GUSTATORY REACTION TIME AND TIME INTENSITY MEASUREMENTS OF TREHALOSE AND SUCROSE SOLUTIONS AND THEIR MIXTURES

Dynamic sweetness perception of commercial food grade trehalose, sucrose solutions and their mixtures were studied for a wide range of concentrations. For gustatory reaction time (GRT), concentrations ranged from 2.3 to 13.8% for sucrose and up to 23.0% for trehalose. For time intensity (T-I) sucrose or trehalose solutions (concentration range 2.3–36.8%) and their combinations (23.0 and 36.8% total solids) were analyzed. Trehalose had bigger GRT along the studied range. Both sugars presented similar values for persistence and times of plateau and to maximum intensity, while a significant difference was observed in intensity and GRT at equal concentrations. Trehalose had longer persistence than sucrose in equi -sweet solutions. Overall sweetness profile of some sucrose solutions (i.e., 29.9% sucrose solution and 0.6 sucrose/trehalose ratio mixture at 36.8% total solids) were perceived as similar to mixtures of sucrose/trehalose or single trehalose solutions, which suggests the possibility of sugar replacement without completely modifying sweetness perception.

PRACTICAL APPLICATIONS

It has been suggested that trehalose may be a potential substitute for sucrose and other sugars used in food formulation because, although its chemical structure is very similar to that of sucrose, it is more stable at low pH and high temperatures. It is not involved in caramelization and does not participate in Maillard reaction with amino acids/proteins. In order to fully establish the potential of trehalose as a functional replacement of sucrose we have determined the sweetness dynamic profile (gustatory reaction time and time-intensity curves) of trehalose solutions and sucrose/trehalose solutions; this aspect is needed for adequately replacing (partially or totally) sucrose in food systems.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

POTENCY OF SWEETNESS OF ASPARTAME, D-TRYPTOPHAN AND THAUMATIN EVALUATED BY SINGLE VALUE AND TIME-INTENSITY MEASUREMENTS

Perceived sweetness of sucrose, aspartame, D-tryptophan and thaumatin in a sour, citric acid background was analyzed in terms of the potency of these compounds relative to sucrose-water combinations. Potencies of the sweeteners were determined from (1) maximum intensity using single value and time-intensity (T-I) measurements and (2) average intensity calculated as the ratio of area under the T-I curve and total perceived time. Stevens' law was applied to sweet responses, either in static or dynamic conditions. It was found that the exponent of the concentration-response function reflected the relative capacity of a compound to sweeten a given food and stressed differences of potency among sweeteners. Aspartame, D-tryptophan and thaumatin exhibited a decrease in sweetness potency relative to sucrose as sweetness increased from 10 to 100% of the full scale of response. Across the entire sweetness range, thaumatin showed the greatest potency but its long persistence time led to differentiate this intense sweetener from the other sweeteners evaluated.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

SOURNESS–SWEETNESS INTERACTIONS IN DIFFERENT MEDIA: WHITE WINE,ETHANOL AND WATER*

The aim of this work was to study the sourness–sweetness interactions in water, white wine and alcoholic environment to interpret sweet/sour perception in low concentrations within the range normally encountered in white wine. Nine trained assessors rated sweetness and sourness intensity in mixtures of fructose (11.1, 25.0 and 38.9 mM) and tartaric acid (pH 3.0, 3.4 and 3.8) in water and wine (experiment 1) or ethanol solutions at 2.0, 4.0 and 12.0% v/v (experiment 2). The range of quantitative responses was larger for sourness than for sweetness in the three media. The global sourness intensity perception in wine mixtures was significantly lower than in water and ethanol mixtures, indicating the effect of other wine components. The suppressive effect of tartaric acid on fructose sweetness was stronger than the suppressive effect of fructose on tartaric acid sourness.     

Revisiting sugar-fat mixtures: sweetness and creaminess vary with phenotypic markers of oral sensation

Genetic variation in oral sensation presumably influences ingestive behaviors through sensations arising from foods and beverages. Here, we investigated the influence of taste phenotype [6-n-propylthiouracil (PROP) bitterness, fungiform papillae (FP) density] on sweet and creamy sensations from sugar/fat mixtures. Seventy-nine subjects (43 males) reported the sweetness and creaminess of water or milk (skim, whole, heavy cream) varying in sucrose (0-20% w/v) on the general Labeled Magnitude Scale. Sweetness grew with sucrose concentration and when shifting from water to milk mixtures--the growth was greatest for those tasting PROP as most bitter. At higher sucrose levels, increasing fat blunted the PROP-sweet relationship, whereas at lower levels, the relationship was effectively eliminated. Perceived sweetness of the mixture exceeded that predicted from the sum of components at low sucrose concentrations (especially for those tasting PROP most bitter) but fell below predicted at high concentrations, irrespective of fat level. Creaminess increased greatly with fat level and somewhat with sucrose. Those tasting PROP most bitter perceived greater creaminess in the heavy cream across all sucrose levels. Perceived creaminess was somewhat lower than predicted, irrespective of PROP bitterness. The FP density generally showed similar effects as PROP on sweetness and creaminess, (but to a lesser degree) and revealed potential taste-somatosensory interactions in weakly sweet stimuli. These data support that taste phenotype affects the nature of enhancement or suppression of sweetness and creaminess in liquid fat/sugar mixtures. Taste phenotype effects on sweetness and creaminess likely involve differential taste, retronasal olfactory, and somatosensory contributions to these perceptual experiences.     

