Purification and structural elucidation of a novel fucoglucan from the fruiting bodies of Phellinus baumii Pilát

BACKGROUND: Recently, much attention has focused on the biological properties of fungal polysaccharides. Polysaccharides extracted from Phellinus baumii Pilát are reported to have antitumor and immunostimulatory properties, as well as anti‐mutagenicity activity, hypoglycemic and free radical scavenging properties. In this paper, we report the chemical and structural characterization of a novel neutral polysaccharide isolated from the fruiting bodies of P. baumii Pilát. RESULTS: PBF4, a purified polysaccharide, was isolated from the fruiting bodies of P. baumii Pilát, and was shown to be composed of L ‐fucose and D ‐glucose in a ratio of 1:4. It was found to be a fucoglucan consisting of a α‐(1 → 4)‐D ‐glucopyranose backbone with some insertions of α‐(1 → 2)‐L ‐Fucp residues. It also contained a minor β‐(1 → 4,6)‐linked D ‐glucopyranosyl and β‐glycosidically linked nonreducing‐end D ‐glucopyranosyl residues. CONCLUSION: Our results add a new polysaccharide to those already described for different fungal groups. This kind of polysaccharide is useful for further study of the structure–function relationships and mechanism responsible for its biological activities. Copyright © 2008 Society of Chemical Industry     

Anti‐inflammatory properties of high pressure‐assisted extracts of Grifola frondosa in lipopolysaccharide‐activated RAW 264.7 macrophages

The aim of this study was to determine the in vitro anti‐inflammatory properties of the shake extract (SE) and the high pressure‐assisted extract (PE) of the mycelia of Grifola frondosa in a lipopolysaccharide‐stimulated RAW 264.7 macrophage model. The content of total polysaccharides and β‐glucans of PE at 600 MPa (PE‐600) was 41.2 and 6.2 mg g^?1 dry weight, respectively, which were significantly higher than SE extracts. The results showed that treatment with 500 μg mL^?1 of PE by 600 MPa (PE‐600) did not reduce RAW 264.7 cell viability but did significantly inhibit the production of LPS‐induced NO, PGE₂ and intracellular ROS. The PE‐600 inhibited the activation of NF‐kB and then reduced the production of LPS‐induced TNF‐α, IL‐6 and IL‐1β in a dose‐dependent manner. Thus, the PE could be used as an alternative extraction method for improving the extraction efficacy of G. frondosa and serve as an alternative source of anti‐inflammatory agents.     

Artificial simulated gastrointestinal digestion of four carbohydrates containing beta‐d‐1 → 4 linkages and new GC‐TQ/MS‐MS method for characterising released monosaccharides

Four types of carbohydrates, including Dendrobium officinale polysaccharide, Dendrobium aphyllum polysaccharide and β‐glucans from yeast and barley, were examined, and their structures were found to mainly contain 1,4‐linked‐β‐d ‐Glcp. Artificially simulated gastrointestinal digestion was conducted to characterise the changes of molecular weight, reducing sugars and released free monosaccharides by high‐performance liquid chromatography, kits and the newly developed gas chromatography (GC)‐mass spectrometry (MS)/MS analysis, which indicated that high molecular weight and complex spatial structures contributed to delayed monosaccharide release following exposure to digestive solution. The spatial structures of carbohydrates were changed during gastric digestion, but their primary structures were destroyed during intestinal digestion. Additionally, for the developed 7890A/7000 GC‐TQ/MS‐MS, the new analytical method was successfully used to analyse very low concentrations of monosaccharides in the simulated gastrointestinal digestive system.     

Antioxidant activity in HepG2 cells,immunomodulatory effects in RAW 264.7 cells and absorption characteristics in Caco‐2 cells of the peptide fraction isolated from Dendrobium aphyllum

DA‐P, fraction of peptides with a molecular weight <1 kDa isolated from Dendrobium aphyllum, was analysed in three types of cell lines to verify its bioactivity and absorptivity. The cellular antioxidant activity of DA‐P in HepG2 cells was used and results revealed an EC50 of 2.88 ± 0.143 mg mL^?1 and a CAA unit of 63.46 ± 2.11 μm QE/100 g peptides. DA‐P treatment enhanced the secretion of cytokines in RAW 264.7 cells. After demonstrating the presence of tight junctions in Caco‐2 monolayers, the absorption was 25.57% ± 0.016% and 19.7% ± 0.012% from different sides. The relatively high absorption indicated that the antioxidant‐relevant immune functions of DA‐P had a greater possibility to be absorbed by Caco‐2 cells. Free amino acids and LC‐MS/MS analysis indicated the degradation and expulsion of components after the absorption of DA‐P, and Ser‐Ser‐Arg was able to come across the monolayers.     

