Isolation and characterization of a bioactive mannose-binding protein from the Chinese chive Allium tuberosum

A mannose-binding protein was isolated from two different cultivars of the Chinese chive Allium tuberosum by extraction with 0.2 M NaCl, ammonium sulfate precipitation, and affinity chromatography on mannose agarose and fetuin agarose. It exhibited hemagglutinating activity toward rabbit erythrocytes. The lectin (agglutinin) was adsorbed on the mannose-agarose column, but not on the fetuin-agarose column. This A. tuberosum lectin (ATL) is unglycosylated, and not sialic acid binding. Lectins isolated from the two cultivars exhibited the same molecular mass of 25 kDa on gel filtration (Superose 12) and 12.5 kDa on SDS-polyacrylamide gel electrophoresis, indicating that they might be a dimeric protein composed of two identical subunits. The N-terminal amino acid sequence analysis of the lectin of various cultivars of A. tuberosum revealed that they were identical and showed 50%, or more, homology to the lectins from Galanthus nivalis (family Amaryllidaceae), Narcissus tazetta (family Amaryllidaceae), and Aloe arborescenes (family Liliaceae).     

Sweetness and enzymatic activity of lysozyme

Hen egg lysozyme elicits a sweet taste sensation for human beings. Effects of reduction of disulfide bonds, heat treatment, and chemical modification of hen egg lysozyme on both sweetness and hydrolytic activity were investigated. Both the sweetness and enzymatic activities were lost when the intradisulfide linkage in a lysozyme molecule was reduced and S-3-(trimethylated amino) propylated. The sweetness and enzymatic activity of lysozyme were lost on heating at 95 degrees C for 18 h. These facts suggest that tertiary structures of lysozyme are indispensable for eliciting a sweet taste as well as enzymatic activity. Although the modification of carboxyl residues in a lysozyme by glycine methylester or aminomethansulfonic acid resulted in the loss of enzymatic activity by blocking the catalytic residues, the sweetness was fully retained. These results indicate that the sweetness of lysozyme was independent of its enzymatic activity. The lysozyme purified from goose egg white similarly elicited a sweet taste, although goose (g-type) lysozyme is quite different from hen egg lysozyme (c-type) on the basis of structural, immunological, and enzymatic properties. These findings indicate that a specific protein property of lysozyme is required for sweetness elicitation and that the enzymatic activity and carbohydrates produced by enzymatic reaction are not related to the sweet taste.     

Purification and characterization of a mannan-binding lectin specifically expressed in corms of saffron plant (Crocus sativus L.)

Despite the economical interest of Crocus sativus, its biochemistry has been poorly studied. Herein, we have isolated a lectin present in saffron corm by gel-filtration, anion-exchange, and reversed-phase chromatography. One- and two-dimensional PAGE, MALDI-MS, and N-terminal amino acid sequence analyses indicated that the native protein forms noncovalently linked aggregates of about 80 kDa apparent molecular mass, mainly composed of two charged heterogeneous (pI's, 6.69-6.93) basic subunits of approximately 12 kDa. Their N-terminal sequences shared 25% similarity and were homologous to the N- and C-terminal domains of monocotyledonous mannose-binding lectins, respectively. An additional polypeptide of around 28 kDa apparent molecular mass was also detected, probably corresponding to a precursor processed into two mature subunits. In addition, the N-terminal domain subunit exhibited 56% similarity with curculin, a sweet protein with taste-modifying activity. The native lectin specifically interacts with a yeast mannan and is a major corm protein specifically expressed in this organ.     

Lectin-erythrocyte interaction with external transmembrane glycophorin saccharides controlling membrane internal cytoskeleta

