Influence of Microbial Transglutaminase on Physicochemical and Cross-Linking Characteristics of Individual Caseins.

The effects of microbial transglutaminase (MTGase) cross-linking on the physicochemical characteristics of individual caseins were investigated. MTGase was used to modify three major individual caseins, namely, κ-casein (κ-CN), αS-casein (αS-CN) and ß-casein (ß-CN). The SDS-PAGE analysis revealed that MTGase-induced cross-linking occurred during the reaction and that some components with high molecular weights (>130 kDa) were formed from the individual proteins κ-CN, αS-CN and ß-CN. Scanning electron microscopy (SEM) and particle size analysis respectively demonstrated that the κ-CN, αS-CN and ß-CN particle diameters and protein microstructures were larger and polymerized after MTGase cross-linking. The polymerized κ-CN (~749.9 nm) was smaller than that of ß-CN (~7909.3 nm) and αS-CN (~7909.3 nm). The enzyme kinetics results showed KM values of 3.04 × 10-6, 2.37 × 10-4 and 8.90 × 10-3 M for κ-CN, αS-CN and ß-CN, respectively, and, furthermore, kcat values of 5.17 × 10-4, 1.92 × 10-3 and 4.76 × 10-2 1/s, for κ-CN, αS-CN and ß-CN, respectively. Our results revealed that the cross-linking of ß-CN catalyzed by MTGase was faster than that of αS-CN or κ-CN. Overall, the polymers that formed in the individual caseins in the presence of MTGase presented a higher molecular weight and larger particles.

Complex Coacervation of Soy Proteins, Isoflavones and Chitosan.

In this study, the chitosan-induced coacervation of soy protein-isoflavone complexes in soymilk was investigated. Most of the soymilk proteins, including ß-conglycinin (7S), glycinin (11S), and isoflavones, were found to coacervate into the soymilk pellet fraction (SPF) following the addition of 0.5% chitosan. The total protein in the soymilk supernatant fraction (SSF) decreased from 18.1 ± 0.3 mg/mL to 1.6 ± 0.1 mg/mL, and the pH values decreased slightly, from 6.6 ± 0.0 to 6.0 ± 0.0. The results of SDS-PAGE revealed that the 7S α', 7S α, 7S ß, 11S A3, and 11S acidic subunits, as well as the 11S basic proteins in the SSF, decreased to 0.7 ± 0.5%, 0.2 ± 0.1%, 0.1 ± 0.0%, 0.2 ± 0.2%, 0.2 ± 0.2% and 0.3 ± 0.2%, respectively. We also found that isoflavones in the SSF, including daidzein, glycitein, and genistein, decreased to 9.6 ± 2.3%, 5.7 ± 0.9% and 5.9 ± 1.5%, respectively. HPLC analysis indicated that isoflavones mixed with soy proteins formed soy protein-isoflavone complexes and were precipitated into the SPF by 0.5% chitosan.

Proteomic analysis of polysaccharide-milk protein interactions induced by chitosan.

The chitosan-induced coacervation of milk proteins was investigated using a proteomic approach. The addition of 0.8% chitosan to milk caused the milk proteins to coacervate after a 1 h incubation period. Approximately 86% of the milk proteins were present in the milk pellet fraction (MPF), and the protein concentration of the milk supernatant fraction (MSF) decreased from 29.4±0.2 to 4.2±0.6 mg/mL. SDS-PAGE analysis showed that the total intensities of serum albumin (BSA), αS-casein (αS-CN), ß-casein (ß-CN), κ-casein (κ-CN) and ß-lactoglobulin (ß-LG) in the MSF decreased to 8.5%±0.2%, 0.9%±0.3%, 0.7%±0.3%, 0.5%±0.2% and 15.0%±0.5%, respectively. Two-dimensional electrophoresis analysis indicated that αS1-, αS2-, ß- and κ-CN and a fraction of the ß-LG and BSA were found in the MSF following incubation with 0.8% chitosan. Isothermal titration calorimetry analysis indicated that binding of chitosan to milk proteins is an exothermic reaction based on binding titration curves of milk proteins dispersions with chitosan, and the enthalpy of binding (ΔH) and binding constant (Ka) were -7.85×10(4) cal/mol and 1.06×10(5)/mol, respectively. These results suggested that the addition of 0.8% chitosan causes milk proteins to coacervate due to polysaccharide-protein interactions.

Improvement of enzymatic cross-linking of ovalbumin and ovotransferrin induced by transglutaminase with heat and reducing agent pretreatment.