Comparison of structural,antioxidant and immuno‐stimulating activities of polysaccharides from Tremella fuciformis in two different regions of China

In this study, we compared the chemical structure, antioxidant and immuno‐stimulating activities of two polysaccharides, TPS–FJ and TPS–SC, extracted from Tremella fuciformis from two of the major cultivation regions of China. TPS–FJ and TPS–SC contained protein and high uronic acid. Average molecular weights of TPS–FJ and TPS–SC were 45 461 and 25 981 kDa, respectively. A triple‐helix conformation was exhibited by TPS–FJ but not by TPS–SC. TPS–FJ and TPS–SC contained same monosaccharides but in different ratios. Both the polysaccharides displayed characteristic polysaccharide bands in their Fourier transform infrared and NMR spectra, shown high water and oil holding capacity, and retained antioxidant potential after simulated gastrointestinal digestion in vitro. TPS–FJ significantly (P < 0.05) stimulated the nitric oxide, tumour necrosis factor‐α and interleukin‐6 production of RAW 264.7 cells with higher reactions than those of TPS–SC in vitro. Thus, Tremella fuciformis polysaccharide can be used as a potential antioxidant and immunomodulatory ingredient in food industry.     

Structural studies of an acidic polysaccharide of Mesona blumes gum

Mesona blumes gum can be isolated into a neutral polysaccharide (NMBG) and an acidic polysaccharide (AMBG). The yield of AMBG can be up to 90% (w/w). AMBG with a molecular weight of 6566 Da is composed of galactose, glucose, mannose, xylose, arabinose, rhamnose and galacturonic acid in the molar ratio 2.66:1:0.37:2.29:12.5:5.99:23.5. Structural features of the purified AMBG were investigated by a combination of chemical and instrumental analysis, such as periodate oxidation, Smith degradation, methylation, and ¹³C and ¹H NMR. It was found that AMBG possessed a (1 → 4)‐α‐galacturonan backbone with some insertions of (1 → 2)‐α‐L ‐Rhap residues. The branches of arabinogalactan, arabinan, galactan and xylan were all attached to the backbone via O‐4 of α‐L ‐Rhap residues. In addition, some α‐L ‐Rhap residues on the backbone terminated with α‐L ‐Araf and some O‐6 in galacturonic acid residues were acetylated or methyl esterified. The molecular structure of AMBG at different concentrations was observed by atomic force microscopy (AFM). It was found that AMBG showed spherical lumps at 1 µg mL^?1 but an irregular worm‐like shape at 10 µg mL^?1, which indicated that the viscosity of AMBG might be caused by its notable molecular aggregation. Copyright © 2007 Society of Chemical Industry     

Anti‐inflammatory and cytotoxic effects of ginseng extract bioconverted by Leuconostoc mesenteroides KCCM 12010P isolated from kimchi

A bioconversion technique using microorganisms has been applied to ginseng to increase content of bioactive ginsenoside and biofunctionality such as anticancer, anti‐obesity and antioxidant activities. The objective of this study was to screen lactic acid bacteria for bioconversion of ginsenosides and to evaluate anti‐inflammatory and cytotoxic effects of bioconverted ginseng extract. Strains isolated from kimchi were screened for their β‐glucosidase activities using esculin agar. Selected strain was identified based on 16S rRNA sequencing and carbohydrate fermentation. During ginseng fermentation, viable cell number and pH were determined. Bioconverted ginsenosides were analysed by HPLC. Anti‐inflammatory effects were evaluated using RAW 264.7 cells, and cytotoxic effects were determined by MTT assay. Among 166 isolates screened, Leuconostoc mesenteroides was selected for ginseng bioconversion, as it showed a higher β‐glucosidase activity and viable cell number than any of the other tested strains. After fermentation for 2 days, viable cell number was 8.8 log CFU mL^?1 and final pH was 4.8. Ginsenoside Rb₂ was bioconverted into ginsenoside Rg₃ (Rb₂ → Rd → Rg₃) by L. mesenteroides. The nitric oxide contents of 2‐day‐fermented extract decreased by as much as 25%, compared to a non‐fermented extract. The cell viabilities of HepG2, HT‐29, HeLa and LoVo treated with fermented ginseng extract also decreased by 49.7%, 20.2%, 21.0% and 8.7%, respectively, compared to those of control cells treated with non‐fermented extract. Ginseng extract bioconverted by L. mesenteroides showed anti‐inflammatory and anticancer effects. Therefore, bioconverted ginseng extract might have applications in the pharmaceutical and/or functional food industry.     