Human red blood cell (RBC, erythrocyte) membranes have internal protein skeletons that govern the cells' distinctive discocyte-echinocyte morphology (shape) changes, seen in conventional microscopy. Glycophorin, the cell's transmembrane protein, presents all of its saccharides outside the cell. The protein sector of glycophorin is linked inside to the RBC cytoskeleton, enabling lectins binding to the external saccharides to gain profound control over internal cytoskeleton behavior, expressed by governance of the visibly seen cell shape. Critical lectin binding stoichiometries ((125)I-labeled lectins) equate to the number of glycophorin monomers per RBC, 7 x 10(5) copies/cell. Wheat germ agglutinin lectin (sialic acid specific) binds to glycophorin's outermost (exo) saccharides and exerts tight control over the cell's morphology. Removal of sialic acid groups (desialation) exposes the endosaccharides of glycophorin, enabling peanut agglutinin and Osage orange lectins to gain equally tight control over the RBC's morphology behavior in simple stoichiometric ratios, bound lectin molecules/glycophorin receptor. Thus, lectin specificities for saccharides are sharply in register with the glycophorin external saccharide composition, the sequence along the chains, and the number of copies of protein (stoichiometry). These relationships were determined via RBC shape change equilibria and also via shape change rates. Rate data are somewhat laborious to determine, but are exquisitely sensitive to lectin specificities and in very small lectin concentrations. Both classes of data enable these interactions to be analyzed in lectin and RBC concentrations approximately 100-fold smaller than agglutinating levels.     

Influence of flavor solvent on flavor release and perception in sugar-free chewing gum

The influence of flavor solvent [triacetin (TA), propylene glycol (PG), medium chained triglycerides (MCT), or no flavor solvent (NFS)] on the flavor release profile, the textural properties, and the sensory perception of a sugar-free chewing gum was investigated. Time course analysis of the exhaled breath and saliva during chewing gum mastication indicated that flavor solvent addition or type did not influence the aroma release profile; however, the sorbitol release rate was statistically lower for the TA formulated sample in comparison to those with PG, MCT, or NFS. Sensory time-intensity analysis also indicated that the TA formulated sample was statistically lower in perceived sweetness intensity, in comparison with the other chewing gum samples, and also had lower cinnamon-like aroma intensity, presumably due to an interaction between sweetness intensity on aroma perception. Measurement of the chewing gum macroscopic texture by compression analysis during consumption was not correlated to the unique flavor release properties of the TA-chewing gum. However, a relationship between gum base plasticity and retention of sugar alcohol during mastication was proposed to explain the different flavor properties of the TA sample.     

Effect of lectins on the transport of food factors in caco-2 cell monolayers

The effect of three plant lectins, soybean lectin (SBA), Japanese jack bean lectin (CGA), and wheat germ lectin (WGA), on the transport of various food factors, such as isoflavones, quercetin, dipeptides, and calcium ions, were investigated by use of an intestinal tract model, Caco-2 cell monolayers. The lectins increased the isoflavone transport but had no effect on aglycon transport. SBA increased the transport of quercetin glycosides, whereas CGA and WGA had no effect. The lectins increased the transport of calcium ions but showed no effect on the transport of dipeptides, carnosine, and anserine. Although SBA did not change the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayers, CGA and WGA decreased the TER value. These results indicate that plant lectins affect the transport of food factors in different manners, presumably due to their specific sugar binding activity.     

THE PATH OF CARBON IN PHOTOSYNTHESIS. XXXI. THE ROLE OF LECTINS

Radish (Raphanus sativus) and Swiss Chard (Beta vulgaris) seedlings, grown hydroponically, exhibited significantly greater increases in height and weight in medium containing α-mannoside supplemented with calcium and manganese salts, than in control treatments lacking one or more of those components. Furthermore, seeds sown on medium containing α-mannoside plus Ca²⁺and Mn²⁺showed accelerated germination. Calcium and manganese are necessary to maintain the protein conformation of mannose-binding lectins. In the presence of those cations, glycosides displace specific sugars bound to lectins, making them available to support plant growth.     

Synthesis and sweetness characteristics of L-aspartyl-D-alanine fenchyl esters

Four isomers of the L-aspartyl-D-alanine fenchyl esters were prepared as potential peptide sweeteners. L-Aspartyl-D-alanine (+)-alpha-fenchyl ester and L-aspartyl-D-alanine (-)-beta-fenchyl ester showed sweetness with potencies 250 and 160 times higher than that of sucrose, respectively. In contrast, L-aspartyl-D-alanine (+)-beta-fenchyl ester and L-aspartyl-D-alanine (-)-alpha-fenchyl ester had the highest sweetness potencies at 5700 and 1100 times that of sucrose, respectively. In particular, L-aspartyl-D-alanine (-)-alpha-fenchyl ester had an excellent sweetness quality; but L-aspartyl-D-alanine (+)-beta-fenchyl ester did not have an excellent quality of sweetness because it displayed an aftertaste caused by the strong sweetness.     