The effects of transglutaminase (TGase, 1.0 unit/mL) with heat (95 °C, 5 min), 2-mercaptoethanol (2-ME, 0.83 %), and l-cysteine (l-Cys, 50 mM) pretreatment on the cross-linking of ovalbumin (OVA) and ovotransferrin (OVT) were investigated. SDS-PAGE revealed that although the polymerization of OVA and OVT did not occur after 3 h of incubation at 40 °C with TGase, OVA polymerized into high molecular weight polymers following TGase with 2-ME and heat pretreatment after 3 h of incubation. The surface hydrophobicity and reactive sulfhydryl (SH) groups of OVA samples significantly increased from 4065.7 ± 136.7 and 89.3 ± 1.2 SH groups (µmol/g) to 31483.6 ± 342.7 and 119.5 ± 3.7 SH groups (µmol/g), respectively. Similar results were obtained for OVT with TGase and l-Cys pretreatment and a 3-h incubation at 40 °C. The use of TGase, a reducing agent, and/or heat pretreatment can be used for the polymerization of OVA and OVT.

Influence of Chymosin on Physicochemical and Hydrolysis Characteristics of Casein Micelles and Individual Caseins.

The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS-PAGE investigation showed that ß-CN, κ-CN, αs-CN, and a portion of ß-lactoglobulin (ß-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS-PAGE analysis suggested that chymosin hydrolyzed individual ß-CN, κ-CN, and αs-CN, but not ß-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed ß-CN, κ-CN, and αs-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed ß-CN and αs-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of ß-CN and αs-CN.

Characterization of Maltase and Sucrase Inhibitory Constituents from Rhodiola crenulata.

The inhibitory properties of epicatechin-(4ß,8)-epicatechingallate (B2-3'-O-gallate), epicatechin gallate (ECG), and epicatechin (EC) isolated from Rhodiola crenulata toward maltase and sucrase were investigated. The half-maximal inhibitory concentration (IC50) values for maltase were as follows: B2-3'-O-gallate (1.73 ± 1.37 µM), ECG (3.64 ± 2.99 µM), and EC (6.25 ± 1.84 µM). Inhibition kinetic assays revealed the inhibition constants (Ki) of the mixed-competitive inhibitors of maltase, as follows: B2-3'-O-gallate (1.99 ± 0.02 µM), ECG (3.14 ± 0.04 µM), and EC (7.02 ± 0.26 µM). These compounds also showed a strong inhibitory activity toward sucrase, and the IC50 values of B2-3'-O-gallate, ECG, and EC were 6.91 ± 3.41, 18.27 ± 3.99, and 18.91 ± 3.66 µM, respectively. Inhibition kinetic assays revealed the inhibition constants (Ki) of the mixed-competitive inhibitors of sucrase as follows: B2-3'-O-gallate (6.05 ± 0.04 µM), ECG (8.58 ± 0.08 µM), and EC (13.72 ± 0.15 µM). Overall, these results suggest that B2-3'-O-gallate, ECG, and EC are potent maltase and sucrase inhibitors.

The conversion and deglycosylation of isoflavones and anthocyanins in black soymilk process.

The conversion of isoflavones and anthocyanins during black soymilk processing including soaking and heating was investigated. During soaking procedure, the ß-glucosidase activity, content of bioactive compounds including daidzein, genistein, delphinidin and cyanidin were significantly increased by deglycosylation reaction. After heating treatment at 90 °C for 1 h, the content of isoflavones ß-glucosides including daidzin, glycitin and genistin were increased through a de-esterification reaction from malonyl-glucosides at high temperature. On the contrary, aglycone content including daidzein and genistein were no significant changed, this result indicated aglycones displayed well thermal stability. Moreover, anthocyanins including delphinidin-3-O-glucoside (D3G) and cyanidin-3-O-glucoside (C3G) were converted into delphinidin and cyanidin by glucose removal. HPLC analysis suggested that daidzein, genistein, D3G and C3G were bound with ß-conglycinin and glycinin. Our results establish the conversion of bioactive components including daidzein, genistein, delphinidin and cyanidin by deglycosylation during soaking and heating progress and benefit to the production of soymilk.

Effect of germination on composition profiling and antioxidant activity of the polysaccharide-protein conjugate in black soybean [Glycinemax (L.) Merr.].

