Extract of Unifloral Camellia sinensis L. Pollen Collected by Apis mellifera L. Honeybees Exerted Inhibitory Effects on Glucose Uptake and Transport by Interacting with Glucose Transporters in Human Intestinal Cells.

Bee pollen possesses potential hypoglycemic effects but its inhibitory mechanisms on glucose absorption and transportation in intestinal cells still need to be clarified. Here, we determined the inhibitory effects of bee pollen extract originating from Camellia sinensis L. (BP-Cs) as well as its representative phenolic compounds on glucose uptake and transport through a human intestinal Caco-2 cell monolayer model. It showed that three representative phenolic compounds, including gallic acid (GA), 3-O-[6'-O-(trans-p-coumaroyl)-ß-d-glucopyranosyl]kaempferol (K1), and 3-O-[2',6'-di-O-(trans-p-coumaroyl)-ß-d-glucopyranosyl]kaempferol (K2), with contents of 27.7 ± 0.86, 9.88 ± 0.54, and 7.83 ± 0.46 µg/mg in BP-Cs extract, respectively, exerted mutual antagonistic actions interacting with glucose transporters to inhibit glucose uptake and transport based on their combination index (CI) and molecular docking analysis. K1, K2, and GA might compete with d-glucose to form hydrogen bonds with the same active residues including GLU-412, GLY-416, GLN-314, and TRP-420 in GLUT2. These findings provide us a deep understanding of the mechanisms underlying the anti-hyperglycemia by bee pollen, which provide a new sight on dietary intervention strategies against diabetes.

Development of mucoadhesive hydrogels based on polyacrylic acid grafted cellulose nanocrystals for local cisplatin delivery.

To fabricate a mucoadhesive hydrogel with superior properties for local delivery of cisplatin (CDDP) to colorectal cancer, a hardcore bottle-brush polymer (HCBBP) was developed through grafting of poly(acrylic acid) (PAA) on cellulose nanocrystals (CNC) at 6, 9 and 12 CNC:PAA w/w ratios. The developed materials were characterized by acid-base titrations, FT-IR, electron microscopy, muco-rheological behaviour in the presence of mucin, in vitro drug release and anticancer activity against human HCT-116 colorectal cancer cells. The results showed CNC-g-PAA9 to have superior rheological behavior in the presence of mucin compared to CNC and other gels under study indicating beneficial mucoadhesive characteristics. CNC-g-PAA9:CDDP complex showed slow CDDP release causing a significant increase in IC 50 of the drug (> 3-fold) against HCT116 cells. The developed CNC-PAA9 hydrogel showed no intrinsic cytotoxicity on its own. The results point to a great promise for CNC-g-PAA9 as mucoadhesive hydrogels for local platinum delivery in colorectal cancer.

Cold non-enzymatic browning of glucosamine in the presence of metmyoglobin induces glucosone and deoxymyoglobin formation.

Glucosamine (GlcN) and GlcN-myoglobin reaction systems were incubated at 4 °C to verify that GlcN can go through non-enzymatic browning at this low temperature, and to test the hypothesis that certain reductones from GlcN non-enzymatic browning can promote the formation of deoxy- and oxymyoglobin from metmyoglobin reduction. Remarkably, alpha-dicarbonyls and self-condensation products, fructosazine and deoxyfructosazine, were produced at this relatively low temperature. The presence of myoglobin shifted GlcN non-enzymatic browning toward the formation of glucosone and fructosazine. When glucosone (250-2000 mg/L) was incubated with myoglobin it contributed to the formation of deoxymyoglobin, indicating its capacity to reduce metmyoglobin. This study opens the possibility of using GlcN in meat products to increase oxy- and deoxymyoglobin and enhance the color of meat.

The effect of amino acids on non-enzymatic browning of glucosamine: Generation of butterscotch aromatic and bioactive health compounds without detectable levels of neo-formed alkylimidazoles.

A mixture of glucosamine (GlcN, 15% w/v) and different amino acids in 1:1 M ratio was incubated at 70 °C for 12 h. The resulting GlcN-amino acid caramels were analysed for α-dicarbonyl compounds, polyhydroxyalkyl pyrazines, heterocyclic compound and alkylimidazoles. All the analyses were performed by using HPLC-MS/MS followed by pooling the variables with principal component analysis (PCA). GlcN-Gly caramels generated the greatest amount of butterscotch aromatic compound diacetyl and polyhydroxyalkyl pyrazines (fructosazine and deoxyfructosazine). The potentially toxic heterocyclic compound, 5-hydroxymethylfurfural (HMF) was generated in greater amounts with the GlcN-Arg caramels. However, the toxic alkylimidazoles (4-MEI and THI) were not present in any of the GlcN-amino acid caramels. The results suggest that caramel with butterscotch aroma and bioactivity can be produced with GlcN-amino acid at 70 °C. The PCA performed discriminated the majority of the GlcN-amino acid combinations; GlcN-Gly and GlcN-Ser were best discriminated.