How proteins bind carbohydrates: lessons from legume lectins

The pioneering studies of Irvin Liener on soybean agglutinin (SBA) in the early 1950s served as the starting point of our involvement in lectin research during the past four decades. Initially we characterized SBA extensively as a glycoprotein and showed that its covalently linked glycan is an oligomannoside commonly present in animal glycoproteins. We have also introduced the use of the lectin to the study of normal and malignant cells and to the purging of bone marrow for transplantation. Our recent work focuses on the combining site of Erythrina corallodendron lectin, closely related to SBA. In this legume lectin, as in essentially all other members of the same protein family, irrespective of their sugar specificity, interactions with a constellation of three invariant residues (aspartic acid, asparagine, and an aromatic residue) are essential for ligand binding. Lectins from other families, whether of plants or animals, also combine with carbohydrates by H-bonds and hydrophobic interactions, but the amino acids involved may differ even if the specificity of the lectins is the same. Therefore, nature finds diverse solutions for the design of binding sites for structurally similar ligands, such as mono- or oligosaccharides. This diversity strongly suggests that lectins are products of convergent evolution.     

Efficiency of mannose-binding plant lectins in controlling a homopteran insect,the red cotton bug

Yield losses of different crops due to the attack of various classes of insects are a worldwide problem. Sucking type homopteran pests causing damage to many crop species are not controlled by commonly known insecticidal proteins, namely, Bacillus thuringiensis delta-endotoxin (Bt). This study describes the purification of mannose-binding lectins from three different monocotyledonous plants (Allium sativum, Colocasia esculenta, and Diffenbachia sequina) and their effects on a homopteran insect, the red cotton bug. All of them had a detrimental effect on the growth and development of the insect, where A. sativum bulb lectin showed the highest mortality of all, in particular. The same bulb lectin not only affected the growth and fecundity of the insect but also imparted drastic changes in the color, weight, and size, even on the second generation of the insects which have been reared on artificial diet supplemented with a sublethal dose of the lectin. Thus, this finding opens up a possibility of using this lectin as an important component in crop management.     

Constituents and their sweetness of food additive enzymatically modified licorice extract

Enzymatically modified licorice extract (EMLE) is a natural sweetener, which is prepared with cyclodextrin glucanotransferase. It is used because of its unique properties such as higher solubility and better taste than those of licorice extract. In the present paper, the structures of six major constituents isolated from EMLE were determined, and their sweetness was studied. The isolated compounds were glycyrrhizin (1), 3-O-[beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronopyranosyl]liquiritic acid (2), and their derivatives glucosylated at the C-4 position of the terminal glucuronopyranose with additional one (3 and 4, respectively) and two (5 and 6, respectively) glucose moieties. Compounds 1 and 2 are the major and minor sweet constituents in licorice extract, respectively. Compounds 3-6 are new compounds isolated for the first time. Compound 2 was sweeter than compound 1. Interestingly, compound 3, which is a monoglucosylated derivative of compound 1, was sweeter than compound 4. The sweetness of both compounds was lower than that of the parent compounds, while the lingering sweet aftertaste was markedly improved. Compounds 5 and 6, which have two additional glucose moieties, showed only slight sweetness.     

Heat-induced formation of intermolecular disulfide linkages between thaumatin molecules that do not contain cysteine residues

Thaumatin, a sweet protein that contains no cysteine residues and eight intramolecular disulfide bonds, aggregates upon heating at pH 7.0 above 70 degrees C, and its sweetness thereby disappears. The aggregate can be solubilized by heating in the presence of both thiol reducing reagent and SDS. This molecular aggregation depended on the protein concentration during heating and was suppressed by the addition of N-ethylmaleimide or iodoacetamide, indicating a thiol-catalyzed disulfide interchange reaction between heat-denatured molecules. An amino acid analysis of the aggregates suggested that the cysteine and lysine residues were reduced, and the formation of a cysteine residue and a lysinoalanine residue was confirmed. The reduction and formation of these residues stoichiometrically satisfied the beta-elimination of a cystine residue. The disulfide interchange reaction was catalyzed by cysteine; that is, a free sulfhydryl residue was formed via beta-elimination of a disulfide bond. Intermolecular disulfide bonds were probably formed between thaumatin molecules upon heating at pH 7.0, which led to the aggregation of thaumatin molecules.     

