Purification and characterization of a novel fungi Se-containing protein from Se-enriched <Emphasis Type="Italic">Ganoderma Lucidum</Emphasis> mushroom and its Se-dependent radical scavenging activity

Our previous studies have established that Ganoderma lucidum (GL) can biotransform inorganic selenium into organic selenium, which was stored preferentially in its water-soluble protein component. In the present study, a novel water-soluble fungi Se-containing protein, named as Se–GL–P, has been purified for the first time from the Se-enriched GL to establish the relationship between the antioxidant activity of protein and its Se content. This protein was isolated and purified to homogeneity using ammonium sulfate precipitation followed by two consecutive anion exchange chromatography and size exclusion chromatography. This protein in its native state was identified as a monomer of 36,600 Da estimated by 19.8% weight carbohydrate. The protein is acidic (pI=4.0) and rich in Asp, Glu, Gly, and Ala. The N-terminal shows a sequence of DINGGGATLPQKLYLTPDVL, suggesting that this protein belongs to a family of D I N G protein. The Se-content of this protein (4.87 mg Se/g protein) is ∼3-fold larger than that of the water-soluble protein extract (1.91 mg Se per protein). Perfectly consistent with its higher Se-content, the protein exhibits approximately three times stronger activity of scavenging superoxide and hydroxyl radicals as compared to the water-soluble protein extract, a finding demonstrating that the increasing antioxidant property of this protein depends quantitatively on its Se content.

Total,cumulative fast-kinetics and cumulative slow-kinetics antiradical activities of juices from clementine (<Emphasis Type="Italic">Citrus clementina</Emphasis>), clementine-hybrids and satsuma (<Emphasis Type="Italic">Citrus unshiu</Emphasis>) cultivars and their utility as discriminant variables

Mandarin juices from three pure clementine (Citrus clementina Hort. ex Tan.) cultivars: Clemenules, Hernandine and Marisol, two clementine-hybrid cultivars: Nova (clementine×tangelo Orlando) and Fortune (clementine× mandarin Dancy), one mandarin-hybrid cultivar: Ortanique (mandarin×orange) and one satsuma (Citrus unshiu Marc) cultivar: Clauselline have been analysed for determination of total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activities. Total antiradical activity followed the order: pure clementine and Nova> Ortanique>Fortune and Clauselline. Cumulative fast-kinetics antiradical activity was the major contributor to total antiradical activity, accounting for about 87% as average. Ascorbic acid content was the major contributor to cumulative fast-kinetics antiradical activity, accounting for about 93.5% as average. A weak correlation (r ²=0.151) between total flavanone-7-O-glycosides (FGs, Narirutin, Hesperidin and Didymin) content and cumulative slow-kinetics antiradical activity was observed. However, ascorbic acid and total FGs contents showed a significant negative correlation (r ²=0.658). By using the cumulative fast-kinetics and slow-kinetics antiradical activities as variables, all the assayed juices were significantly discriminated (<0.05) by statistical multivariate discriminant analysis.     

Dark chocolates supplemented with <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

Dark chocolate masses and chocolates were supplemented with viable cells of two bacterial strains Lactobacillus caseii and Lactobacillus paracasei with potential probiotic properties, which were lyophilized in milk. Total number of live bacteria in the lyophilizate was 7.9×10⁹ cfu/g. Sucrose or isomalt and aspartame were used as bulking substances and sweeteners. Sensory attributes of these chocolates were not different from that of traditional chocolates. Calorie value of sucrose-free chocolate was lower by approximately 11.1–14.6% (dependent on their formulation) relative to chocolate sweetened with sucrose. Chocolate, which contains isomalt and aspartame can be consumed by diabetics. Numbers of live L. casei and L. paracasei cells in the examined batches of chocolate were very high and approached 10⁶–10⁷ cfu/g after 12 months of keeping at 4 and 18 °C. Neither the texture nor the total and volatile acidity of chocolate masses were changed by addition of the lyophilized preparation of Lactobacillus cells. Casson yield values of dark sucrose-free chocolate masses supplemented with this lyophilizate were decreased by approximately 3–55% (dependently on fat contents in these masses) as compared to that of analogous chocolate masses sweetened with sucrose.     