Modeling sucrose hydrolysis in dilute sulfuric acid solutions at pretreatment conditions for lignocellulosic biomass

Agricultural and herbaceous feedstocks may contain appreciable levels of sucrose. The goal of this study was to evaluate the survivability of sucrose and its hydrolysis products, fructose and glucose, during dilute sulfuric acid processing at conditions typically used to pretreat lignocellulose biomass. Solutions containing 25g/l sucrose with 0.1-2.0% (w/w) sulfuric acid concentrations were treated at temperatures of 160-200 degrees C for 3-12min. Sucrose was observed to completely hydrolyze at all treatment conditions. However, appreciable concentrations of fructose and glucose were detected and glucose was found to be significantly more stable than fructose. Different mathematical approaches were used to fit the kinetic parameters for acid-catalyzed thermal degradation of these sugars. Since both sugars may survive dilute acid pretreatment, they could provide an additional carbon source for production of ethanol and other bio-based products.     

Comparison of the hydrolytic activity of commercial cellulase preparations

Summary Two different quality types of sugar-cane molasses containing a total sugar content of 48%–50% (w/v) and 35%–42% (w/v) were investigated for Zymomonas biothanol production. Molasses concentrations of up to 250 g/l (1:3 dilution) were successfully fermented within 24 h despite a higher salt concentration in the lower grade molasses. Higher molasses concentrations (300 g/l) led to fructose accumulation. The addition of sucrose to a final sugar concentration of 15% (w/v) led to 10% (v/v) ethanol with conversion efficiencies up to 96%. Sorbitol levels were negligible, but increased up to tenfold upon addition of invertase. Offprint requests to: H. W. Doelle     

Differential adaptation of membranes of two osmotolerant fungi,Aspergillus chevalieri and Penicillium expansum to high sucrose concentrations

Aspergillus chevalieri and Penicillium expansum were able to tolerate sucrose concentrations in the growth media up to 80% (w/v). At 50% sucrose the growth rate is approximately 1.4 and 1.2 times, respectively, higher than in the control. While at 80% sucrose it drops to 35% and 45% of the control level for both fungi. Lipids and proteins in plasma membranes increased with increasing sucrose concentrations in the growth medium. Phospholipid content in membranes of both organisms being also increased, phosphatidyl glycerol was the major detected phospholipid and represented the highest increase. The fatty acid composition of fraction enriched plasma membrane of both fungi changed when they were grown in high sucrose concentrations. Some fatty acids which had not been detected in control cultures were present and the proportions of other fatty acids changed. At 50% sucrose the unsaturation index of membranes decreased by 20-25% in both fungi, indicating that the plasma membrane is less fluid at this concentration. At 80% sucrose a similar trend was observed for P. expansum but for A. chevalieri the unsaturation index was little changed compared with the control. The fluorescence polarization values of 1,6-diphenyl 1,3,5-hexatriene (DPH) in membranes of both fungi grown at 80% sucrose increased, indicating a decrease in membrane fluidity. At 50% sucrose the increase in saturation of membrane fatty acids would tend to reduce membrane fluidity but in A. chevalieri at 80% sucrose fatty acids did not become more saturated. In this case the marked increase in sterols at this sucrose concentration may be responsible for low membrane fluidity.     

Effect of fructose and glucose supplementation on invertase mediated synthesis of oligosaccharides from sucrose

Summary The total amount of novel oligosaccharides synthesized by -D-fructofuranosidase at pH 7.5 increased three-fold using a medium composed of 1.2M sucrose, 0.5M fructose and 0.1M glucose, as compared to that with only 1.8M sucrose solution. Using 0.6M of the three sugars did not increase yield but reduced rate of sucrose hydrolysis by 72.7%. Synthesis of fructosyl/glucosyl oligosaccharides based on -fructofuranosidase mediated transglycosylation is enhanced by supplementation of sucrose solution with appropriate concentrations fructose and glucose.     

Effect of Temperature on the Nitrogenase Activity of Intact and Detached Nodules in Lotus and Stylosanthes

Temperature and plant age influenced the nitrogenase activityof Lotus and Stylosanthes nodules. Time course studies usingnodulated plants in closed vials showed a decline in activityafter 48 h; regassing with 10% (v/v) acetylene in air partiallyrestored the activity. Transfer of plants from 15, 20, 25, and30 ?C to 40 ?C immediately stopped activity; this was completelyrestored within 1 h after return to original temperatures. Detached nodules cultured on nitrogen-free agar medium exhibiteda sucrose concentration (2–8%, w/v)-dependent nitrogenaseactivity at each temperature. With 6% sucrose prolonged activity,up to 96 h, was obtained. Decline in nitrogenase activity indetached nodules was due partly to exposure to air during excisionand transfer to the medium.     

