Isolation and allergenicity evaluation of glycated α-lactalbumin digestive products and identification of allergenic peptides.

This study aimed to isolate and evaluate the allergenicity of glycated α-lactalbumin (ALA) digestive products and identify its allergenic peptides. The digestive products of native-, alone glycated- and ultrasound-assisted glycated ALA (ALA-D, ALA-gal-D, 100ALA-gal-D) were isolated into three fractions (F1, F2 and F3). High-resolution mass spectrometry showed that the digestion-resistant peptides of F2 and F3 mainly distributed in amino acid sequence (AA) 25-31, AA32-53, AA40-53, AA54-60, AA80-90, AA94-104. The allergenicity of the three fractions of glycated ALA was lower than that in ALA-D, indicating glycation of ALA could indeed reduce its allergenicity after digestion. Furthermore, most fractions isolated from high glycation-degree ALA had the lowest allergenicity. The IgG/IgE binding abilities of synthesized peptides indicated that AA94-104 firstly identified by us embodied the strongest allergenicity and might be the potential allergenic peptide. This will provide a theory for preparing hypoallergenic products based on the identified allergenic peptides.

Flavor, antimicrobial activity and physical properties of gelatin film incorporated with of ginger essential oil.

Adding essential oil into the gelatin-based film can enhance the antibacterial activity of the film, but excessive amounts of addition will bring the film an unpleasant flavor and reduce its mechanical performance. Hence, we prepared functional gelatin-based films by incorporating low content of ginger essential oil (GEO). The flavor of GEO was not detected from the films containing less than 1% GEO. The antimicrobial activity of films was found to be proportional to GEO content. As GEO content increased from 0 to 1%, the value of water vapor permeability (WVP) and elongation at break (EAB) increased, whereas the value of tensile strength (TS) of film decreased. The Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy analysis revealed the vibration of gelatin film was affected by adding low content of essential oil. Surface morphologies demonstrated oil droplets and a discontinuous structure, and cross-section morphologies proved the formation of a loose structure as GEO was incorporated in the film through SEM. Sensory evaluation revealed that composite films incorporated with 0.5% GEO exhibited the best performance. The resulting films can be used as antimicrobial packaging materials with good physical properties and sensory performance.

Mechanism on the Allergenicity Changes of α-Lactalbumin Treated by Sonication-Assisted Glycation during In Vitro Gastroduodenal Digestion.

Physical-assisted chemical modification is effective to reduce the allergenicity of α-lactalbumin (ALA). However, there are few in-depth studies on the allergenicity changes of physical-assisted chemical-modified ALA during digestion. The effect of gastroduodenal digestion on the allergenicity changes of ALA treated by sonication-assisted glycation was assessed. Digestion of both ALA and its glycated forms generated peptide fractions, and intact undigested glycated ALA in the hydrolysates still covalently bound to d-galactose. High-resolution mass spectrometry revealed that a higher glycation degree was discovered in sonication-preprocessed ALA compared to native ALA. Enzyme-linked immunosorbent assay and basophil degranulation showed that sonication-assisted glycation could significantly reduce ALA allergenicity. The allergenicity of both gastric and gastroduodenal hydrolysates was further increased, and the hydrolysates of sonication-assisted glycated ALA showed the lowest allergenicity. The reason could be the shielding effect of the linear epitope found to be caused by a higher glycation degree; although linear epitopes were exposed, d-galactose covalently bound to intact undigested glycated ALA in the hydrolysates retained its masking role. These results indicated that sonication-assisted glycation could be a promising method to prepare immunotherapeutic agents for allergen immunotherapy to achieve the purpose of allergy desensitization.

Mechanism of the Reduced IgG/IgE Binding Abilities of Glycated ß-Lactoglobulin and Its Digests through High-Resolution Mass Spectrometry.

Glycation between proteins and reducing sugars is the common chemical modification in food protein, and many studies have focused on the allergenicity of the glycated protein. However, a systemic study on the allergenicity change of its digests is lacking. In this work, we explored the change rule of the digestibility and allergenicity of glycated ß-Lg during in vitro gastrointestinal digestion and interpreted the mechanism using high-resolution mass spectrometry. Glycation with arabinose increased the resistance of ß-Lg to digestive enzyme, with a low hydrolysis value. Indirect competitive ELISA showed that the IgG/IgE binding rates of ß-Lg were reduced after glycation and further reduced after digestion, in comparison with the digests of unglycated ß-Lg. There are two reasons for this phenomenon. On the one hand, 11 glycated sites were determined in the lowest allergenicity arabinose-ß-Lg conjugation (Ara-ß-Lg), which was distributed in the IgG and IgE linear allergic epitopes of ß-Lg. On the other hand, glycation masking linear allergenic epitopes had a more significant effect on reducing allergenicity in comparison to digestive enzyme hydrolysis. These results indicated that the allergenicity of Ara-ß-Lg in the human body might be lower than that of unglycated ß-Lg.