Antioxidant synergism between fruit juice and <Emphasis Type="Italic">α</Emphasis>-tocopherol. A comparison between high phenolic black chokeberry (<Emphasis Type="Italic">Aronia melanocarpa</Emphasis>) and high ascorbic blackcurrant (<Emphasis Type="Italic">Ribes nigrum</Emphasis>)

Black chokeberry juice (Aronia melanocarpa, Elliot), blackcurrant juice (Ribes nigrum, Ben Lomond) and α-tocopherol were found to protect phosphatidyl choline against oxidation in a peroxidating liposome system as evidenced by lag phases for formation of conjugated dienes. When present together, black chokeberry juice and α-tocopherol showed a clear synergistic effect on the length of the lag phase, while effects of blackcurrant juice and α-tocopherol were additive. The concentration of total phenolics in black chokeberry juice was six times higher than in blackcurrant juice (gallic acid equivalents). Ascorbic acid corresponded to 1% of total phenolics in black chokeberry juice and 10% in blackcurrant juice. Based on the length of the lag phase, the phenolics present in black chokeberry were on an average, twice efficient as scavengers of lipid peroxyl radicals as phenolics in blackcurrant. Black chokeberry was by HPLC analysis of peroxidating liposomes, in contrast to blackcurrant, found to protect α-tocopherol efficiently against oxidation to the end of the lag phase. The phenolics present in black chokeberry juice were concluded to be more efficient in regenerating or protecting α-tocopherol than ascorbic acid or the phenolics in blackcurrant. As for the phenolics, this was further evidenced by ranking of their radical scavenging activity as studied by ESR-spectroscopy.     

Antioxidant activity of half <Emphasis Type="Italic">N</Emphasis>-acetylated water-soluble chitosan in vitro

Different molecular weight water-soluble chitosan (half N-acetylated chitosan) was prepared and the structure of water-soluble chitosan was characterized by FT-IR. The pH dependence of water solubility of water-soluble chitosan was evaluated from turbidity. Total antioxidant activity, reducing power, superoxide anion radical and hydroxyl radical quenching assay, metal chelating activity, and H₂O₂ scavenging activity were used for the evaluation of different molecular weight half N-acetylated chitosan in vitro. Low-molecular weight water-soluble chitosan (WSC4) exhibited high reductive capacity and expressed good inhibition of linoleic acid peroxidation in the linoleic acid model system. WSC4 (0.25 mg/mL) scavenged 78.8% of superoxide radical. At 5 mg/mL, scavenging percentage of WSC1, WSC2, WSC3, and WSC4 against hydroxyl radical was 49.3%, 66.8%, 77.1%, and 83.7%, respectively. These results indicate that water-soluble chitosan is an ideally natural antioxidant, and its antioxidant activity depends on its molecular weight.     

Antitumor activities of liposome-incorporated aqueous extracts of <Emphasis Type="Italic">Anodonta </Emphasis><Emphasis Type="Italic">woodiana</Emphasis> (Lea, 1834)

To acquire the effectiveness of oral treatments, aqueous extracts of Anodonta woodiana (HB) were incorporated into liposome and its antitumor activities evaluated in vivo. The HB-loaded liposomes (HBL) were prepared at a mean size of 14.85 μm by reverse-phase evaporation method. Referring to the maximum tolerated doses test, mice with orally administrated HBL, at a 3 g/kg body weight dosage, showed no obvious acute toxic sign. Furthermore, the tumoricidal activities of HBL against C26 murine colon carcinoma, Lewis lung tumor, human QGY hepatic carcinoma and MKN-45 gastric tumor were examined, respectively. In contrast to free HB, HBL possessed remarkable antitumor activity. Simultaneously, the effect of HBL was observed in a dose-dependent manner. For C26 murine colon carcinoma and Lewis lung tumor, the inhibitory ratios of HBL were 54.36 and 51.97% at a dose of 400 mg/kg, respectively. The suppression of tumor growth and the reduction in body weight were more pronounced in human QGY hepatic carcinoma and MKN-45 gastric tumors inoculated mice by treatment of HBL during 30 days. All these promising results implied that liposome-incorporated aqueous extracts of Anodonta woodiana had a more potential application as a natural antitumor and immunomodulator formulation.     