Structural characterization and immunomodulatory activity of a new polysaccharide isolated from the radix of Platycodon grandiflorum

In this study, a novel polysaccharide fraction (PGPIV-1-a) was isolated from Platycodon grandiflorum through the combination of DEAE-52 and Sephadex G-100 chromatography. PGPIV-1-a was a neutral heteropolysaccharide with an average molecular weight of about 6462 Da. PGPIV-1-a was composed of glucose, arabinose and mannose in the relative molar ratio of 64.79%, 31.16% and 4.05%. The main linkage types in PGPIV-1-a consisted of → 6)-β-Glcp-(1 → and →5)-α-L-Ara-(1→. Bioactivity assay results showed that PGPIV-1-a significantly stimulated the proliferation of RAW264.7 cells and enhanced their phagocytic capacity in a dose-dependent manner. In addition, treatment with higher dosage of PGPIV-1-a (800 μg mL⁻¹) remarkably induced the secretion of NO, TNF-α and IL-6. This study provides a theoretical basis for further systematic investigation and utilisation of Platycodon grandiflorum polysaccharides.     

Health-promoting effects of dietary polysaccharide extracted from Dendrobium aphyllum on mice colon,including microbiota and immune modulation

The health-promoting effects of a polysaccharide (DAP) purified from Dendrobium aphyllum, on mice colon were investigated. The results indicated that consumption of DAP facilitated the differentiation degree of CD4⁺ T cells to Th₁, Th₁₇ and Treg cells; and depressed the differentiation degree to Th₂ cells, since DAP up-regulated IL-6 expression (from 1.08 ± 0.24 to 3.40 ± 0.36). Besides, oral administration of DAP reduced the gastrointestinal transit time from 255 ± 1.21 to 164 ± 0.46 min, decreased the pH from 6.70 ± 0.27 to 5.54 ± 0.31, improved faecal water-binding capability from 59.4% ± 0.35 to 63.5% ± 0.22. Additionally, the increase in four health-promoting short chain fatty acids were observed, which might result from the enrichment of the relative abundance of health-promoting microbiota genera, including Porphyromonadaceae and Ruminococcaceae. The DAP-induced up-regulation of the genus Erysipelotrichaceae might be related to the homeostasis in both Th₁/Th₂ and Treg/Th₁₇.     

Isolation,characterisation and sulphation of soluble polysaccharides isolated from Cucurbita maxima

Using hot water extraction, a large number of polysaccharides were obtained from Cucurbita maxima. A DEAE‐Sepharose CL‐6B chromatography column was used to isolate the major polysaccharides from C. maxima. Two fractions were obtained (LP2‐1 and LP2‐2). LP2‐1 and LP2‐2 consisted of neutral polysaccharides (MW: 1.02 × 10⁴ and 4.32 × 10⁸ g mol^?1, respectively) comprised mainly of galactose units. Analyses by FT‐IR spectrometry, partial acid hydrolysis, periodate oxidation, Smith degradation and GC‐MS indicated that LP2‐1 consisted of 85.3% (1→4) glycosidic linkages and 1.7% (1→3) or (1→6) glycosidic linkages. The LP2‐1 backbone consisted of (1→4)‐linked galactose units, which occasionally branched at O6 or O3. The branches were composed of (1→4)‐linked galactose and terminated with galactose (13%). Two sulphated derivatives (SLP2‐1 and SLP2‐2) with variable degrees of sulphation (DS) were obtained by the sulphur trioxide–pyridine method, without degradation of the polysaccharide. DS of PL2‐1 and PL2‐2 was 0.19 and 0.20, respectively.     

Structural analysis of a polysaccharide from <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis> viscera

A water-soluble polysaccharide, SVP-2, was obtained from Patinopecten yessoensis viscera. Its major structural features were elucidated using composition analysis, methylation analysis, periodate oxidation and Smith degradation, partial acid hydrolysis, and NMR spectroscopy. The results indicated that SVP-2, with an average molecular weight of 170 kDa, was composed of rhamnose, fucose, arabian, xylose, mannose, galactose, and glucose in molar ratios of 1.65:2.54:4.05:5.60:1.48:4.90:1.00. Furthermore, it could deduce that the backbone of SVP-2 consisted of 1,3,4-linked pyran fucose, 1,3,4-linked pyran galactose, and 1,3-linked pyran arabian residues.     