Flavor, antimicrobial activity, and physical properties of composite film prepared with different surfactants.

Different surfactants (lecithin, Tween-20, and Tween-80) were added in composite film during the preparation. Flavor, antimicrobial activity, and physical properties of ginger essential oil -gelatin film were investigated, in order to study the effect of surfactants on the properties of film. The flavor of GEO was not detected in the film prepared with Tween-20 and film prepared with Tween-80, and these two films exhibited stronger antimicrobial activity; film prepared with lecithin possessed higher value in thickness, elongation at break, water solubility, ΔE and opacity, lower value in water vapor property, and tensile strength; attenuated total reflectance-Fourier transform infrared spectrum results suggested, Tween-20 and Tween-80 enhanced the strength of covalent bond, and lecithin weakened the strength of hydrogen bond; and the result of scanning electron microscope showed that Tween-20 and Tween-80 improved the dispersion of oil droplets in film. Therefore, this study suggested that surfactants had an influence on the physical properties and molecular structure of a resulting film; in addition, Tween-20 and Tween-80 could reduce the flavor of GEO in film, improving the antimicrobial activity of film.

Effects of γ-polyglutamic acid on the gelling properties and non-covalent interactions of fish gelatin.

The effects of γ-polyglutamic aid (γ-PGA) on the gelling properties and non-covalent interactions of fish gelatin were investigated. The gel strength and melting temperature of fish gelatin gradually increased, with increasing γ-PGA concentration, although there was no significant change when the γ-PGA concentration was greater than 0.04%. As the concentration of γ-PGA increased, the electrostatic interaction of fish gelatin increased and the hydrophobic interaction between gelatin molecules decreased. The fish gelatin system was comprised of γ-PGA concentrations of 0.04 and 0.06% showing a strong hydrogen bond. When the γ-PGA concentration increased from 0 to 0.04%, more phenolic hydroxyl groups in the tyrosine residue tended to form hydrogen bonds with the protein. However, an additional increase in γ-PGA concentration to 0.1% led to enhanced hydrogen bonding with water molecules. The results of this study showed that hydrogen bonds played an important role in improving the gelling properties of gelatin by γ-PGA.

Mitigation of isoquercitrin on ß-lactoglobulin glycation: Insight into the mechanisms by mass spectrometry and interaction analysis.

Formation of advanced glycation end products (AGEs) on foods imposes threats to human health after intaking. This research firstly evaluated the inhibition of isoquercitrin on ß-lactoglobulin (ß-Lg) glycation, the mechanisms were elucidated by fluorescence spectroscopy, Orbitrap MSn and molecular docking. Fluorescence spectra indicated that isoquercitrin effectively alleviated the formation of AGEs, it could stabilize the conformation structure of glycated ß-Lg (G-ß-Lg), change the micro-environment in the vicinity of chromophores. SDS-PAGE analysis revealed the suppressed cross-linking of G-ß-Lg induced by isoquercitrin. The number of glycation site detected on G-ß-Lg was reduced from ten to eight after the addition of isoquercitrin, and the relative glycation degree of substitution of per site (RGDSP) of most glycation sites were also greatly decreased. As indicated by intermolecular interaction, isoquercitrin quenched the fluorescence of ß-Lg via a static mechanism, and their combination is an endothermic processing mainly derived by hydrophobic interaction, hydrogen bonds, and van der Waals forces. Isoquercitrin interacted with ß-Lg to form an equimolar complex, and one hydrogen bond was formed between isoquercitrin and Lys69 (4.96 Å). Above results proved that isoquercitrin can be a promising anti-glycation agent used in food system to prevent the formation of harmful glycation products.

Effect and Mechanism of Elaeagnus angustifolia Flower and Its Major Flavonoid Tiliroside on Inhibiting Non-enzymatic Glycosylation.

