico-chemical properties and toxicology in vitro of collagens obtained by the conventional acid-assisted and pepsin-assisted extraction to an improved physical-aided extraction process. By increasing physical intervention, the production yield increased significantly compared to the conventional extraction processes (p < .05). Collagen extracted using the improved process was found to possess similar proximate and amino acids composition to those extracted using pepsin (p > .05) while retaining high molecular weight distributions and polypeptide profiles similar to those extracted using only acid. Moreover, they exhibited better appearance, instrumental colour and were found to be non-toxic in vitro and free of heavy metal contamination.

PEGylated Nanoemulsions for Oral Delivery: Role of the Inner Core on the Final Fate of the Formulation.

Since the past decade, there has been growing interest to grant nanoparticles with diffusion properties across mucosae. In this sense, the nonionic block copolymer Pluronic F127 (PF127) has emerged as a promising coating agent to formulate mucus-penetrating particles. In the journey to find efficient coating agents, researchers have focused more on the effect of the coating agent architecture rather than on the role of the physicochemical properties of the nanoparticle used as the substrate. The current knowledge about mucodiffusive particles is in general based on model-like nanoparticles, such as polystyrene or poly(lactic-co-glycolic) acid nanoparticles, but there is a lack of information about the potential of PF127 on other colloidal systems. This work aims to shed some light on this issue by selecting three oils, palm (solid), coconut (semisolid), and wheat germ (liquid), with different physicochemical properties to formulate PF127-coated nanoemulsions. The obtained nanoemulsions were characterized, and their colloidal stability was tested. Their diffusion capacity was determined by particle tracking after challenging the nanoemulsions across an intestinal porcine mucus layer. In accordance with the evidence of model-like nanoparticles, our results state that PF127 allows mucodiffusion, but its effectiveness as a coating agent clearly depends on the physicochemical properties of the nanostructure core over which PF127 is placed. Among other physicochemical properties, the results certainly showed that the hydrophobic character of the nanostructure core emerges as a critical factor in the formulation of successful PF127 coatings.

A Novel Antihypertensive Derived from Adlay (Coix larchryma-jobi L. var. ma-yuen Stapf) Glutelin.

Our previous studies have shown that Coix glutelin pepsin hydrolysate can effectively inhibit angiotensin converting enzyme (ACE) activity in vitro. The main purpose of this study was to obtain potent anti-hypertensive peptides from Coix glutelin. The Coix glutelin hydrolysates (CGH) were prepared by pepsin catalysis and further separated by an ultrafitration (UF) system, gel filtration chromatography (GFC) and reversed-phase high performance liquid chromatography (RP-HPLC). As a result, the sub-fraction F5-3 had the highest ACE-inhibitory activity. Six ACE inhibitory peptides were identifiedusing nano-liquid chromatography coupled to tandem mass spectrometry. The most potent peptide GAAGGAF (IC50 = 14.19 µmol·L-1) was finally obtained by further molecular simulation screening and a series of division and optimization. Single oral administration of synthesized GAAGGAF at 15 mg/kg body weight (BW) in spontaneously hypertensively rats (SHR) could reduce the systolic blood pressure (SBP) around 27.50 mmHg and blood pressure-lowering effect lasted for at least 8 h. The study demonstrated for the first time that the ACE inhibitory peptide GAAGGAF from Coix glutelin has a significant antihypertensive effect, and it could be a good natural ingredient for pharmaceuticals against hypertension and the related diseases.

Lectin Digestibility and Stability of Elderberry Antioxidants to Heat Treatment In Vitro.

Elderberry contains healthy low molecular weight nutraceuticals and lectins which are sequence-related to the elderberry allergen Sam n1. Some of these lectins are type II ribosome-inactivating proteins. The sensitivity of native lectins present in elderberry fruits and bark to the proteolysis triggered by in vitro simulated gastric and duodenal fluids has been investigated. It was found that these lectins are refractory to proteolysis. Nonetheless, incubation for 5-10 min in a boiling water bath completely sensitized them to the hydrolytic enzymes in vitro. Under these conditions neither total Folin-Ciocalteau's reagent reactive compounds, total anthocyanins and the mixture of cyanidin-3-glucoside plus cyanidin-3-sambubioside, nor antioxidant and free-radical scavenging activities were affected by more than 10% for incubations of up to 20 min. Therefore, short-time heat treatment reduces potential allergy-related risks deriving from elderberry consumption without seriously affecting its properties as an antioxidant and free-radical scavenging food.

In vitro digestibility and changes in physicochemical and structural properties of common buckwheat starch affected by high hydrostatic pressure.

