A Preliminary Study on Metabolome Profiles of Buffalo Milk and Corresponding Mozzarella Cheese: Safeguarding the Authenticity and Traceability of Protected Status Buffalo Dairy Products.

The aim of this study is to combine advanced GC-MS and metabolite identification in a robust and repeatable technology platform to characterize the metabolome of buffalo milk and mozzarella cheese. The study utilized eleven dairies located in a protected designation of origin (PDO) region and nine dairies located in non-PDO region in Italy. Samples of raw milk (100 mL) and mozzarella cheese (100 g) were obtained from each dairy. A total of 185 metabolites were consistently detected in both milk and mozzarella cheese. The PLS-DA score plots clearly differentiated PDO and non-PDO milk and mozzarella samples. For milk samples, it was possible to divide metabolites into two classes according to region: those with lower concentrations in PDO samples (galactopyranoside, hydroxybuthyric acid, allose, citric acid) and those with lower concentrations in non-PDO samples (talopyranose, pantothenic acid, mannobiose, etc.,). The same was observed for mozzarella samples with the proportion of some metabolites (talopyranose, 2, 3-dihydroxypropyl icosanoate, etc.,) higher in PDO samples while others (tagatose, lactic acid dimer, ribitol, etc.,) higher in non-PDO samples. The findings establish the utility of GC-MS together with mass spectral libraries as a powerful technology platform to determine the authenticity, and create market protection, for "Mozzarella di Bufala Campana."

Plant gum identification in historic artworks.

We describe an integrated and straightforward new analytical protocol that identifies plant gums from various sample sources including cultural heritage. Our approach is based on the identification of saccharidic fingerprints using mass spectrometry following controlled enzymatic hydrolysis. We developed an enzyme cocktail suitable for plant gums of unknown composition. Distinctive MS profiles of gums such as arabic, cherry and locust-bean gums were successfully identified. A wide range of oligosaccharidic combinations of pentose, hexose, deoxyhexose and hexuronic acid were accurately identified in gum arabic whereas cherry and locust bean gums showed respectively PentxHexy and Hexn profiles. Optimized for low sample quantities, the analytical protocol was successfully applied to contemporary and historic samples including 'Colour Box Charles Roberson &Co' dating 1870s and drawings from the American painter Arthur Dove (1880-1946). This is the first time that a gum is accurately identified in a cultural heritage sample using structural information. Furthermore, this methodology is applicable to other domains (food, cosmetic, pharmaceutical, biomedical).

The structure of the LPS O-chain of Fusobacterium nucleatum strain 25586 containing two novel monosaccharides, 2-acetamido-2,6-dideoxy-l-altrose and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism, and thus we have initiated studies to examine the bacterium's surface glycochemistry. We isolated lipopolysaccharide (LPS) from F. nucleatum strain 25586 and purified and performed structural analysis on the O-antigen polysaccharide. The polysaccharide contained two novel sugars, 2-acetamido-2,6-dideoxy-l-altrose (l-6dAltNAc) and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid (Non5Am), which was tentatively assigned the l-glycero-l-gluco configuration. The polysaccharide was found to have a trisaccharide repeating unit, which is phosphorylated with phosphocholine (PCho), and the following structure was established: -[-4-ß-Nonp5Am-4-α-l-6dAltpNAc3PCho-3-ß-d-QuipNAc-]- We propose the trivial name 'fusaminic acid' for the novel nonulosonic acid. It is the first occurrence of a 9-deoxynonulosonic acid with a hydroxyl group at C-7, which is occupied by an amino group in all monosaccharides of this class described so far.

Structural and immunological feature of rhamnogalacturonan I-rich polysaccharide from Korean persimmon vinegar.

The crude polysaccharide (KPV-0) isolated from Korean persimmon vinegar was fractionated using gel filtration chromatography to enhance the immunostimulatory activity and to identify the structural features of active fraction. Among three fractions, KPV-I obtained in a void volume, demonstrated the potent production of macrophage-stimulating mediators, including tumor necrosis factor-α, interleukin (IL)-6, IL-12, and nitric oxide. KPV-I showed a combined single peak with high molecular weight of 55,000Da by high performance size exclusion chromatography. Component sugar analysis revealed that KPV-I contained mainly of arabinose, mannose, galactose, rhamnose and galacturonic acid. Single radial gel diffusion assay using ß-glucosyl Yariv reagent showed that KPV-I contained arabinogalactan protein with 13.7%. Methylation analysis indicated that KPV-I contained 21 kinds of neutral glycosidic linkages, which seemed to be composed three kinds of polysaccharide; that is a rhamnogalacturonan-I (65-70%) derived from persimmon as a raw material, a mannan (20-25%) derived from fermentation-associated microorganisms, and a linear glucans (less than 10%). In conclusion, polysaccharide isolated from persimmon vinegar could augment the macrophage stimulation, and a large amounts of RG-I polysaccharide derived from persimmon is likely a crucial role in expression of the activity in persimmon vinegar.