This study aimed to evaluate the antiglycation ability of Elaeagnus angustifolia flower extract and to elucidate the mechanism with its major compound. The results indicated that E. angustifolia flower extract and its major compound tiliroside (24.2 mg/g of extract) exhibited excellent antiglycation ability with inhibition rates of 92.1 and 78.9% at 37.5 µg/mL, which are much higher than that of aminoguanidine (55.3% at 37.5 µg/mL). The stable tiliroside-ovalbumin (OVA) complexes were formed through a spontaneous exothermic progress in an equimolar manner, and hydrophobic interaction, hydrogen bond, and van der Waals forces were the major driving forces. Tiliroside could significantly ameliorate the conformation changes of OVA induced by the glycation reaction, quench its fluorescence by a static mechanism, and change the microenvironment adjacent to tryptophan and tyrosine. Molecular docking revealed that tiliroside inserted into the OVA hydrophobic pocket resulted in the formation of five hydrogen bonds. Orbitrap tandem mass spectrometry showed that tiliroside significantly suppressed the glycation of OVA, and the number of glycation sites was reduced from 9 to 5 after tiliroside was added. The above results indicated that E. angustifolia flowers and tiliroside have a good antiglycation effect and can be used as food additives to suppress the undesired glycation reaction during food processing.

Observation of the structural changes of α-lactalbumin induced by ultrasonic prior to glycated modification.

Bovine α-lactalbumin (BLA) was treated by ultrasonic at 150 W/cm2 for different times and subsequently glycated with mannose by dry-heating. Molecular weight, intrinsic fluorescence spectra, glycation sites and degree of modified BLA were observed. The proteinaceous high molecular weight components were formed after ultrasonic prior to glycated modification, while the conformational changes were obvious. Prior to ultrasonic pretreatment, K62, K114, and K122 of BLA were identified. After treated by ultrasound at 150 W/cm2 for 5, 10, 15, and 20 min, the sites were increased to four, four, five, and five, respectively. All glycated sites of modified BLA exhibited a higher degree of substitution per peptide (DSP) values compared to native BLA. Ultrasonic at 150 W/cm2 for 20 min revealed the most significant change in the BLA structure. Therefore, conformational changes, the intensified glycation site, and DSP value were responsible for the structural changes of BLA. Practical applications BLA is suitable as an ingredient for infant nutrition in food, and has immune-modulating, antioxidant, antibacterial, and antitumor activity etc. This study revealed that the structural changes of BLA induced by ultrasonic prior to glycated modification. It will be beneficial to understand the mechanism of the functional changes of modified BLA. Ultrasonic prior to glycated modification will be more likely to develop a practical technology to modify protein in the food industry, and improve the functional characteristics of food, such as produce hypo-allergenic cow's milk in future.

Reduced IgE/IgG binding capacities of bovine α-Lactalbumin by glycation after dynamic high-pressure microfluidization pretreatment evaluated by high resolution mass spectrometry.

Dynamic high-pressure microfluidization (DHPM) pretreatment and glycation with lactose were employed to modify α-Lactalbumin (α-LA) with respect to the IgE/IgG binding capacities. No significant difference on incorporation ratio value of glycated α-LA was observed with and without DHPM pretreatment. However, IgE/IgG binding capacities of α-LA were decreased after glycation and DHPM pretreatment promoted the reduction. The lowest IgE/IgG binding capacities of glycated α-LA were obtained by DHPM pretreatment at 110 MPa. Native α-LA was mainly glycated at K62, K94, K98, whereas glycation sites and degree of substitution per peptide (DSP) were added after DHPM treatment. Therefore, the reduced IgE/IgG binding capacities of α-LA was attributed to the characteristics of glycated sites, including the amount, location, and DSP values. Interestingly, K98 played the most important role in decreasing IgE/IgG binding capacities of α-LA. The study revealed that glycation combined with DHPM was a promising way to decrease the allergenicity of proteins.

HPLC-QTOF-MS/MS profiling, antioxidant, and α-glucosidase inhibitory activities of Pyracantha fortuneana fruit extracts.

