Carbohydrates in the hypothallus and areolae of the crustose lichen <Emphasis Type="Italic">Rhizocarpon geographicum</Emphasis> (L.) DC.

Carbohydrate concentrations in the marginal hypothallus and areolae of the crustose lichen Rhizocarpon geographicum (L.) DC. were measured in north Wales, U.K. using gas chromatography. Ribitol, arabitol, and mannitol were the most abundant carbohydrates while α- glucose β-glucose, fructose, sucrose, and trehalose were present in smaller amounts. The concentrations of arabitol, ribitol, mannitol, fructose, and α-glucose were greater in the areolae while the concentration of trehalose was greater in the hypothallus. Concentrations of carbohydrates varied little between sample days. Concentrations of polyols in the hypothallus were not correlated with those in the areolae. These results suggest: 1) the hypothallus has a lower demand for carbohydrates than the areolae or there is limited transport from areolae to hypothallus, 2) increased trehalose in the non-lichenised hypothallus may be an adaptation to withstand stress and desiccation, and 3) polyols are partitioned differently in the hypothallus and areolae.     

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose), and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures — highly homogeneous with regard to the respective cell form produced — contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5–2% to the biomass of M. rouxii and 1.5–6% to that of C. albicans; the values for trehalose ranged from 0.2–11% in the former and 1–3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions — regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.     

Transport of acyclic polyols inSaccharomyces cerevisiae

Acyclic polyols (erythritol, xylitol, ribitol, D-arabinitol, mannitol, sorbitol and galactitol) are not metabolized by Saccharomyces cerevisiae. They are taken up by a fast non-active process, reaching 40-70% distribution referred to total cell water. The uptake is insensitive to temperature, pH (between 4 and 8), 2,4-dinitrophenol and uranyl ions. Its initial rate rises linearly with concentration from 10(-5)M to 1M. The process resembles simple diffusion through large pores or the trapping of the whole solution on the surface. Protoplasts behave like whole cells in this respect. Only erythritol shows a second type of uptake which is inhibitor-insensitive but temperature-dependent.     

Polyol metabolism by Rhizobium trifolii.

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.     

Distribution of the Products of Photosynthesis between Powdery Mildew and Barley

The photosynthetic assimilation of ¹⁴CO₂ has been studied in healthy and mildew-infected barley. The parasite was separated from the host by removing the mycelium with a camel's hair brush. The ethanol soluble metabolites of the parasite, infected host and healthy host were extracted, separated by paper chromatography and individually identified. From this work it appears that there is a rapid movement of label from host to parasite mainly in the form of sucrose which is then quickly metabolized into many compounds. The majority is converted into mannitol, and lesser amounts are converted into trehalose, arabitol, aspartic acid, and glutamic acid. In conidia the major carbon reserve is arabitol instead of mannitol, with lesser amounts of trehalose and mannitol.

Photosynthetic uptake of ¹⁴CO₂ by the complex decreases steadily after inoculation as compared with healthy leaves. However, the ethanol soluble metabolites of the infected host tissue differ only slightly from those of healthy host tissue. The major differences are a slight decrease in the amount of sucrose and increases in malic acid and serine.

     

Differences in polyols content among fermentations of the same must with several yeasts

Summary Changes in polyols contents of must after fermentation with different yeasts were determined by gas-liquid chromatography (GLC). A decrease in sorbitol, mannitol and inositol as well as the formation of erythritol, arabitol and xylitol were observed in all samples. Maximum decrease in sorbitol and mannitol was detected when flor yeast was employed.     

Transplacental carbohydrate and sugar alcohol concentrations and their uptakes in ovine pregnancy

The concentrations of glucose, fructose, sorbitol, glycerol, and myo-inositol in sheep blood and tissues have been reported previously (1--5). However, the other polyols that are at low concentrations have not been investigated in pregnant sheep due to technical difficulties. By using HPLC and gas chromatography-mass spectrometry, seven polyols (myo-inositol, glycerol, erythritol, arabitol, sorbitol, ribitol, and mannitol) and three hexoses (mannose, glucose, and fructose) were identified and quantified in four blood vessels supplying and draining the placenta (maternal artery, uterine vein, fetal artery, and umbilical vein). Uterine and umbilical blood flows were measured, and uptakes of all the polyols and hexoses in both maternal and fetal circulations were calculated. There was a significant net placental release of sorbitol to both maternal and fetal circulations. Fructose was also taken up significantly by the uterine circulation. Maternal plasma mannose concentrations were higher than fetal concentrations, and there was a net umbilical uptake of mannose, characteristics that are similar to those of glucose. Myo-inositol and erythritol had relatively high concentrations in fetal plasma (697.8 plus minus 53 microM and 463.8 plus minus 27 microM, respectively). The ratios of fetal/maternal plasma arterial concentrations were very high for most polyols. The concentrations of myo-inositol, glycerol, and sorbitol were also high in sheep placental tissue (2489 plus minus 125 microM/kg wet tissue, 2119 plus minus 193 microM/kg wet tissue, and 3910 plus minus 369 microM/kg wet tissue), an indication that these polyols could be made within the placenta.     