Limited hydrolysis of β‐casein by cell wall proteinase and its effect on hydrolysates's conformational and structural properties

Optimisation of enzymatic hydrolysis of β‐casein with cell envelope proteinase (CEP) from Lactobacillus acidophilus JQ‐1 to produce the angiotensin‐I‐converting enzyme (ACE) inhibitory peptides using response surface methodology (RSM). Under optimal conditions (enzyme‐to‐substrate ([E]/[S]) ratio (w/w) of 0.132 and pH of 8.00 at 38.8 °C), the ACE inhibitory activity of hydrolysates was 72.06% and the total peptides was 11.75 mg mL^?1. Scanning electron microscopy (SEM) micrographs indicated that the tightness of the β‐casein surface structure was gradually weakened and small holes appeared after enzymatic treatment, while Fourier transform infrared spectroscopy (FTIR) spectra indicated remarkable changes in the chemical composition and macromolecular conformation of β‐casein after enzymatic hydrolysis. Differential scanning calorimetry (DSC) analysis indicated that the corresponding hydrolysates had higher thermal stability. The enzymatic hydrolysis also led to an increase in the free sulfhydryl content of β‐casein hydrolysates compared with raw β‐casein, which led to the increase in the antioxidant activity of β‐casein hydrolysates.     

Purification and characterisation of soluble acid invertase from mango fruits

Soluble acid invertase (SAI) was purified from mango fruits (Mangifera indica L.) by ammonium sulphate fractionation and anion‐exchange chromatography (DEAE‐Sepharose Fast Flow). Molecular mass of the enzyme is 45 kDa estimated by SDS–PAGE. Dynamic light scattering analysis suggests the hydrodynamic radius of SAI distributes from 4 to 20 nm with a peak at 6.68 nm. Transmission electron microscopy shows that SAI is a globulin with diameter of 10–30 nm. Its optimal pH and temperature are 4.0 and 60 °C, respectively. The enzyme is not stable at high temperature (≥60 °C) or in alkaline (pH ≥ 8) environment. Using sucrose as substrate, its K_M and V_max are 25.55 mm and 1.002 mmol min^?1 mL^?1, respectively. Its circular dichroism spectrum shows a negative band at 220 nm and a positive band at 195 nm, suggesting a β‐sheet structure. The fluorescence spectra reflect that the tryptophan and tyrosine residues of SAI are partially exposed.     

Preparation and Use of Poly(hydroxyethyl methacrylate) Cryogels Containing L‐Histidine for β‐Casein Adsorption

The objective of this study was to determine β‐casein adsorption by using supermacroporous poly(2‐hydroxyethyl methacrylate‐N‐methacryloyl‐(l) ‐histidine methyl ester) [p(HEMA‐MAH)] cryogel. β‐Casein adsorption properties of p(HEMA‐MAH) cryogel were studied for the application of β‐casein purification. The cryogel was produced by free radical polymerization initiated by N,N,N’,N’‐tetramethylene diamine and ammonium persulfate pairs in an ice bath. P(HEMA‐MAH) cryogel was characterized by swelling tests, Fourier transform infrared spectroscopy, and scanning electron microscopy. The effects of the flow rate, pH, temperature, initial β‐casein concentration, and ionic strength on the adsorption efficiency of cryogel were studied. The equilibrium swelling degree of the p(HEMA‐MAH) cryogel was 6.73 g H₂O/g cryogel. β‐Casein adsorption capacity of p(HEMA‐MAH) cryogel from aqueous solution was estimated as 31.17 mg/g cryogel. It was also observed that β‐casein could be repeatedly adsorbed and desorbed with p(HEMA‐MAH) cryogel without significant loss in the adsorption capacity.     

Antioxidant,immunomodulatory and antiproliferative effects of gelatin hydrolysates from seabass (Lates calcarifer) skins

This study investigated the antioxidant, immunomodulatory and antiproliferative potentials of gelatin hydrolysates from seabass skins in cell model systems. Gelatin hydrolysates were extracted from seabass skins using different processes and enzyme concentrations. The ability of the hydrolysates to protect against H₂O₂‐induced DNA damage was assessed on U937 cells using the Comet assay, and one of the samples showed DNA protective effects. All samples showed immunomodulatory potential by significantly (P < 0.05) reducing interleukin‐6 (IL‐6) and IL‐1β production in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. Antiproliferative activities of seabass skin hydrolysates were measured using human colon cancer (Caco‐2) and liver cancer (HepG2) cell lines as the model cell cultures. The inhibition of cell proliferation of Caco‐2 and HepG2 cancer cells occurred in a dose‐dependent manner at concentrations of 1–25 mg mL^?1. Therefore, seabass skin hydrolysates prepared using an appropriate process could serve as a potential functional food ingredient with various health benefits.     