Influence of yeast macromolecules on sweetness in dry wines: role of the saccharomyces cerevisiae protein Hsp12

Yeast autolysis during lees contact influences the organoleptic properties of wines especially by increasing their sweet taste. Although observed by winemakers, this phenomenon is poorly explained in enology. Moreover, the compounds responsible for sweetness in wine remain unidentified. This work provides new insights in this way by combining sensorial, biochemical and genetic approaches. First, we verified by sensory analysis that yeast autolysis in red wine has a significant effect on sweetness. Moderate additions of ethanol or glycerol did not have the same effect. Second, a sapid fraction was isolated from lees extracts by successive ultrafiltrations and HPLC purifications. Using nano-LC-MS/MS, peptides released by the yeast heat shock protein Hsp12p were distinctly identified in this sample. Third, we confirmed the sweet contribution of this protein by sensorial comparison of red wines incubated with two kinds of yeast strains: a wild-type strain containing the native Hsp12p and a deletion mutant strain that lacks the Hsp12p protein (Δ°HSP12 strain). Red wines incubated with wild-type strain showed a significantly higher sweetness than control wines incubated with Δ°HSP12 strains. These results demonstrated the contribution of protein Hsp12p in the sweet perception consecutive to yeast autolysis in wine.     

Effect of selenium on the yield and quality of green tea leaves harvested in early spring

Foliar applications of a fertilizer of selenite or selenate were carried out to determine the influence of selenium on the yield and quality of green tea leaves harvested in early spring. Numbers of sprouts and the yield were significantly increased by the application of selenium. The sweetness and aroma of green tea leaves were also significantly enhanced, and bitterness was significantly decreased by the application of selenium. However, no significant differences were found in sweetness, bitterness, and aroma between tea leaves fertilized with selenite and selenate. Se concentration was significantly increased by selenium fertilization, and tea enriched by sodium selenate had a significantly higher selenium content than did tea enriched by sodium selenite. Total amino acid and vitamin C contents were significantly enhanced by the application of selenium. Tea polyphenol contents were significantly decreased by fertilization with selenium. The marked difference of tea polyphenols was also found between applications of selenite and selenate.     

Discovery and structure determination of a novel Maillard-derived sweetness enhancer by application of the comparative taste dilution analysis (cTDA)

Application of a novel screening procedure, the comparative taste dilution analysis (cTDA), on the non-solvent-extractable reaction products formed in a thermally processed aqueous solution of glucose and l-alanine led to the discovery of the presence of a sweetness-enhancing Maillard reaction product. Isolation, followed by LC-MS and 1D- and 2D-NMR measurements, and synthesis led to its unequivocal identification as N-(1-carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol inner salt. This so-called alapyridaine, although being tasteless itself, is the first nonvolatile, sweetness-enhancing Maillard reaction product reported in the literature. Depending on the pH value, the detection thresholds of sweet sugars, amino acids, and aspartame, respectively, were found to be significantly decreased when alapyridaine was present; for example, the threshold of glucose decreased by a factor of 16 in an equimolar mixture of glucose and alapyridaine. Studies on the influence of the stereochemistry on taste-enhancing activity revealed that the (+)-(S)-alapyridaine is the physiologically active enantiomer, whereas the (-)-(R)-enantiomer did not affect sweetness perception at all. Thermal processing of aqueous solutions of alapyridaine at 80 degrees C demonstrated a high thermal and hydrolytic stability of that sweetness enhancer; for example, more than 90 or 80% of alapyridaine was recovered when heated for 5 h at pH 7.0, 5.0, or 3.0, respectively.     