Glucose metabolism in resting cells ofCandida albicans as studied by13C NMR

The metabolism of glucose in resting cells ofCandida albicans was studied by¹³C NMR spectrometry using¹³C-labeled glucose. Under aeration, the formation of ethanol, glycerol, arabitol, mannitol, and trehalose was observed. The addition of inhibitors of the respiratory chain or the omission of aeration resulted in a total loss of formation of those polyols and trehalose, with ethanol being the only detectable product. Thus, respiration seems to favor the production of polyols including glycerol, as well as that of trehalose. With regard to glycerol, this finding is in contrast with the previous observation inSaccharomyces cerevisiae that oxygenation represses its production.     

Natural abundance 13C-nuclear magnetic resonance spectroscopic analysis of acyclic polyol and trehalose accumulation by several yeast species in response to salt stress

Analysis of seventeen yeast strains by 13C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii, although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species.     

Detection and characterization of sorbitol dehydrogenase from apple callus tissue

Sorbitol dehydrogenase (l-iditol:NAD(+) oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, l-arabitol, ribitol, and l-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, d-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of d-fructose or l-sorbose. No detectable activity was observed with d-tagatose. NADPH could partially substitute for NADH.Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2-methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The K(m) for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The K(m) for fructose reduction was 1.5 molar.Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO(4) (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity.     

Natural abundance 13C-nuclear magnetic resonance spectroscopic analysis of acyclic polyol and trehalose accumulation by several yeast species in response to salt stress

Abstract: Analysis of seventeen yeast strains by ¹³C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii , although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species.     

Absorption and metabolism of galactose and galactitol in Anthonomus grandis

Galactitol is a significant metabolite of galactose in fasted adults of Anthonomus grandis; this is the first time that galactitol has been found to be a metabolite of galactose in an insect in vivo. Galactitol is not metabolized, and galactose, at best, is poorly metabolized by the boll weevil to trehalose and glycogen. Because sizeable quantities appear in the haemolymph when fed, galactose and galactitol must be readily absorbed through the gut.     

Simultaneous measurement of urinary polyols using gas chromatography/mass spectrometry

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry-positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 microL of a urine sample were diluted with 3 ml of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5-1.0 ng/microL, and we made the calibration curves to be linear from 0.25 to 250 ng/microL (r(2)>0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.     

Resistance of food spoilage yeasts to sorbic acid

Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were grown on Sabouraud Dextrose Agar (SDA) modified with KCl to give a range of water activity (a_w) from 0.938 to 0.998. Growth of all three species was optimal at 0.983 a_w and growth occurred over the a_w range tested. Acyclic sugar alcohol (polyol) and trehalose content of conidia was determined by HPLC and found to vary with species and a_w. Conidia of B. bassiana and P. farinosus were found to contain totals of 1.5% and 2.3% polyols respectively at 0.998 a_w, and double these amounts at <0.950 a_w. Conidia of M. anisopliae contained from 5.7% to 6.8% polyols at each a_w tested. In conidia of all three species the predominant polyol was mannitol. The lower molecular weight polyols, arabitol and erythritol, were found to accumulate at reduced a_w. Small amounts of glycerol were present in conidia of each species; <15% total polyols. Conidia of B. bassiana and M. anisopliae contained about 0.5% trehalose from 0.970 to 0.998 a_w, but only trace amounts below 0.950 a_w. Conidia of P. farinosus contained 2.1% trehalose at 0.998 a_w and this decreased to <0.1% below 0.950 a_w. Potential to manipulate the endogenous reserves of conidia of these biological control agents to enhance viability and desiccation toierance is discussed     

Perspectives of engineering lactic acid bacteria for biotechnological polyol production

Polyols are sugar alcohols largely used as sweeteners and they are claimed to have several health-promoting effects (low-caloric, low-glycemic, low-insulinemic, anticariogenic, and prebiotic). While at present chemical synthesis is the only strategy able to assure the polyol market demand, the biotechnological production of polyols has been implemented in yeasts, fungi, and bacteria. Lactic acid bacteria (LAB) are a group of microorganisms particularly suited for polyol production as they display a fermentative metabolism associated with an important redox modulation and a limited biosynthetic capacity. In addition, LAB participate in food fermentation processes, where in situ production of polyols during fermentation may be useful in the development of novel functional foods. Here, we review the polyol production by LAB, focusing on metabolic engineering strategies aimed to redirect sugar fermentation pathways towards the synthesis of biotechnologically important sugar alcohols such as sorbitol, mannitol, and xylitol. Furthermore, possible approaches are presented for engineering new fermentation routes in LAB for production of arabitol, ribitol, and erythritol.     