Molecular structure, chemical properties and biological activities of Pinto bean pod polysaccharide.

Pinto bean pod polysaccharide (PBPP) was successfully extracted with yield of 38.5g/100g and the PBPP gave total carbohydrate and uronic acid contents of 286.2mg maltose equivalent/g and 374.3mgGal/g, respectively. The Mw of PBPP was 270.6kDa with intrinsic viscosity of 0.262dm(3)/g, which composed of mannose (2.5%), galacturonic acid (15.0%), rhamnose (4.0%), glucose (9.0%), galactose (62.2%), xylose (2.9%) and arabinose (4.3%) with trace amount of ribose and fucose. The result suggested that PBPP has a spherical conformation with a highly branched structure. Fourier Transform Infrared analysis showed that PBPP has a similar structure as commercial pectin with an esterification degree of 59.9%, whereas scanning electron microscopy study showed that the crude polysaccharide formed a thin layer of film that was made of multiple micro strands of fibre. PBPP exhibited substantial free radical scavenging activity (7.7%), metal reducing capability (2.04mmol/dm(3)) and α-amylase inhibitory activity (97.6%) at a total amount of 1mg. PBPP also exhibited high water- and oil-holding capacities (3.6g/g and 2.8g/g, respectively). At a low concentration, PBPP exhibited emulsifying activity of 39.6% with stability of 38.6%. Apart from that, PBPP was able to show thickening capability at low concentration (0.005kg/dm(3)).

Efficient enzymatic synthesis of L-rhamnulose and L-fuculose.

L-Rhamnulose (6-deoxy-L-arabino-2-hexulose) and L-fuculose (6-deoxy-L-lyxo-2-hexulose) were prepared from L-rhamnose and L-fucose by a two-step strategy. In the first reaction step, isomerization of L-rhamnose to L-rhamnulose, or L-fucose to L-fuculose was combined with a targeted phosphorylation reaction catalyzed by L-rhamnulose kinase (RhaB). The by-products (ATP and ADP) were selectively removed by silver nitrate precipitation method. In the second step, the phosphate group was hydrolyzed to produce L-rhamnulose or L-fuculose with purity exceeding 99% in more than 80% yield (gram scale).

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories.

Chiral separation of D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent and a phenylboronic acid complex.

A novel method was developed for D/L-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of D/L isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300 nM), and the repeatabilities of the migration times and peak area responses were 0.8 % and 7.9 % (relative standard deviation; n = 6), respectively. The method was applied to the determination of D/L- galactose in red seaweed.

Enhanced fermentable sugar production from kitchen waste using various pretreatments.

The kitchen waste fraction in municipal solid waste contains high organic matter particularly carbohydrate that can contribute to fermentable sugar production for subsequent conversion to bioethanol. This study was carried out to evaluate the influence of single and combination pretreatments of kitchen waste by liquid hot water, mild acid pretreatment of hydrochloric acid (HCl) and sulphuric acid (H2SO4) and enzymatic hydrolysis (glucoamylase). The maximum total fermentable sugar produced after combination pretreatment by 1.5% HCl and glucoamylase consisted of 93.25 g/L glucose, 0.542 g/L sucrose, 0.348 g/L maltose, and 0.321 g/L fructose. The glucose released by the combination pretreatment method was 0.79 g glucose/g KW equivalent to 79% of glucose conversion. The effects of the pre-treatment on kitchen waste indicated that the highest solubilization was 40% by the combination method of 1.5% HCl and glucoamylase. The best combination pre-treatment gave concentrations of lactic acid, acetic acid, and propionic acid of 11.74 g/L, 6.77 g/L, and 1.02 g/L, respectively. The decrease of aliphatic absorbance bands of polysaccharides at 2851 and 2923 cm(-1) and the increase on structures of carbonyl absorbance bands at 1600 cm(-1) reflects the progress of the kitchen waste hydrolysis to fermentable sugars. Overall, 1.5% HCl and glucoamylase treatment was the most profitable process as the minimum selling price of glucose was USD 0.101/g kitchen waste. Therefore, the combination pretreatment method was proposed to enhance the production of fermentable sugar, particularly glucose from kitchen waste as the feedstock for bioethanol production.