This study was carried out to optimize the solvent for extracting the antioxidants and α-glucosidase inhibitors (AGIs) from Pyracantha fortuneana fruit (PFF) and the major chemical components were characterized by HPLC-QTOF-MS/MS. The results showed that 50% and 70% acetone (v/v, ml/ml) gave the best extraction efficiency on phenolics and total flavonoids, while 70% acetone and 50% methanol possess better recovery on protein and polysaccharides, respectively. In addition, the 50% and 70% acetone extracts gave the strongest radical scavenging ability and α-glucosidase inhibitory activity (p > 0.05), but the Fe3+ reducing power of the 50% acetone extract was higher than that of 70% acetone. Correlation analysis indicated that phenolic acids and flavonoids were connected to the antioxidant activity and α-glucosidase inhibitory activity closely. Moreover, 25 compounds including 7 flavonoids, 6 phenolic acids, 7 organic acids, 3 tannins, 1 terpene, and 1 alkaloid were identified or tentatively identified in the 50% acetone extract. Overall, 50% acetone can be a proper solvent for extracting antioxidants and AGIs from PFF. PRACTICAL APPLICATIONS: Imbalance between production and clearance of reactive oxygen species (ROS) in the body could induce various chronic diseases. PFF is an edible fruit beneficial to human health; it is reported to be capable of optimizing blood glucose levels and may prevent premature aging. In the present study, PFF was found to be excellent in antioxidant activities and α-glucosidase inhibitory ability; 50% acetone was found to be the best extraction solvent. In addition, the predominant phytochemical components of the 50% acetone extract were characterized. This study can promote further research of Pyracantha fortuneana in natural functional products, especially in the prevention of type II diabetes and its complication.

Insights into the Mechanism of Quercetin against BSA-Fructose Glycation by Spectroscopy and High-Resolution Mass Spectrometry: Effect on Physicochemical Properties.

Quercetin has been reported to suppress protein glycation or the formation of advanced glycation end-products (AGEs), but the inhibition mechanism related to protein structure and glycation sites and the influence on physicochemical properties remain unclear. The aim of the current research was to investigate the mechanism of quercetin against glycation with BSA-fructose as model by spectroscopic and spectrometric techniques. Changes in physicochemical properties were evaluated by antioxidant activity and emulsifying properties. The results indicated that quercetin dose-dependently inhibited the glycation of BSA by attenuating the alteration of conformational structure and microenvironment induced by glycation. It could also suppress the cross-linking or aggregation of glycated BSA, which reflected in the decreased molecular weight determined by SDS-PAGE and MALDI-TOF. Nanoliquid chromatography coupled to Q-Exactive tandem mass spectrometry analysis revealed the mapping of 20, 23, 19, and 19 glycation sites in glycated BSA with 0, 0.5, 1.5, and 3.0 mM quercetin, respectively. Quercetin changed the glycation sites of BSA, but it could not reduce the number greatly. In addition, quercetin reduced the antioxidant ability and increased the emulsifying properties of BSA, while negligible efficiency was observed on the antioxidant activity and emulsifying activity index of glycated BSA.

The Mechanism of Decreased IgG/IgE-Binding of Ovalbumin by Preheating Treatment Combined with Glycation Identified by Liquid Chromatography and High-Resolution Mass Spectrometry.

Ovalbumin is one of the most important sensitizing ingredients in allergens of egg albumin, which restricts the application of egg in the field of food processing. Previous research has indicated that glycation could cause the protein to partially expand, which may bring about the destruction of the structural IgG and IgE epitopes and induce the decline of the IgG- and IgE-binding ability of ovalbumin. In this research, the effect of a preheating treatment integrated with glycation on the IgG- and IgE-binding capability and the conformation changes of ovalbumin was studied by detecting the glycated sites and the values of degree of substitution per peptide (DSP) by liquid chromatography and high-resolution mass spectrometry (LC-HRMS). Interestingly, we found that a glycation site (K227) attached by two ribose molecules was detected in glycated ovalbumin with preheating treatment. In addition, a new glycation site (K323) appeared in G-60. The results displayed that preheating treament could strengthen the changes in the secondary and tertiary structure of ovalbumin by enhancing glycation and further reduce the IgG/IgE-binding ability by integrating with glycation because of the cover of IgG and IgE epitopes. Therefore, preheating treatment integrated with glycation may offer a way for ovalbumin to reduce sensitization.

[Chemical constituents from the tuber of Cremastra appendiculata].