Bee Pollen Extracts Modulate Serum Metabolism in Lipopolysaccharide-Induced Acute Lung Injury Mice with Anti-Inflammatory Effects.

Bee pollen (BP) collected from different floras possesses various potential bioactivities, but the mechanism-related research on anti-inflammatory effects is limited. Here, three types of BP originating from Camellia sinensis L. (BP-Cs), Nelumbo nucifera Gaertn. (BP-Nn), and Brassica campestris L. (BP-Bc) were assessed using molecular and metabolomics methods to determine their anti-inflammatory effects. The differences in polyphenolic abundance of three types of BP extracts were determined by HPLC-DAD/Q-TOF-MS. In vitro anti-inflammatory effects of three BP extracts were evaluated in a lipopolysaccharide (LPS)-induced RAW 264.7 cells model. BP-Cs extract with the most abundant polyphenols was found to be the most effective in reducing inflammation by downregulating inflammatory-related genes expression and blocking the activation of MAPK and NF-κB signaling pathways. Polyphenol-rich BP-Cs was further evaluated for their in vivo anti-inflammatory effect in a LPS-induced acute lung injury mouse model. An UPLC-Q-TOF/MS-based metabolomics approach was applied to analyze metabolite changes in mouse serum. Weshowed that the pretreated BP-Cs extract alleviated inflammation and regulated glycerophospholipid metabolism significantly. Our findings provide a foundation for developing and justifying BP as a potential anti-inflammatory ingredient in functional foods or nutraceutical formulations.

Improved bactericidal capacity of UV-B radiation against E. coli strains by photosensitizing bacteria with fructosazine - An advanced Maillard reaction product.

This study investigated the effect of UV-B irradiation and the combinational effect with glucosamine caramel, fructosazine and riboflavin on the antimicrobial activities against Bacillus subtilis (ATCC 6633) and two strains of Escherichia coli (AW 1.7 and ATCC 25922). The quantum yield of fructosazine was two times less than that of tryptophan, indicating its ability to emit fluorescent light but less efficiently than tryptophan. UV-B treatment alone was efficient to achieve a bactericidal effect for both E. coli stains tested, however no effect was found for Bacillus subtilis for up to 80 mJ/cm2 UV-B. The combination of UV-B with photosensitizers fructosazine, glucosamine caramel and riboflavin enhanced the UV-B efficacy against E. coli strains at lower UV-B doses, while Bacillus subtilis ATCC 6633 was more resistant to the treatment combinations. High-performance liquid chromatography showed the production of different fructosazine reaction products occurred during irradiation, including the possible formation of endoperoxides.

Sous-Vide Nonenzymatic Browning of Glucosamine at Different Temperatures.

Sous-vide is an increasingly popular method of cooking under controlled conditions of temperature and time inside vacuumed pouches to preserve the nutritional and sensory qualities of food. Sous-vide nonenzymatic browning of glucosamine (GlcN) was investigated at 50, 60, and 70 °C for 12 h. Changes investigated were pH, color, level of browning, and the concentrations of the key Maillard and caramelization reaction products, including α-dicarbonyls and pyrazines. The concentrations of undesired 4-methylimidazole (4-MEI), 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), and 5-hydroxymethylfurfural (5-HMF) were also determined. Six types of caramels were produced of unique composition with no detectable levels of 4-MEI. GlcN caramels produced under vacuum were more acidic and lighter in color, containing significantly less flavorful diacetyl, but more fructosazine (FR) as compared to nonvacuum caramels. THI concentration was well below the toxicity levels for all studied caramels. Principal component analyses showed that the incubation temperature played a key role in determining the composition of caramels.

Inhibitory activity of a Concanavalin-isolated fraction from a glucosamine-peptides reaction system against heat resistant E. coli.