Five natural carbohydrates from the leaves of Sauropus rostratus.

Three new hexose derivatives (1-3), together with two known ones (4,5), were isolated from the leaves of Sauropus rostratus. The structures of new compounds were elucidated on the basis of the spectroscopic methods including 1D and 2D NMR (HSQC, (1)H-(1)H COSY, HMBC and NOESY) and HR-MS analyses. The absolute configurations of three new compounds were established by the modified Mosher's method.

Structure of a novel α-glucan substitute with the rare 6-deoxy-D-altrose from Lactarius lividatus (mushroom).

A novel α-glucan substituted rare 6-deoxy-D-altropyranose was isolated from edible fruiting bodies of a mushroom (Lactarius lividatus) grown in Okinawa, Japan. The polysaccharide consists of D-glucose, D-galactose and 6-deoxy-D-altrose in a molar ratio of 3.0:1.0:1.0. The specific rotation [α](589) was estimated as +64.3° (0.2% in water) at 25 °C. Based on results of IR, NMR ((1)H, (13)C, 2D-COSY, 2D-HMQC, 2D-ROESY and 2D-HMBC), and methylation analyses, the structure of the polysaccharide was determined as [formula, see text] This work is the first demonstration of rare 6-deoxy-D-altropyranose moiety on polysaccharides.

Hamamelitannin from witch hazel (Hamamelis virginiana) displays specific cytotoxic activity against colon cancer cells.

Hamamelis virginiana (witch hazel) bark is a rich source of condensed and hydrolyzable tannins reported to exert a protective action against colon cancer. The present study characterizes different witch hazel tannins as selective cytotoxic agents against colon cancer. To cover the structural diversity of the tannins that occur in H. virginiana bark, the hydrolyzable tannins, hamamelitannin and pentagalloylglucose, together with a proanthocyanidin-rich fraction (F800H4) were selected for the study. Treatment with these compounds reduced tumor viability and induced apoptosis, necrosis, and S-phase arrest in the cell cycle of HT29 cells, with hamamelitannin being the most efficient. Owing to polyphenol-mediated H(2)O(2) formation in the incubation media, the antiproliferative effect was determined in the presence and absence of catalase to rule out any such interference. The presence of catalase significantly changed the IC(50) only for F800H4. Furthermore, at concentrations that inhibit the growth of HT29 cells by 50%, hamamelitannin had no harmful effects on NCM460 normal colonocytes, whereas pentagalloylglucose inhibited both cancerous and normal cell growth. Using the TNPTM assay, we identified a highly reactive phenolic position in hamamelitannin, which may explain its efficacy at inhibiting colon cancer growth.

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation.

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.

Development of a chemical strategy to produce rare aldohexoses from ketohexoses using 2-aminopyridine.

Rare sugars are monosaccharides that are found in relatively low abundance in nature. Herein, we describe a strategy for producing rare aldohexoses from ketohexoses using the classical Lobry de Bruyn-Alberda van Ekenstein transformation. Upon Schiff-base formation of keto sugars, a fluorescence-labeling reagent, 2-aminopyridine (2-AP), was used. While acting as a base catalyst, 2-AP efficiently promoted the ketose-to-aldose transformation, and acting as a Schiff-base reagent, it effectively froze the ketose-aldose equilibrium. We could also separate a mixture of Sor, Gul, and Ido in their Schiff-base forms using a normal-phase HPLC separation system. Although Gul and Ido represent the most unstable aldohexoses, our method provides a practical way to rapidly obtain these rare aldohexoses as needed.

HPLC separation of all aldopentoses and aldohexoses on an anion-exchange stationary phase prepared from polystyrene-based copolymer and diamine: the effect of NaOH eluent concentration.

To investigate the separations of all aldopentoses (ribose, arabinose, xylose and lyxose) and aldohexoses (glucose, galactose, allose, altrose, mannose, gulose, idose and talose) on the D6 stationary phase prepared by the reaction of chloromethylated styrene-divinylbenzene copolymer and N,N,N',N'-tetramethyl-1,6-diaminohexane, we examined the effect of varying the concentration of the NaOH eluent on the elution orders. Separations of these aldoses were achieved using a 20 mM NaOH eluent. The elution behaviors of the aldoses were probably due to not only the individual pK(a) values, but also the chemical structures of the cyclic aldoses.

1, 2 di-substituted idopyranose from Vitex negundo L. protects against streptozotocin-induced diabetes by inhibiting nuclear factor-kappa B and inducible nitric oxide synthase expression.