Black soybeans are commonly consumed as health foods and used in traditional Chinese medicine, but they are rarely cultivated as edible sprouts. During germination, the composition of seeds undergoes distinct changes that cause variations in bioactivities. In this study, the water-soluble black soybean polysaccharide (BSPS) was isolated from sprouts harvested at two-day intervals during the first week of seedling growth. The chromatographic profiles of the BSPS in ungerminated seeds showed fraction 1 (F1, about 64kDa) and fraction 2 (F2, <1kDa) that degraded during germination. The polysaccharide in F1 fraction of ungerminated seeds was covalently associated with the protein and mainly contained arabinose, galactose, glucose, and galacturonic acid at various levels during germination. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) scavenging activities and the reducing power of F1 were highest on the seventh day of germination. The phenolic and flavonoid content significantly increased after the fifth day of germination, suggesting that these ingredients also contributed to the antioxidant activities. During long-term germination, the polysaccharide-protein conjugate in the F1 fraction with enhanced antioxidant activities is regarded as a potential natural antioxidant for the development of functional foods.

Galactomannan from Antrodia cinnamomea Enhances the Phagocytic Activity of Macrophages.

Galactomannan with an octasaccharide-repeating unit (ACP) was isolated from Taiwan medicinal mushroom Antrodia cinnamomea, and its chemical structure was determined herein. ACP significantly enhanced the phagocytosis and bactericidal activity of J774A.1 murine macrophages against Escherichia coli, with prospects for developing a new immunomodulatory compound or adjuvant in immunotherapy and vaccination.

A screening platform for compounds with potential immuno-regulatory activities using human cord blood mononuclear cells.

A systematic and combinatorial approach was adopted using human umbilical cord blood mononuclear cells (hUCB-MNCs) to screen for potential immuno-regulatory compounds. The hUCB-MNCs contain several types of immunogenic cells, which are a suitable material to mimic the in vivo immuno-response after drug treatment. hUCB-MNCs were treated with various natural products such as quercetin, astaxanthin, caffeic acid, bilobalide, eugenol, rutin and gamma-dodecalactone (gamma-DDL). Phenotypic expression analysis revealed that the subpopulation of CD3(+) T cells, CD56(+) NK cells and CD1a(+) dendritic cells apparently increased after being treated with gamma-DDL for 6 days. The expression of CD56 reached a maximum at 72 h with a dose-dependent relationship. The NK cells activation marker (CD69) also elevated following gamma-DDL treatment. These results demonstrated that the gamma-DDL has immuno-regulatory effects to enhance cord blood NK cells population and bioactivities. Such a high-throughput methodology using hUCB-MNCs may be an effective platform for systematically screening potential immuno-regulatory compounds.

Microwave-assisted cross-linking of milk proteins induced by microbial transglutaminase.

We investigated the combined effects of microbial transglutaminase (MTGase, 7.0 units/mL) and microwave irradiation (MI) on the polymerization of milk proteins at 30 °C for 3 h. The addition of MTGase caused the milk proteins to become polymerized, which resulted in the formation of components with a higher molecular-weight (>130 kDa). SDS-PAGE analysis revealed reductions in the protein content of ß-lactoglobulin (ß-LG), αS-casein (αS-CN), κ-casein (κ-CN) and ß-casein (ß-CN) to 50.4 ± 2.9, 33.5 ± 3.0, 4.2 ± 0.5 and 1.2 ± 0.1%, respectively. The use of MTGase in conjunction MI with led to a 3-fold increase in the rate of milk protein polymerization, compared to a sample that contained MTGase but did not undergo MI. Results of two-dimensional gel electrophoresis (2-DE) indicated that κ-CN, ß-CN, a fraction of serum albumin (SA), ß-LG, α-lactalbumin (α-LA), αs1-casein (αs1-CN), and αs2-casein (αs2-CN) were polymerized in the milk, following incubation with MTGase and MI at 30 °C for 1 h. Based on this result, the combined use of MTGase and MI appears to be a better way to polymerize milk proteins.

Aggregation of soy protein-isoflavone complexes and gel formation induced by glucono-δ-lactone in soymilk.

This study investigated the glucono-δ-lactone (GDL)-induced aggregation of isoflavones and soy proteins in soymilk. High-performance liquid chromatography (HPLC) analysis indicated that isoflavones mixed with ß-conglycinin (7S) and glycinin (11S) proteins formed 7S-isoflavone and 11S-isoflavone complexes in soymilk supernatant fraction (SSF). Most of the soy protein-isoflavone complexes then precipitated into the soymilk pellet fraction (SPF) following the addition of 4 mM GDL, whereupon the pH value of the soymilk dropped from 6.6 to 5.9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis suggest that the addition of 4 mM GDL induced the aggregation of most 7S (α', α and ß subunits), 11S acidic and 11S basic proteins as well as isoflavones, including most aglycones, including daidzein, glycitein, genistein and a portion of glucosides, including daidzin, glycitin, genistin, malonyldaidzin and malonylgenistin. These results provide an important reference pertaining to the effects of GDL on the aggregation of soy protein-isoflavone complexes and could benefit future research regarding the production of tofu from soymilk.