Short anoxic treatments to enhance <Emphasis Type="Italic">trans</Emphasis>-resveratrol content in grapes and wine

In a search for a method to improve the (endogenous) trans-resveratrol content in post harvested grapes, several short anoxic treatments with dry nitrogen have been assayed. A significant enhancement of the trans-resveratrol content was obtained, especially for treatments under 24 h; moreover, with shorter treatments (e.g. 6 or 15 h) the induced higher content of trans-resveratrol was maintained during several days with no appreciable anoxic damage of the grapes. These results allowed the design of an anoxic treatment protocol for wine grapes prior to the vinification process that led to a trans-resveratrol enriched wine.     

Evaluation of novel lactose-positive and exopolysaccharide-producing strain of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> for fermented foods

A novel strain of lactic acid bacteria Pediococcus pentosaceus P 773 was isolated from spoiled beer and identified by means of 16S rDNA sequence analysis. The ability to assimilate lactose as a sole carbon source as a specific feature for this strain was detected and confirmed on dairy substrates. In the presence of sucrose containing substrates (sucrose, raffinose) this P. pentosaceus P 773 lactose-positive strain produced a complex of extracellular polysaccharides (Qp = 0.08 g/l/h) with a molecular mass about 2,000 kDa composed by glucose and fructose residues at a ratio 3:1, respectively. These exopolysaccharides were capable to stimulate the growth rate and biomass productivity of common constituent cultures of probiotic dairy starters (Bifidobacterium lactis, Lactobacillus acidophilus, Streptococcus thermophilus) as well as were assimilated as a sole carbon source by these strains. The present study confirmed the presence of lactose-positive and exopolysaccharide-producing strain of P. pentosaceus in natural environment which could be used as a starter culture to impart more functional attributes to fermented food.     

Treatment of slices from carrot (<Emphasis Type="Italic">Daucus carota</Emphasis>) using high intensity white pulsed light

Experiments with washing of carrot slices using distilled water and 0.9% (w/w) NaCl in combination with varying number and pulses of high intensity white were carried out. Slices of carrot 3.5 cm in diameter and 2 mm thick were inoculated using diluted dispersions of yeasts (Saccharomyces cerevisiae) for varying time (0–240 min) and then the slices were treated with high intensity pulsed white light (one pulse = 0.7 J/cm²) using from none to 24 pulses. The major part of the yeast cells were killed using two pulses of light. The studies included washing of inoculated slices up to five times using salt 0.9% (w/w) NaCl and distilled water and inoculation at 22 °C for between 5 and 120 min. It was concluded that treatment of carrot slices with high intensity white light may reduce the load of yeast cells with up to 6 log cycles. Inoculation time at 22 °C had no effect on the maximum load of yeast cells.     

Cholesterol lowering activity of mango ginger (<Emphasis Type="Italic">Curcuma amada</Emphasis> Roxb.) in induced hypercholesterolemic rats

Mango ginger (Curcuma amada Roxb.) is a spice commonly consumed in Asian countries. Health beneficial hypotriglyceridemic property of mango ginger has been reported earlier. In this investigation, the anti-hypercholesterolemic influence of dietary mango ginger was evaluated in experimental rats. Dietary mango ginger powder (10%) or its equivalent of 10 mg% curcumin-containing portion was fed along with 1% cholesterol supplemented diet in Wistar rats for 5 weeks. The treatment countered the liver and serum total and LDL + VLDL associated cholesterols and increased the HDL associated cholesterol while it had no influence on cholesterol levels in animals, maintained on normal diet. Dietary C. amada and its curcumin-free portion were effective in lowering liver cholesterol in animals, maintained on basal diet, while the curcumin-containing component of C. amada was ineffective. The biliary secretion of total lipids and bile acids was increased by dietary C. amada and both of its components. While biliary cholesterol was increased in animals fed with whole mango ginger, no such increases were noticed in groups fed with either components of mango ginger. Cholesterol absorption in ligated rat intestinal loops was not affected by mango ginger or either of its components. Thus, the present study has evidenced that the spice mango ginger possesses beneficial anti-hypercholesterolemic activity in hypercholesterolemic situation. This information is complementary to the earlier report on the health beneficial hypotriglyceridemic influence of this spice.     