Control of arabitol formation in Schizophyllum commune development

Summary Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.     

Monoclonal antibody screening: two methods using antigens immobilized on nitrocellulose

Aldose reductase is an enzyme that plays an important role in diabetic complications such as cataract and neuropathy. The best way to estimate the enzymatic activity of this enzyme in vivo consists of measuring the accumulation of sorbitol or galactitol in various types of cells or tissues. A sensitive method to measure the polyols in biological samples by high-performance liquid chromatography has been developed. This method is based on the fact that polyols like sorbitol and galactitol react with phenylisocyanate to yield uv-absorbing derivatives at 240 nm. Applications to the separation and determination of polyols in biological samples of various origins, such as lenses, sciatic nerves, human skin fibroblasts, and red cells, are described and illustrated.     

Electricity generation from polyalcohols in single-chamber microbial fuel cells

Six polyalcohols derived from lignocellulosic carbohydrates were investigated as carbon sources for electricity generation in single-chamber mediator-less microbial fuel cells (MFCs) for the first time. Electricity was directly generated from all polyalcohols tested, including pentitols (xylitol, arabitol, and ribitol) and hexitols (galactitol, mannitol, and sorbitol). Bacterial cultures initially enriched using acetate could be adapted to these substrates with varied adaptation times. The resultant maximum power density ranged from 1490+/-160 mW/m(2) to 2650+/-10 mW/m(2) at current densities between 0.58 mA/cm(2) and 0.78 mA/cm(2). Galactitol generated the highest maximum power density, while mannitol resulted in the lowest one. The estimated maximum voltage output at an external resistance of 120 Omega ranged between 0.24 V and 0.34 V with half saturation kinetic constants varied from 298 mg/L to 753 mg/L. The removal of chemical oxygen demand (COD) was above 91% for all polyalcohols except sorbitol (71%). Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments of the anode biofilms showed the influence of substrates (polyalcohols) on the anode microbial populations.     

Temporal accumulation of mannitol and arabitol in Geotrichum candidum

The temporal depletion and accumulation of polyols were investigated in the fungus Geotrichum candidum. The major intracellular polyols were tentatively identified by paper chromatography as mannitol and arabitol. Inositol was also present in small quantities, and trehalose was also detected in appreciable concentrations.Germination and vegetative growth depended on the type and concentration of the sole exogenous carbon source. Mannitol occurred in arthrospores at 9.4% of the dry weight after several days growth in 2% (w/v) glucose solid medium, and became depleted during germination and vegetative growth in liquid medium containing 2% (w/v) glucose, 2% (w/v) sodium acetate or 25% (w/v) glucose as sole carbon source. This hexitol latter accumulated during arthrosporulation. The depletion and accumulation of ethanol-soluble carbohydrate believed to be primarily trehalose was temporally similar to that of mannitol. Arabitol accumulated intracellularly during germination and vegetative growth in sodium acetate medium and 25% glucose medium. This pentitol was not detected intracellularly at any culture age during growth in 2% glucose medium.Prolonged incubation of the culture in 25% glucose medium after stationary phase was reached resulted in the gradual disappearance of arabitol from the arthrospores simultaneously with an increase in intracellular mannitol. In comparison, ethanol-soluble carbohydrate did not change with prolonged incubation in this medium.     

Improving water stress tolerance of the biocontrol yeast Candida sake grown in molasses-based media by physiological manipulation

The biocontrol agent Candida sake was cultured on either an unmodified molasses-based medium (water activity, a(w) 0.996) or on water stressed media produced by the addition of glycerol, glucose, NaCl, sorbitol, or proline to 0.98, and 0.96 a(w) for 24, 48, and 72 h, to study their impact on subsequent cell viability, and on concentrations of endogenous sugars (trehalose and glucose) and polyols (glycerol, erythritol, arabitol, and mannitol). The viability of cells of different ages cultured on these media was evaluated on NYDA medium with freely available water (a(w) 0.995), and on medium modified with polyethylene glycol to a(w) 0.95. Regardless of solute used, viable counts of cells grown on molasses-based medium (a(w) 0.98) were equal to or higher than those obtained from the medium with water freely available. The amino acid proline stimulated growth at 10% concentration. In contrast, water stress induced by addition of NaCl, glucose, or sorbitol at a(w) 0.96 caused a significant reduction in viable counts. Older cultures were more resistant to water stress. Glycerol and arabitol were the main solutes accumulated by C. sake cells in response to lowered a(w). Intracellular concentration of these polyols depended more on the solute used to adjust the a(w) than on the a(w) itself. Candida sake was more resistant to water stress with higher intracellular concentration of glycerol and erythritol.