Coacervation of ß-conglycinin, glycinin and isoflavones induced by propylene glycol alginate in heated soymilk.

This study investigated the propylene glycol alginate (PGA)-induced coacervation of ß-conglycinin (7S), glycinin (11S) and isoflavones in heated soymilk. The addition of 0.9% PGA caused 7S, 11S, daidzein and genistein to coacervate following a 1h incubation period. SDS-PAGE showed that the protein bands corresponding to the 7S α', 7S α, 7S ß, 11S A3, and 11S acidic subunits and the 11S basic proteins in the soymilk supernatant fraction (SSF) decreased to 37.7 ± 12.7%, 24.7 ± 3.9%, 4.9 ± 1.8%, 8.5 ± 2.7%, 18.1 ± 1.8% and 6.0 ± 1.6%, respectively. In addition, isoflavones including daidzein and genistein were also coacervated from the SSF into the soymilk pellet fraction (SPF) following incubation with 0.9% PGA for 1h. The amounts of daidzein and genistein in the SSF decreased to 8.6 ± 1.6% and 2.0 ± 1.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound to the 7S and 11S proteins. These results suggested that daidzein and genistein were co-precipitated with the 7S and 11S proteins into the SPF by 0.9% PGA. Our results demonstrated that PGA is a potent coagulant for the coacervation of 7S, 11S, daidzein and genistein.

Anti-diabetic effects of Ganoderma lucidum.

Ganoderma lucidum is a white rot fungus widely used as a tonic for the promotion of longevity and health. Extracts of G. lucidum have been recognized as an alternative adjuvant treatment for diabetes. Among the many biologically active constituents of G. lucidum, polysaccharides, proteoglycans, proteins and triterpenoids have been shown to have hypoglycemic effects. G. lucidum polysaccharides have been reported to have hypoglycemic activity by increasing plasma insulin levels and decreasing plasma sugar levels in mice. Protein tyrosine phosphatase 1B is a promising therapeutic target in diabetes, and G. lucidum proteoglycan can inhibit this enzyme in vitro. Moreover, G. lucidum triterpenoids were shown to have inhibitory activity on aldose reductase and α-glucosidase that can suppress postprandial hyperglycemia. In addition, a protein Ling Zhi-8 extracted from G. lucidum significantly decreased lymphocyte infiltration and increased the antibody detection of insulin in diabetic mice. This review summarizes most of the research about the hypoglycemic action effects of polysaccharides, proteoglycans, proteins and tritrerpenoids from G. lucidum as a guide for future research.

Coagulation of ß-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk.

The coagulation of ß-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α', α and ß), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

Catalysis of rice straw hydrolysis by the combination of immobilized cellulase from Aspergillus niger on ß-cyclodextrin-Fe3O4 nanoparticles and ionic liquid.

Cellulase from Aspergillus niger was immobilized onto ß-cyclodextrin-conjugated magnetic particles by silanization and reductive amidation. The immobilized cellulase gained supermagnetism due to the magnetic nanoparticles. Ninety percent of cellulase was immobilized, but the activity of immobilized cellulase decreased by 10%. In this study, ionic liquid (1-butyl-3-methylimidazolium chloride) was introduced into the hydrolytic process because the original reaction was a solid-solid reaction. The activity of immobilized cellulase was improved from 54.87 to 59.11 U g immobilized cellulase(-1) at an ionic liquid concentration of 200 mM. Using immobilized cellulase and ionic liquid in the hydrolysis of rice straw, the initial reaction rate was increased from 1.629 to 2.739 g h(-1) L(-1). One of the advantages of immobilized cellulase is high reusability--it was usable for a total of 16 times in this study. Compared with free cellulase, magnetized cellulase can be recycled by magnetic field and the activity of immobilized cellulase was shown to remain at 85% of free cellulase without denaturation under a high concentration of glucose (15 g L(-1)). Therefore, immobilized cellulase can hydrolyze rice straw continuously compared with free cellulase. The amount of harvested glucose can be up to twentyfold higher than that from the hydrolysis by free cellulase.