Alcalase-derived gelatin hydrolysates were glycated with glucosamine in the presence (+) or absence (-) of transglutaminase (TGase), and their antimicrobial activities toward Escherichia coli AW 1.7 were studied. Glycation treatments were subjected to concanavalin A affinity chromatography to selectively collect the glycopeptide-enriched fractions and the changes in antimicrobial activity were determined. The minimum inhibitory concentration of glycated hydrolysates decreased by 1.2 times compared to the native hydrolysate, with no differences between (+) or (-) TGase treatments. No difference was observed in the dicarbonyl compound concentration between the two glycation methods except that 3-deoxyglucosone was greater in the TGase-mediated reaction. Concanavalin A-retentate, but not the flow-through fractions, significantly improved the antimicrobial activity, however there was no difference between +TGase and -TGase glycated treatments. Purification of the retentate fraction from fluorescent compounds did not improve its antimicrobial activity.

Transport of the Glucosamine-Derived Browning Product Fructosazine (Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco-2 Cell Monolayer: Role of the Hexose Transporters.

The transport mechanism of fructosazine, a glucosamine self-condensation product, was investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1 and 2, phloretin and quercetin, inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters was further studied using downregulated or overexpressed cell lines. The apparent permeability (Pa,b) of fructosazine was 1.30 ± 0.02 × 10-6 cm/s. No significant (p > 0.05) effect was observed in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin, phloretin, and quercetin decreased fructosazine transport. The downregulated GLUT cells line was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or GLUT2 and SGLT are mainly responsible for fructosazine transport.

Transglutaminase-catalyzed amination of pea protein peptides using the biogenic amines histamine and tyramine.

BACKGROUND: Biogenic amines (BAs) are produced by the enzymatic decarboxylation of amino acids, and are well-known for their toxicity to humans. This study describes a new method using microbial transglutaminase (MTGase) to covalently link BAs such as histamine (HIS) and tyramine (TYR) to the glutamine residues of alcalase-hydrolyzed pea protein (PPH). RESULTS: The incubation of PPH and HIS and TYR in the presence of MTGase at 37 °C led to the formation of conjugates, as determined by liquid chromatography, after derivatization with dansyl chloride. Seventy-six % of HIS and 65% of TYR were covalently incorporated to PPH by MTGase. The incubation of PPH and TYR in the presence of MTGase exhibited a 52% DPPH radical scavenging activity at 10 mg mL-1 . Conjugation via MTGase improved the antioxidant status by reducing lipid peroxidation. CONCLUSION: This study emphasizes that the application of MTGase can effectively reduce histamine and tyramine content while simultaneously enhancing antioxidative capacity of PPH. © 2016 Society of Chemical Industry.

Fructosazine, a Polyhydroxyalkylpyrazine with Antimicrobial Activity: Mechanism of Inhibition against Extremely Heat Resistant Escherichia coli.

Fructosazine is a polyhydroxyalkylpyrazine recently reported to have antimicrobial activity against heat-resistant Escherichia coli AW 1.7. This study investigated fructosazine's antimicrobial mechanism of action and compared it to that of riboflavin. Fructosazine-acetic acid was effective in permeabilizing the outer membrane based on an evaluation of bacterial membrane integrity using 1-N-phenyl-1-naphthylamine and propidium iodide. The uptake of fructosazine by E. coli was pH-dependent with a greater uptake at pH 5 compared to pH 7 for all times throughout 16 h, except 2, 3, and 10 h. Fructosazine generates 1O2, which is partially why it damages E. coli. DNA fragmentation was confirmed by fluorescence microscopy, and the fructosazine-acetic acid was the second most intense treatment after riboflavin-acetic acid. Electron microscopy revealed membrane structural damage by fructosazine at pH 5 and 7. This study provides evidence that fructosazine exerts antimicrobial action by permeabilizing the cell membrane, damaging membrane integrity, and fragmenting DNA.

Iron (Fe(2+))-Catalyzed Glucosamine Browning at 50 °C: Identification and Quantification of Major Flavor Compounds for Antibacterial Activity.

Glucosamine browning at 50 °C with (GlcN/Fe(2+)) or without iron (GlcN) was studied over time from 0 to 48 h. Generation of reactive oxygen species (ROS), H2O2, and (1)O2, along with α-dicarbonyls, fructosazine, and deoxyfructosazine, was evaluated. Singlet oxygen generation increased over time and was greater in GlcN/Fe(2+) caramel solution. The presence of iron significantly increased the concentration of α-dicarbonyls at an early incubation time (3 h). Fructosazine and deoxyfructosazine were the major degradation products at 48 h comprising together up to 37 and 49% in GlcN and GlcN/Fe(2+), respectively. GlcN/Fe(2+) (48 h) exhibited a MIC50 against highly heat-resistant Escherichia coli AW 1.7 at pH 5, but not at pH 7. Despite several antimicrobial compounds being produced during browning, GlcN/Fe(2+) created a synergistic environment for the fructosazine-organic acids to confer their antimicrobial activity. GlcN caramel solutions have the potential to serve as both flavoring compounds and antimicrobial agents in formulated food systems.