Volatile flavor analysis and sensory evaluation of custard desserts varying in type and concentration of carboxymethyl cellulose

The influence of type and concentration of carboxymethyl cellulose (CMC) on flavor and textural properties of custard desserts was examined. A synthetic strawberry flavor mixture was used to flavor the custards; it comprised 15 volatile flavor compounds. The viscosity of the custards was determined using rheometric measurements. Static headspace gas chromatography and in-nose proton transfer reaction-mass spectrometry analyses were conducted to determine the custards' volatile flavor properties. Perceived odor, flavor, and textural properties were assessed in sensory analysis experiments using magnitude estimation against a fixed modulus. Both type and concentration of CMC altered the viscosity of the custards. Softer custards had higher static headspace flavor concentrations. On the contrary, firmer custards demonstrated higher in-nose flavor concentrations. In sensory analysis, firmer custards showed higher thickness and lower sweetness intensities than their low-viscosity counterparts. The thickness perception corresponded to the viscosity of the custards. Removal of sucrose from the custards affected sweetness intensity only and not the intensity of other attributes. Therefore, the influence of the viscosity of the custards on the release of sweet-tasting components is held responsible for the effect on perceived sweetness intensity. Odor intensities were generally higher for the low-viscosity custard, whereas fruity flavor intensities were higher for the firmer custards. Odor intensities correlated with static headspace concentrations and flavor intensities related reasonably well with in-nose concentrations. Opening and closing of the nasal cavity is regarded as an important factor determining the discrepancy between static and in-nose measurements.     

New mannose-binding lectin isolated from the rhizome of Sarsaparilla Smilax glabra Roxb. (Liliaceae)

A new mannose-binding lectin, designated SGM2, was isolated from the rhizome of a Chinese medicinal herb Smilax glabra (also known as sarsaparilla in general) by saline extraction, ammonium sulfate precipitation and fractionation, and affinity chromatography on fetuin- and mannose-agarose. SGM2 is shown to have a molecular mass of 37 kDa on gel filtration and 12.5 kDa on SDS-PAGE, indicating that it is a trimeric protein composed of three identical subunits. When the first 30 amino acid residues at the N-terminal were compared, SGM2 had approximately 40% homology with those of some other monocots. SGM2 had the property of hemagglutinating activity toward rabbit erythrocytes, which could be reversed by mannose and mannose polymers. SGM2 exhibited antiviral activities against both herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV) with the same EC(50) of 8.1 microM.     

Hypoglycemic health benefits of D-psicose

Diabetes is an emerging health problem worldwide. The incidence of type 2 diabetes has dramatically increased and is expected to increase more rapidly in the future. Most patients with type 2 diabetes suffer from obesity and diabetes-related complications, including cardiovascular disease and hepatic steatosis. It has been proposed that simple sugar consumption is one of the major risk factors in the development of diabetes. Hence, the replacement of sugars with a low glycemic response would be an effective strategy to prevent type 2 diabetes. Accumulating evidence demonstrates that D-psicose, which has 70% the sweetness of sucrose and no calories, is a functional sugar exerting several health benefits preventing the development of diabetes. Although D-psicose presents in small amounts in natural products, a recent new technique using biocatalyst sources enables large-scale D-psicose production. More importantly, several clinical and animal studies demonstrated that D-psicose has hypoglycemic, hypolipidemic, and antioxidant activities, which make it an ideal candidate for preventing diabetes and related health concerns. This review will summarize the protective effects of D-psicose against type 2 diabetes and its complications, suggesting its potential benefits as a sucrose substitute.     

棉花根际促生细菌的研究进展

综述了根际促生细菌(PGPR)的概念、研究手段、基因标记技术和荧光原位杂交技术在研究棉花根际促生细菌中的应用.植物根系可以分泌各种对微生物有益的物质,吸引微生物在根际的聚集.棉花凝集素对根际促生细菌菌株的胞外多糖具有凝集作用,在棉花根际促生细菌与棉花根部相互识别过程中具有重要作用,可以利用棉花凝集素作为筛选工具筛选对棉花具有促生作用的根际促生细菌菌株.基因标记技术证明棉花根际促生细菌能高密度地定殖在棉花根部,对棉花的生长起促进作用,并能抑制某些植物病原菌及根际有害微生物.分子生物学技术在微生物生态学领域的应用促进了棉花根际促生细菌的研究和应用,可以利用棉花根际促生细菌研发微生物肥料.