Antioxidant activity and inhibition of α-glucosidase by hydroxyl-functionalized 2-arylbenzo[b]furans.

This study synthesized a series of hydroxyl-functionalized 2-arylbenzo[b]furans based on the structure of tournefolic acid A and evaluated them for antioxidant and α-glucosidase inhibitory activities. Compounds 5a, 5e, and 5n showed remarkable inhibition of α-glucosidase (IC50 values of 1.9-3.0 µM), and they appear to be even more potent than quercetin. A kinetic binding study indicated that compounds 5a and 5n used a mechanism of mixed-competition to inhibit α-glucosidase. This study also revealed that compounds 5a and 5n bind to either the α-glucosidase or α-glucosidase-4-NPGP complex. Using the crystal structure of the Saccharomyces cerevisiae α-glucosidase, the molecular docking study has predicted the binding of compounds 5a and 5n to the active site of α-glucosidase through both hydrophobic and hydrogen interactions. A DPPH radical scavenging assay further showed that most hydroxyl-functionalized 2-arylbenzo[b]furans possess antioxidant activity. The exception was compound 5p, which has only one hydroxyl group on the 2-phenyl ring of 2-arylbenzo[b]furan. Our results indicate that hydroxyl-functionalized 2-arylbenzo[b]furans possess both antidiabetic as well as antioxidant properties.

Interactive effects of microbial transglutaminase and recombinant cystatin on the mackerel and hairtail muscle protein.

Interactive effects of microbial transglutaminase (MTGase) and recombinant cystatin on the mackerel and hairtail water soluble protein (WSP), salt soluble protein (SSP), and muscle protein (MP) were investigated. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymic activity analyses, cross-linking of mackerel and hairtail myosin heavy chain and low molecular mass compounds and formation of epsilon-(gamma-glutamyl)lysine cross-links were observed on samples with MTGase, while the recombinant cystatin could effectively inhibit the cathepsins and subsequently prevent degradation of proteins during setting. The cathepsins and MTGase activities in WSP, SSP, and MP solutions decreased, but the recombinant cystatin activity increased during setting at 45 degrees C.

Inhibition of xanthine oxidase by Rhodiola crenulata extracts and their phytochemicals.

Using a fractionation technique, four phytochemicals were isolated from Rhodiola crenulata extracts. These compounds were identified as 4'-hydroxyacetophenone (4-HAP), epicatechin-(4ß,8)-epicatechin gallate (B2-3'-O-gallate), salidroside, and p-tyrosol using mass spectrometry and nuclear magnetic resonance spectroscopy. The inhibition of xanthine oxidase (XO) activity by these purified compounds was then evaluated and compared to that of a known XO inhibitor (allopurinol; IC50 = 12.21 ± 0.27 µM). Both 4-HAP and B2-3'-O-gallate showed an XO inhibitory effect, for which the half maximal inhibitory concentration (IC50) values were 15.62 ± 1.19 and 24.24 ± 1.80 µM, respectively. However, salidroside and p-tyrosol did not show significant inhibitory effects on XO at 30 µM. Furthermore, an inhibition kinetics study indicated that 4-HAP and B2-3'-O-gallate are mixed competitive inhibitors. The inhibition constants (Ki) of 4-HAP and B2-3'-O-gallate were 8.41 ± 1.03 and 6.16 ± 1.56 µM, respectively. These results suggest that 4-HAP and B2-3'-O-gallate are potent XO inhibitors.

Proteomic profiling of the coagulation of milk proteins induced by chymosin.

Chymosin-induced coagulation of individual milk proteins during incubation at 30 °C was investigated using a proteomic approach. The addition of chymosin (0.006 units/mL) caused the milk proteins to coagulate after a 3 h incubation period. Approximately 88% of the milk proteins were coagulated into the milk pellet fraction, and the protein concentration of the milk supernatant fraction (MSF) decreased from 29.88 ± 0.12 to 3.74 ± 0.13 mg/mL. SDS-PAGE analysis showed that α(S)-, ß- and κ-caseins in the MSF were almost depleted and that the total intensity of the protein bands corresponding to α(S)-caseins (α(S1) and α(S2)), ß-casein, and κ-casein decreased from 1088.0, 901.5, and 617.0 area units to 6.9, 6.1, and 5.2 area units, respectively. Two-dimensional electrophoresis analysis indicated that α(S1)-, α(S2)-, ß-, and κ-casein and a fraction of the ß-lactoglobulin and serum albumin were found in the MSF following incubation with chymosin.