High hydrostatic pressure (HHP), a non-thermal processing technology, was applied at 120, 240, 360, 480, and 600MPa to assess its effect on the in vitro digestibility, physicochemical, and structural properties of common buckwheat starch (CBS). HHP treatment resulted in CBS granules with more rough surfaces. With the increasing pressure level, amylose content, pasting temperature, and thermal stability substantially increased and relative crystallinity, hardness, swelling power, and viscosity decreased. At 120-480MPa, HHP did not affect the 'A'-type crystalline pattern of CBS. However, at 600MPa, HHP contributed to a similar 'B'-type pattern. Compared with native starch, HHP-modified CBS samples had lower in vitro hydrolysis, reduced content of rapidly digestible starch, and increased levels of slowly digestible starch and resistant starch. These results revealed that the in vitro digestibility, physicochemical, and structural properties of CBS are effectively modified by HHP.

Extraction optimization of pepsin-soluble collagen from eggshell membrane by response surface methodology (RSM).

Response surface methodology (RSM) was used to investigate the effect of extraction-process variables on pepsin-soluble collagen (PSC) from eggshell membrane. A central composite design (CCD) was employed for experimental design and analysis of the results to obtain the best possible combination of NaOH concentration (X1: 0.4-1.2 mol/l), alkali treatment time (X2: 6-30 h), enzyme concentration (X3: 15-75 U/mg) and hydrolysis time (X4: 12-60 h) for maximum PSC extraction. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and analyzed by appropriate statistical methods. According to the results, optimum extraction conditions were as follows: NaOH concentration of 0.76 mol/l, alkali treatment time of 18 h, enzyme concentration of 50 U/mg and hydrolysis time of 43.42 h. The experimental extraction yield under optimal conditions was found to be 30.049%, which is in good agreement with the predicted value of 30.054%.

Effects of heat treatment on oil-binding ability of rice flour.

Heat-treated (120 °C for 120 min) rice flour showed high affinity to oil (oil-binding ability). This oil-binding ability could be observed by shaking the heat-treated rice flour (2.0 g), oil (4.0 mL), and water (20 mL) vigorously in a test tube, and the oil bound to the rice flour sank into the water. To examine the time-dependent levels of the oil-binding ability, rice flour was heat-treated at 120 °C for 10, 20, 40, 60, and 120 min, and the precipitated volume of oil/rice flour complex increased with an increase of the heating time. The oil-binding ability of the rice flour was not affected by the treatments with diethyl ether or boiled chloroform/methanol (2:1) solutions, which suggested no relationship to the oil in the rice flour, but was lost upon alkali (0.2% NaOH solution) or pepsin treatment, which suggested its relationship to the rice proteins.

Concentrated protein body product derived from rice endosperm as an oral tolerogen for allergen-specific immunotherapy--a new mucosal vaccine formulation against Japanese cedar pollen allergy.

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.

Casein/pectin nanocomplexes as potential oral delivery vehicles.

Delivery systems prepared with natural biopolymers are of particular interests for applications in food, pharmaceutics and biomedicine. In this study, nanocomplex particles of sodium caseinate (NaCas) and pectin were fabricated and investigated as potential oral delivery vehicles. Nanocomplexes were prepared with three mass ratios of NaCas/pectin by acidification using glucono-δ-lactone and thermal treatment. NaCas/pectin at 1:1 mass ratio resulted in dispersions with the lowest turbidity and the smallest and most uniform nanocomplexes. Thermal treatment at 85 °C for 30 min facilitated the formation of stable, compact, and spherical nanocomplexes. Heating not only greatly increased the yield of nanocomplexes but also significantly improved the encapsulation capability of rutin studied as a model compound. Pectin in nanocomplexes delayed the hydrolysis of NaCas by pepsin at gastric conditions and enabled the controlled release of most rutin in simulated intestinal conditions. The nanocomplexes based on food-sourced biopolymers have promising features for oral delivery of nutrients and medicines.

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna (Katsuwonus pelamis).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the spine (ASC-SP and PSC-SP) and skull (ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were (2.47 ± 0.39)%, (5.62 ± 0.82)%, (3.57 ± 0.40)%, and (6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly (330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH (1-5) and lost their solubilities when the NaCl concentration was above 2% (W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.

Enzymes in the dissolution testing of gelatin capsules.