The antioxidant capacity of polysaccharide from Laminaria japonica by citric acid extraction

An optimal citric acid extraction condition (pH 2.0; extraction temperature: 120 °C; extraction time: 3 h) was developed to obtain polysaccharide from Laminaria japonica. The yield of polysaccharide was 13.31 ± 0.08%, with IC₅₀ value of DPPH radical scavenging activity of 0.98 ± 0.01 mg mL^?1. The viscosity of polysaccharide extracted by citric acid (LJPA) was eight times lower than that of polysaccharide extracted by hot water (LJPW), which may be attributed to the low average molecular weight of LJPA (17.12 kDa). Gas chromatography analysis indicated that LJPA was composed of rhamnose, fucose, xylose, manose, glucose and galactose with relative molar percentages of 4.51%, 20.27%, 12.43%, 12.81%, 10.29% and 39.69% respectively. Furthermore, LJPA exhibited significantly higher antioxidant capacities including oxygen radical absorbance capacity (ORAC), ABTS radical scavenging activity and reducing power than LJPW. Citric acid extraction showed a positive influence on the polysaccharide degradation and antioxidant capacities of L. japonica.     

Optimisation of the formulation for barley–milk composite‐based fermented drink

The effect of barley flour concentration, Lactobacillus plantarum NCDC344 (Lp344) and co‐culture (Streptococcus thermophilus 20) inoculum levels on the sensory quality, Lp344 count, β‐glucan content and viscosity of barley–milk composite‐based fermented drink was investigated. A central composite rotatable design of response surface methodology was used for optimisation of the formulation. Of the three formulation variables, barley flour concentration was found to be the most critical as it significantly affected overall acceptability, Lp344 count and β‐glucan content (P < 0.01). The optimised drink rated 7.80 on a 9‐point hedonic scale, and contained 8.59 log cfu/mL of Lp344 cells and 0.144 g/100 g of β‐glucan.     

Antioxidant and nitric oxide inhibitory activities of tilapia (Oreochromis niloticus) protein hydrolysate: effect of ultrasonic pretreatment and ultrasonic‐assisted enzymatic hydrolysis

Ultrasound was incorporated to processing of fish protein hydrolysate to facilitate homogenate pretreatment and enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein. Their effects on Flavourzyme hydrolysis and biological activities of the tilapia hydrolysate were examined. The ultrasound‐assisted hydrolysis caused reduction in degree of hydrolysis ranging from 23% to 35% relative to that of the conventional process. The 70 W ultrasound‐assisted hydrolysis process increased DPPH radical‐scavenging activity and reducing power of tilapia hydrolysate prepared from the non‐pretreatment homogenate by 33% and 45%, respectively. All hydrolysates have no cytotoxicity on RAW264.7 cell lines at the maximum concentration of 20 mg protein mL^?1. The 70 W ultrasound pretreatment at 30 and 45 min combined with conventional hydrolysis is the suitable condition for producing tilapia hydrolysate with nitric oxide inhibitory and antioxidative activities on RAW264.7 cell lines, respectively. As a result, ultrasound could be applied to enzymatic protein hydrolysis either as pretreatment or during the hydrolysis.     

Phosphorylation of peptidoglycan from Lactobacillus acidophilus and its immunoregulatory function

Peptidoglycan (PG) is available from a wide variety of lactic acid bacteria (LAB) and is the main structure of cell wall components. Phosphorylated modification would bring new properties such as the potential antioxidant activities and antiviral capability to an organic molecule. In the present work, small molecular fragments of PG (derived from Lactobacillus acidophilus) hydrolysed by mutanolysin were phosphorylated under optimal conditions. P‐PG had a monomer molecular structure of GlcNAC[PO₃]–MurNAC–Ala–Glu–Lys–Ala, with a molecular mass of 884 Da and a phosphorus content of 8.9%. P‐PG displayed some immunoregulatory activity in lipopolysaccharides (LPS) stimulated RAW 264.7 macrophages. Compared with the LPS‐stimulated group, the addition of P‐PG inhibited the secretion of GM‐CSF, TNF‐α and IL‐1 in a dose‐dependent manner. The effect of 50 μg mL^?1 of P‐PG was more significant than 50 μg mL^?1 of PG. Lower fluorescence of lysosomes was observed in P‐PG‐treated RAW 264.7 cells may also reveal some immune defence function in the LPS‐induced macrophages.