The aim of this study is to investigate the mechanism of an action of compound isolated from Vitex negundo in streptozotocin-induced diabetic mice. Light microscopic examination of liver, kidney and pancreatic sections of streptozotocin-induced diabetic mice showed changes like coarsening of acinar cells of endoplasmic reticulum, destruction of ß-cells, and alteration in their secretory function were observed in the pancreas. Changes like dilation of vein, unusual concentric arrangement of hepatocytes, and liver fibrosis were observed in the liver. Thickening of tubules and expansion of glomerulus were observed in kidneys. All these altered parameters were reversed close to normal condition upon treatment using idopyranose. The results show the antidiabetic potential of idopyranose. Interestingly, liver, kidney, and pancreatic sections of diabetic mice fed with the isolated 1, 2 di-substituted idopyranose showed regeneration of hepatocytes, nephrocytes, as well as ß-cells and acinar region appeared normal with increased numbers of ß-cells. To understand the probable mechanism of action of 1, 2 di-substituted idopyranose, we analyzed proinflammatory inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) expression by immunohistochemistry and the results showed an increased iNOS and NF-κB levels in streptozotocin-induced diabetic liver, kidney and pancreas. Such high iNOS and NF-κB levels were inhibited in 1, 2 di-substituted idopyranose treated mice. The results suggest that 1, 2 di-substituted idopyranose helps in the protection of hepatocytes, nephrocytes and pancreatic ß-cells probably by its action against NF-κB and iNOS mediated inflammation in streptozotocin-induced diabetes.

L-valine assisted distinction between the stereo-isomers of D-hexoses by positive ion ESI tandem mass spectrometry.

The binary mixtures of 7 hexoses and 20 amino acids were investigated by electrospray ionization ion trap mass spectrometry (ESI-ITMS). The adduct ions of the amino acid and the hexose were detected for 12 amino acids but not for the other 8 amino acids which are basic acidic amino acids and amides. The ions of amino acid-hexose complexes were further investigated by tandem mass spectrometry (MS/MS), and some of them just split easily into two parts whereas the others gave rich fragmentation, such as the complex ions of isoleucine, phenylalanine, tyrosine, and valine. We found that hexoses could be complexed by two molecules of valine but only by one molecule of the other amino acids. Among seven kinds of valine-hexose complexes coordinated by potassium ion, the MS(2) spectra of the ion at m/z 453 yielded unambiguous differentiation. And the fragmentation ions are sensitive to the stereochemical differences at the carbon-4 of hexoses in the complexes, as proved by the MS(2).

A comprehensive HPLC analytical system for the identification and quantification of hexoses that employs 2-aminobenzamide coupling.

Rare sugars are defined as monosaccharides with extremely low natural abundances. Their natural distributions and biological functions remain to be clarified. To establish a robust analytical system that can separate, identify and quantify rare sugars, 12 d-hexoses-including five rare aldohexoses and three rare ketohexoses-were labelled with 2-aminobenzamide (2-AB), and the fluorescently tagged monosaccharides were separated by high-performance liquid chromatography (HPLC). Because the ketohexoses were much less reactive than were the aldohexoses, the reaction conditions were optimized to achieve the maximum yields (>75%) for both aldohexoses and ketohexoses. The calibration curve determined for the rare ketohexose, d-psicose (Psi), was linear between 1 pmol and 1 micromol and had a correlation coefficient of 0.9999. Using the developed method, the psicose content in a leaf of Itea virginica, in which the presence of psicose has previously been reported, was found to be 2.7 mg psicose/g leaf. The result proved feasibility of the method even for natural products. Because the system is a comprehensive tool, it should help answer questions concerning the biosyntheses and functions of rare hexose sugars.

[Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan].

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4-14% and a yield of 60-80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coil. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02-0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.

In vitro anti-influenza screening of several Euphorbiaceae species: structure of a bioactive Cyanoglucoside from Codiaeum variegatum.

A bio-guided screening against influenza A virus (FLUAV) was carried out with seven Euphorbiaceae species. The results showed that chromatographic fractions from Phyllantus niruri, Euphorbia pulcherrima and Codiaeum variegatum had relevant anti-FLUAV activity, although only chromatographical subfractions from C. variegatum kept the activity. From this plant, the active compound against FLUAV was isolated. Its structure was assigned as 2-(3,4,5)-trihydroxy-6-hydroxymethyltetrahydropyran-2-yloxymethyl)acrylonitrile (1) on the basis of NMR, mass spectrometry and X-ray diffraction analysis. The compound displayed virucidal activity without impairment of haemagglutination properties of the used virus strain. This is the first report indicating antiviral activity of a cyanoglucoside.