Gelatin capsules are a widely used dosage form both for pharmaceutical drug products as well as dietary supplements. Gelatin in the presence of certain compounds, mainly aldehydes, or in high humidity and high temperature conditions can cross-link. Cross-linking involves covalent bonding of the amine group of a lysine side chain of one gelatin molecule to a similar amine group on another molecule. The covalent bonding is, for practical purposes, irreversible. Cross-linking results in the formation of a pellicle on the internal or external surface of the gelatin capsule shell that prevents the capsule fill from being released. In vitro dissolution testing of cross-linked gelatin capsules can result in slower release of the drug or no release at all. The data obtained by the Gelatin Capsule Working Group, created in the early 90s to investigate noncompliance of gelatin capsules, was used to establish the type and amounts of enzymes that can be added to the dissolution medium in the case of test failure to the presence of cross-linking in the gelatin. The two-tier dissolution testing was included in the US Pharmacopeia and it recommends the addition of pepsin (pH below 6.8) or pancreatin (pH above 6.8) to the medium depending on its pH. Pepsin shows good protease activity up to pH 4 and pancreatin above pH 6 leaving a gap where neither one has good activity. Possible proteolytic enzymes that could be used for the pH range 4-6.8 could be papain or bromelain.

Effects of short-term heating on total polyphenols, anthocyanins, antioxidant activity and lectins of different parts of dwarf elder (Sambucus ebulus L.).

Dwarf elder (Sambucus ebulus L.) berries are rich in health-promoting phytochemicals such as polyphenols and anthocyanins, and display a significant antioxidant activity. They are also rich in two lectins (ebulin f and SELfd) that share amino acid sequence homology with the elderberry allergen Sam n1 present in Sambucus nigra pollen and fruits. Ebulin f displays toxicity by oral ingestion. This study was aimed at eliminating the toxicity of these lectins whilst having little or no effect on the antioxidant properties of dwarf elder berries. We thus investigated the potential effects of incubation in a boiling water bath of extracts from several parts of the plant on total polyphenol content, antioxidant activity, total anthocyanins, cyanidin-3-glycoside content, and the sensitivity of purified dwarf elder fruit lectins to a simulated gastric fluid. The study shows that five minutes of said heat treatment fully sensitized both lectins to pepsin digestion, whilst minimally reducing phenol and antioxidant as well as free radical scavenging activities to below 13%. It proved possible to eliminate the potential risks derived from the presence of lectins in dwarf elder juices without any significant reduction in the content of the antioxidant compounds. Dwarf elder berries may thus be a valuable nutritional source.

Characterisation of acid-soluble and pepsin-solubilised collagen from jellyfish (Cyanea nozakii Kishinouye).

Annual outbreaks of the Jellyfish (Cyanea nozakii Kishinouye) in the waters of the Yellow Sea and the East China Sea are regarded as a nuisance. Thus, utilizing this jellyfish species is of great significance to reduce harm to fisheries and marine environments. The yield of the acid-soluble collagens (ASCs) from the C. nozakii umbrella was 13.0% (dry weight) and that of the pepsin-solubilised collagens (PSCs) was 5.5% (dry weight). The SDS-PAGE patterns of the ASCs and PSCs differed from that of type I collagen, which indicate the presence of (α1)3. The denaturation temperature (Td) of the collagens was approximately 23.8°C. Fourier transform infrared spectroscopy proved that the ASCs and PSCs retained their helical structures and the As, Pb, and Hg content of the collagens, detected by ICP-MS, were considerably lower than the national standards. The results suggest that collagens isolated from C. nozakii can potentially be used as an alternative source of collagen for use in various applications.

Intragastric gelation of whey protein-pectin alters the digestibility of whey protein during in vitro pepsin digestion.

The aim of this work is to investigate the effect of pectin on in vitro digestion of whey protein. Digestion of heated whey protein isolate (WPI) and pectin solutions (WPI-pectin) as influenced by pectin concentration and pH was studied under simulated gastric conditions. Electrophoresis, dynamic light scattering, colorimetric measurements, and gel microstructures were used to study the digestion pattern. At low pectin concentration (0.25% w/w), pectin did not significantly influence the degradation of whey protein. Increasing the pectin concentration to 1% led to extensive intragastric gelation immediately after mixing with simulated gastric fluid. The microstructure of intragastric gel from WPI-pectin at pH 6.0 showed a more interconnected and denser gel network than that at pH 7.0. More protein and pectin were involved in the gelation at pH 6.0 than pH 7.0. The digesta of samples at pH 6.0 was mainly composed of peptides, while that at pH 7.0 mostly consisted of aggregates and crosslinked peptides. This study suggests that WPI-pectin at high biopolymer ratio formed intragastric gel in simulated gastric models, which could delay protein digestion and potentially slow gastric emptying and promote satiety.