To study the chemical constituents of "Shan-Ci-Gu" (the tuber of Cremastra appendiculata (D. Don) Makino), the compounds were isolated with silica gel and reverse phase silica gel as well as Sephadex column chromatographic method. Their structures were elucidated on the basis of modern spectra technology. Seven compounds were isolated and identified as 5-methoxybibenzyl-3, 3'-di-O-beta-D-glucopyranoside (1), militarine (2), loroglossin (3), protocatechuic acid (4), succinic acid (5), gastrodin (6), and daucosterol (7). Compound 1 is a new compound. Compounds 2 -6 were isolated from this plant for the first time.

New prenylchalcones from the hops of Humulus lupulus.

Two new prenylchalcones, xanthohumol C and D, together with two known prenylchalcone derivatives, xanthohumol (1) and 5''-(2''-hydroxyisopropyl)-dihydrofurano-[2'',3''-b]-4,4'-dihydroxy-6'-methoxy-chalcone (4) were isolated and identified from the hops of Humulus lupulus L. The structures of xanhohumol C and D were elucidated as 5''-hydroxy-6'',6''-dimethyl-dihydropyrano-[2'',3''-b]-4,4'-dihydroxy-6'-methoxy-chalcone (2) and 5''-hydroxy-6'',6''-dimethyl-dihydropyrano-[2'',3''-b]-4',6'-dimethoxy-4-hydroxy-chalcone (3) on the basis of HRFAB-MS, 1D and 2D NMR (HMQC, HMBC) spectroscopic data. Among the new prenylchalcones, compound 2 showed marginal cytotoxic activity against human stomach carcinoma BGC-823 and human hepatic carcinoma HepG2 cells.

[Studies on flavones in of Lavandula augustifolia].

OBJECTIVE: To study on the chemical constituents of Lavandula augustifolia. METHOD: The compounds were extracted with 95% ethyl alcohol, isolated by various column chromatography and identified by spectroscopic methods. RESULT: Seven flavanoids were isolated and identified as apigenin (1), ladanein (2), apigenin-7-O-beta-D-(6'-p-hydoxy-cinnamoyloxy) -mannoside (3), luteolin (4), apigenin-7-O-beta-D-glucoside (5), luteolin-7-O-beta-D-glucoside (6), 5, 4'-dihydroxy flavonoid-7-O-beta-D-pyranglycuronate buthyl ester (7). CONCLUSION: All of these seven compounds were obtained from this plant for the first time.

DEPBT as an efficient coupling reagent for amide bond formation with remarkable resistance to racemization.

3-(Diethoxyphosphoryloxy)-1, 2, 3-benzotriazin-4(3H)-one (DEPBT) is an effective coupling reagent for synthesis of linear and cyclic peptides by both solution and solid-phase peptide synthesis. DEPBT mediates amide bond formation with remarkable resistance to racemization. When DEPBT is used as a coupling reagent, it is not necessary to protect the hydroxy group of the amino component (such as tyrosine, serine, and threonine) and the imidazole group in the case of histidine. The high efficiency of DEPBT and its utility have been demonstrated in the syntheses of complex natural products such as ramoplanin A2, ramoplanose aglycon, ustiloxin, and teicoplanin aglycon.

[Studies on the flavonoids in herb from Scutellaria barbata].

OBJECTIVE: To study flavonoids in Scutellaria barbata and elucidate their structures. METHOD: Compounds were isolated and purified by normal column chromatography, paper thin layer chromatography and Sephadex chromatography. The chemical structures were elucidated by ESI mass and NMR spectra. RESULT: Five flavonoids were isolated from S. barbata and identified as apigenin (1), luteolin (2), ethyl-7-O-apigenin-glucuronate (3), apigenin-7-O-beta-glucoside (4), and apigenin-7-O-neohesperidoside (5), in addition to benzyaldehyde (6). CONCLUSION: Flavonoidglycoside (3), (4) and (5) were isolated from S. barbata for the first time.

Studies on the synthesis of cyclic pentapeptides as LHRH antagonists and the factors that influence cyclization yield.

Six cyclic pentapeptides containing two or three non-protein amino acids have been synthesized by cyclization of linear precursors in dilute solution and characterized by TLC. HPLC, NMR, melting point. specific rotation etc. A total of 72 cyclization reactions were carried out to study the factors that influence head-to-tail cyclization: linear precursor sequence, coupling reagent, residue configuration, the proportion of DMAP additive, concentration, reaction temperature and reaction time. The cyclic pentapeptides will be modified by active moieties and evaluated as LHRH antagonists.