Reevaluation of bioactivity and antioxidant activity of <Emphasis Type="Italic">Myrtus communis</Emphasis> extract before and after encapsulation in liposomes

The antioxidant and antimicrobial activity of Myrtus communis extract was determined before and after its encapsulation in liposomes of different composition. Evaluation of the sunflower oil oxidation by the Rancimat stability test and malondialdehyde formation by HPLC were used to measure the antioxidant action in comparison with common commercial antioxidants, such as butylated hydroxytoluene and α-tocopherol. The thermal-oxidative decomposition of the samples, the modification of the main transition temperature for the lipid mixture and the splitting of the calorimetric peak in the presence of the antioxidants were studied by differential scanning calorimetry. The size and the surface charge of liposomes were also studied. The extract showed antioxidant and antimicrobial activity. Αt concentrations up to 160 ppm, the extract showed superior activity than α-tocopherol. When the extract was encapsulated in liposomes, its antioxidant as well as its antimicrobial activity proved to be superior from that of itself in pure form.     

Production of malic and succinic acids by sugar-tolerant yeast <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

Zygosaccharomyces rouxii V19 was grown in YPG medium (yeast extract, 0.5%, peptone, 1.0%, glucose, 10%). Fermented broth was purified through a series of ion-exchange columns and ODS column and the purified sample was TMS-esterified. Malic and succinic acids were identified with GC-MS analysis. The yeast was cultivated under various cultural conditions and quantitative determination of the organic acids was carried out with HPLC on Shodex column. Glucose concentration of 30%, initial pH 5.0, and 25 °C incubation temperature were the optimum conditions. Inclusion of glutamic, malic, and succinic acid precursors in the medium increased the production of malic acid. On the other hand, only addition of malic acid enhanced the production of succinic acid. Maximum amount of malic acid produced was 74.9 g/L (32.8% yield, based on glucose consumption) in the medium with 0.5% glutamic acid supplement, and that of succinic acid was 7.7 g/L (8.1% yield) when 0.3% malic acid was added in the medium.     

Selection of elderberry (<Emphasis Type="Italic">Sambucus nigra</Emphasis> L.) genotypes best suited for the preparation of juice

Experiments with elderberry genotypes, ripening stage and pectinolysis were carried out in order to investigate the possibility to improve different quality characteristics of elderberry juice. Raw juice was processed using either an optimised method with respect to enzyme concentration and treatment time or produced directly without any enzymatic treatment. Neochlorogenic acid, chlorogenic acid, cyanidin-3-sambubioside-5-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-sambubioside, cyanidin-3-glucoside, quercetin-3-rutinoside (rutin) and quercetin-3-glucoside (isoquercitrin) were identified in the raw juice by liquid chromatography-mass spectrometry (LC-MS) and quantified by high-performance liquid chromatography-diode array detection (HPLC-DAD). The content of phenolic acids and flavonoids in elderberries were shown to be affected by berry development stage at harvest (ripening stage). Furthermore, a considerable variation in juice yield and physicochemical properties such as turbidity, soluble solids, titratable acidity, and content of phenolic acids and flavonoids was found between juices processed from elderberries by pectinolysis of different elderberry genotypes. Pectinolysis appeared not to have any major effect on juice yield and the content of phenolic acids and flavonoids in the juice compared to non-enzymatic processed juice. Factor analysis revealed correlations among quality characteristics such as soluble solids, titratable acidity, turbidity, and content of phenolic acids and flavonoids and it is shown that these correlations may be useful for the selection of elderberry genotypes best suited for the preparation of juice.     

Phytochemical and biological investigation of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni; 1-labdane-type diterpene

Five labdane diterpenoids, austroinulin, iso-austroinulin, sterebin E, sterebin E acetate, and sterebin A acetate, along with hydrocarbons, aliphatic alcohols, β-amyrin, β-sitosterol and stigmasterol were isolated from the chloroform soluble fraction of the methanol extract of Stevia rebaudiana leaves. Chlorogenic and caffeic acids were isolated from the EtOAc fraction. All the isolated compounds were identified using spectral tools. The chloroform and methanol extracts proved significant anti-inflammatory effect and caused marked inhibition of carrageenan-induced paw oedema in rats.     

Use of supercritical CO<Subscript>2</Subscript> to obtain extracts with antimicrobial activity from <Emphasis Type="Italic">Chaetoceros muelleri</Emphasis> microalga. A correlation with their lipidic content

Supercritical CO₂ extracts of the marine diatom Chaetoceros muelleri (gracilis) have been investigated for their potential use as food preservatives, namely, as antimicrobials. A screening of different pressures and temperatures for supercritical CO₂ extraction was assayed in order to determine the main factors controlling the yield and antimicrobial activity of the extracts. Since the potential antimicrobial activity of these CO₂ extracts is mainly induced by the lipidic fraction, HPLC with evaporative light scattering detection (HPLC-ELSD) and GC with flame ionization detection (GC-FID) were used to identify lipid families and fatty acids, respectively. Antimicrobial activity of the extracts was measured against Staphyloccocus aureus, Escherichia coli and Candida albicans. Possible correlations between antimicrobial activity of extracts and their chemical composition were investigated, concluding that the total triglycerides and the DPA content seem to be the main parameters controlling the antimicrobial activity of the studied extracts.     