Growth and hyoscyamine production of ‘hairy root’ cultures of Datura stramonium in a modified stirred tank reactor

Summary The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg·g^–1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g^–1 wet weight). Growth at 30°C was initially faster than at either 25°C or 35°C and by day 12, the drained weight of roots in the fermentor at 30°C was about fourfold greater than at 25°C and twice that at 35°C. The ultimate hyoscyamine levels attained (approximately 0.5 mg·g^–1 wet weight) were similar at both 25°C and 30°C but some 40% lower at 35°C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25°C and the highest production rate of 8.2 mg hyoscyamine l^–1 per day was obtained for roots grown at 30°C. In continuous fermentation at 25°C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30°C or 35°C. Offprint requests to: M. G. Hilton     

An assessment of binary mixture interactions for nine sweeteners

Binary mixtures of acesulfame K, aspartame, sodium cyclamate,fructose, glucose, stevioside, sodium saccharin, sucrose andxylitol were assessed using factorial mixture designs. A simpleadditive model was used to generate predictions for the sweetnessof the mixtures and these predicted responses were comparedto the observed sweetness ratings of the mixtures. It was foundthat the mixtures tended to exhibit superadditivity at low concentrations,additivity at intermediate concentrations and subadditivityat high concentrations. Synergistic and suppressive effectsin the mixtures were evaluated by comparing mixture responsesto the sweetness ratings of ‘self-mixtures’. Self-mixturedata were generated by treating a mixture of a substance withitself as if it were a mixture of two different substances.Synergism was defined as a mixture response that was greaterthan the sweetness of the component self-mixtures, and suppressionwas defined as a mixture response that was less than the sweetnessof the component self-mixtures. Of the 31 binary mixtures studied,18 showed synergism, two showed suppression and 11 did not differsignificantly from their components. It is hypothesized thatmultiple sweetness receptors or release from bitter suppressionmay account for the synergistic effects.     

Genome shuffling to improve thermotolerance,ethanol tolerance and ethanol productivity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we improved the thermotolerance and ethanol tolerance of an industrial yeast strain SM-3 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by protoplast ultraviolet irradiation and then subjected for the recursive protoplast fusion. The positive colonies from the library, created by fusing the inactivated protoplasts were screened for growth at 35, 40, 45, 50 and 55°C on YPD-agar plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of three phenotypes of thermotolerance, ethanol tolerance and ethanol yields enhancement. After three rounds of genome shuffling, the best performing strain, F34, which could grow on plate cultures up to 55°C, was obtained. It was found capable of completely utilizing 20% (w/v) glucose at 45–48°C, producing 9.95% (w/v) ethanol, and tolerating 25% (v/v) ethanol stress.     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.     

The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger

Summary The influence of various carbon sources and their concentration on the production of citrate by Aspergillus niger has been investigated. The sugars maltose, sucrose, glucose, mannose and fructose (in the given order) were carbon sources giving high yields of citric acid. Optimal yields were observed at sugar concentrations of 10% (w/v), with the exception of glucose (7.5%). No citric acid was produced on media containing less than 2.5% sugar. Precultivation of A. niger on 1% sucrose and transference to a 14% concentration of various other sugars induced citrate accumulation. This could be blocked by the addition of cycloheximide, an inhibitor of de novo protein synthesis. This induction was achieved using maltose, sucrose, glucose, mannose and fructose, and also by some other carbon sources (e.g. glycerol) that gave no citric acid accumulation in direct fermentation. Precultivation of A. niger at high (14%) sucrose concentrations and subsequent transfer to the same concentrations of various other carbohydrates, normally not leading to citric acid production, led to formation of citrate. Endogenous carbon sources were also converted to citrate under these conditions. A 14%-sucrose precultivated mycelium continued producing some citrate upon transfer to 1% sugar. These results indicate that high concentrations of certain carbon sources are required for high citrate yields, because they induce the appropriate metabolic imbalance required for acidogenesis.     

Survival Rate and Ultrastructure of Vitrified Bovine in vitro and in vivo Developed Embryos

The capacity of different vitrification media and methods was tested onto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification solutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/l sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plunged and stored in LN2 for 10–25 days, and then thawed in a 20° C waterbath. The content of the straws was rediluted in 1M sucrose solution in PBS and later cocultured with BOEC for 48 h. The overall survival rates for in vitro and in vivo embryos were 36% (12 of 33) and 20% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15 ) after 48 h. The survival rates for precooled embryos were significantly higher than for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespective of the vitrification media used. The in vivo developed embryos showed a rather modified post-thaw ultrastructure, with clear signs of osmotic changes at both the trophoblastic and embryonic cells. The results indicated that in vitro and in vivo developed bovine embryos can survive vitrification using ethylene glycol as a cryoprotectant.     

Synthesis of levan in water-miscible organic solvents

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.