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation.

The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a ß-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a ß-sheet structure.

Studies on the Formation of Maillard and Caramelization Products from Glucosamine Incubated at 37 °C.

This experiment compared the in vitro degradation of glucosamine (GlcN), N-acetylglucosamine, and glucose in the presence of NH3 incubated at 37 °C in phosphate buffer from 0.5 to 12 days. The reactions were monitored with UV-vis absorption and fluorescence emission spectroscopies, and the main products of degradation, quinoxaline derivatives of α-dicarbonyl compounds and condensation products, were determined using UHPLC-UV and Orbitrap mass spectrometry. GlcN produced two major dicarbonyl compounds, glucosone and 3-deoxyglucosone, ranging from 709 to 3245 mg/kg GlcN and from 272 to 4535 mg/kg GlcN, respectively. 3,4-Dideoxyglucosone-3-ene, glyoxal, hydroxypyruvaldehyde, methylglyoxal, and diacetyl were also detected in lower amounts compared to glucosone and 3-deoxyglucosone. Several pyrazine condensation products resulting from the reaction between dicarbonyls and GlcN were also identified. This study determined that GlcN is a significantly unstable molecule producing a high level of degradation products at 37 °C.

Non-enzymatic glycation of natural actomyosin (NAM) with glucosamine in a liquid system at moderate temperatures.

Muscle protein functionality plays an important role in routine applications in the food industry. Glycation by the Maillard reaction is a naturally occurring process, which can be used to develop new ingredients with improved functionality using a food grade approach. Actomyosin was conjugated with glucose or glucosamine in a liquid system at moderate temperatures (40°C). Sugar to protein conjugation was evident by UV-Vis spectral changes, with the glycation level determined by matrix assisted laser desorption/ionisation mass spectrometry. Parameters for glycation of muscle protein were optimised using the bidimensional hierarchical clustering analyses. The best glycation conditions were 40°C for 8 h at 1:3 protein:sugar ratio. Solubility and emulsifying properties of glycoconjugates were significantly improved as compared to non-glycated proteins. At pH 7 glycated actomyosin was on average 31% more soluble compared to non-treated protein. Glucosamine was found to be more effective for glycation and provided higher protein functionality as compared to glucose.

Effect of acid- and alkaline-aided extractions on functional and rheological properties of proteins recovered from mechanically separated turkey meat (MSTM).

UNLABELLED: Functional and rheological characteristics of acid- and alkali-extracted proteins from mechanically separated turkey meat (MSTM) have been investigated. Extractions were carried out at 4 pH values (2.5, 3.5, 10.5, and 11.5). The study demonstrated that alkali and acid extractions resulted in significant (P < 0.0001) decreases of cooking and water loss compared to raw MSTM; however, the cooking loss was found to be similar (P = 0.5699) among the different protein isolates. Proteins extracted at pH 10.5 showed the lowest (P = 0.0249) water loss. Emulsion and foaming properties were found to be slightly higher in alkali-extracted proteins compared to those for acid extractions. The myofibrillar protein fraction showed better ability to form and stabilize emulsions compared to sarcoplasmic proteins. Myofibrillar proteins also showed better foam expansion; however, foam volume stability was similar for both myofibrillar and sarcoplasmic protein fractions. Textural characteristics (hardness, chewiness, springiness, and cohesiveness) of recovered proteins were found to be unaffected (P > 0.05) by different extraction pH. The protein extracted at pH 3.5 formed a highly viscoelastic gel network as evidenced by storage modulus (G') values, whereas the gel formed from proteins extracted at pH 10.5 was found to be the weakest. The work also revealed that acid treatments were more effective for removal of total heme pigments from MSTM. Color characteristics of protein isolates were markedly improved compared to the initial material and tended to be better when subjected to acid extractions. PRACTICAL APPLICATION: Mechanically separated meat is one of the cheapest sources of protein obtained by grinding meat and bones together and forcing the mixture through a perforated drum. The use of mechanically separated turkey meat (MSTM) for the production of further processed poultry products is limited due to its undesirable color and textural properties. Recovery of proteins from MSTM using pH shifting process will help the poultry processors to get better returns and also create opportunity to produce functional food ingredients.