Sensitive and rapid detection of freshwater crustacean <Emphasis Type="Italic">Spiroplasmas</Emphasis> by ISRs-sequence-targeted species-specific primers

For the rapid and sensitive identification of the Spiroplasmas in the Chinese mitten crab Eriocheir sinensis, a species-specific PCR primer-pair was designed according to Spiroplasma sp. strain CRAB-specific segments of the previously sequenced 16S–23S rRNA intergenic spacer region (ISR). The PCR-mediated assay allowed identification of Spiroplasmas from different aquatic crustacean hosts, E. sinensis and red swamp crayfish Procambarus clarkia, in different geographical areas in China. No cross-reaction could be observed with other three available terrestrial plants/insects Spiroplasma strains (Spiroplasma sp. CH-1, Spiroplasma sp. CR-1 and Spiroplasma sp. M10). The sensitivity of detection of the Spiroplasma sp. strain CRAB could get as little as 100 fg of DNA template. The presented PCR method is useful for sensitive and specific detection of Spiroplasma from freshwater cultured crustaceans in the trade of aquatic products.     

Influence of polyphenols isolated from <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi and <Emphasis Type="Italic">Crataegus oxyacantha</Emphasis> on the oxidative stability of cholesterol in butter stored in various conditions

The aim of the present study was to determine whether the exposure of two types of butter (A1, A2) to different conditions (4 °C and 30 °C in darkness and exposed to light 3600–3900 lx at 50 °C) could be stabilized with natural polyphenols. The polyphenols (500 ppm) added to butter included flavones of skullcap in the form of root powder, 95% pure baicalin and procyanidins from the bark of hawthorn. The rate of cholesterol oxide products (COPs) as well as the storage periods were closely connected with sample storage conditions and the type of butter. Among the polyphenols used, the flavones of skullcap proved to be stronger inhibitors of oxidation process than procyanidins of hawthorn. The storage of samples with the addition of flavones at 50 °C and exposed to light resulted in preserving 50% of cholesterol in both types of butter. The lowest amounts of COPs were formed in samples stored at 4 °C with no light exposure and, under the other conditions, in samples with flavones of skullcap added.     

A neutral polysaccharide from the abalone pleopod<Emphasis Type="Italic">, Haliotis discus hannai</Emphasis> Ino

For the first time, a water-soluble neutral polysaccharide, APP, was obtained from abalone pleopod (Haliotis Discus Hannai Ino) by proteolytic method with papain followed by precipitation with ethanol and purification with gel chromatography. The results of HPLC with TSK-gel column and gel filtration chromatography with Sepharose CL-6B indicated APP was uniform in polarity and its molecular weight (MW) was about 6 kDa. According to chemical methods, IR spectroscopy, and NMR spectroscopy, APP consists of 1-, 1,4-, 1,6-, 1,4,6-linked glucose and 1-, 1,3-, 1,6-, 1,4,6-linked galactose.     

ACE-inhibitory activity and gamma-aminobutyric acid content of fermented skim milk by <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> isolated from Xinjiang koumiss in China

Eighty-one strains of Lactobacillus were isolated from the koumiss collected in Xinjiang, China. The strains were cultivated in skim milk medium, ACE inhibitory activity and GABA concentrations in the culture supernatants were measured. Screening results revealed that ACE inhibitory activity of 16 strains was higher than 50% and two strains produced GABA. The Lactobacillus—ND01 strain produces both the high ACE inhibitory activity and GABA. The sequence of 16S rDNA of the Lactobacillus—ND01 showed 99% homology to L. helveticus. The first identification of the newly isolated Lactobacillus—ND01 strain which produces both high ACE inhibitory activity and GABA revealed differences from reported species in our study. The L. helveticus ND01 was resistant to acidic condition. The results suggest that L. helveticus ND01 showed good potential for application in the management of hypertension.