D-Fructose/D-Glucose test kit, an enzymatic UV-method for the measurement and analysis of D-fructose and/or D-glucose in plant and food products.

Extended cofactors stability. Dissolved cofactors stable for > 1 year at4^oC.

Suitable for manual, auto-analyser and microplate formats.

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5. Link to Article Read Abstract It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as \"stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

Megazyme \"advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5. Link to Article Read Abstract Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

Lactose fermentation by Kombucha – a process to obtain new milk–based beverages.

Iličić, M., Kanurić, K., Milanović, S., Lončar, E., Djurić, M. & Malbaša, R. (2012). Romanian Biotechnological Letters, 17(1), 7013-7021. Link to Article Read Abstract This paper focuses on fermentation of lactose from a model system (black tea) and from two types of milk (0.9% w/w and 2.2% w/w of fat) by application of Kombucha. Quantities of the applied Kombucha starter were 10% v/v and 15% v/v. All fermentations were performed at 42°C. The process to achieve a desirable pH=4.5 was slower in the model system (16 h) than in milks (9 - 10 h). Regarding starter quantity, 10% v/v proved the optimal. Regarding types of milk, higher fat content guarantees shorter fermentation and higher yield of metabolites. Utilization of lactose was found at a level of ≈20% and ≈30% in milks with 0.9% w/w and 2.2% w/w of fat, respectively. This was correlated with an appearance of intermediates and/or products. Glucose underwent further transformations almost entirely, while galactose showed much lower reactivity. Seven to twelve times higher contents of lactic acid were found compared to acetic acid. Milk-based beverage from the reduced fat sample, inoculated with 10% v/v of Kombucha starter, has the best physical characteristics (syneresis and water holding capacity). It also developed a good texture (especially cohesiveness and index of viscosity). Milk lactose fermentation was a process that could have been used for obtaining new milk-based products.

Sourdough-leavened bread improves postprandial glucose and insulin plasma levels in subjects with impaired glucose tolerance.

Maioli, M., Pes, G. M., Sanna, M., Cherchi, S., Dettori, M., Manca, E. & Farris, G. A. (2008). Acta Diabetologica, 45(2), 91-96. Link to Article Read Abstract Sourdough bread has been reported to improve glucose metabolism in healthy subjects. In this study postprandial glycaemic and insulinaemic responses were evaluated in subjects with impaired glucose tolerance (IGT) who had a meal containing sourdough bread leavened with lactobacilli, in comparison to a reference meal containing bread leavened with baker yeast. Sixteen IGT subjects (age range 52–75, average BMI 29.9 ± 4.2 kg/m²) were randomly given a meal containing sourdough bread (A) and a meal containing the reference bread (B) in two separate occasions at the beginning of the study and after 7 days. Sourdough bread was leavened for 8 h using a starter containing autochthonous Saccharomyces cerevisiae and several bacilli able to produce a significant amount of D-and L-lactic acid, whereas the reference bread was leavened for 2 h with commercial baker yeast containing Saccharomyces cerevisiae. Plasma glucose and insulin levels were measured at time 0, 30, 60, 120, and 180 min. In IGT subjects sourdough bread induced a significantly lower plasma glucose response at 30 minutes (p = 0.048) and a smaller incremental area under curve (AUC) Δ 0–30 and Δ 0–60 min (p = 0.020 and 0.018 respectively) in comparison to the bread leavened with baker yeast. Plasma insulin response to this type of bread showed lower values at 30 min (p = 0.045) and a smaller AUC Δ 0–30 min (p = 0.018). This study shows that in subjects with IGT glycaemic and insulinaemic responses after the consumption of sourdough bread are lower than after the bread leavened with baker yeast. This effect is likely due to the lactic acid produced during dough leavening as well as the reduced availability of simple carbohydrates. Thus, sourdough bread may potentially be of benefit in subjects with impaired glucose metabolism.

Endocrine and metabolic effects of consuming fructose- and glucose-sweetened beverages with meals in obese men and women: Influence of insulin resistance on plasma triglyceride responses.

Teff, K. L., Grudziak, J., Townsend, R. R., Dunn, T. N., Grant, R. W., Adams, S. H., Keim, N. L., Cummings, B. P., Stanhope, K. L. & Havel, P. J. (2009). Journal of Clinical Endocrinology & Metabolism, 94(5), 1562-1569. Link to Article Read Abstract Context: Compared with glucose-sweetened beverages, consumption of fructose-sweetened beverages with meals elevates postprandial plasma triglycerides and lowers 24-h insulin and leptin profiles in normal-weight women. The effects of fructose, compared with glucose, ingestion on metabolic profiles in obese subjects has not been studied.Objective: The objective of the study was to compare the effects of fructose- and glucose-sweetened beverages consumed with meals on hormones and metabolic substrates in obese subjects.Design and Setting: The study had a within-subject design conducted in the clinical and translational research center. Participants: Participants included 17 obese men (n = 9) and women (n = 8), with a body mass index greater than 30 kg/m².Interventions: Subjects were studied under two conditions involving ingestion of mixed nutrient meals with either glucose-sweetened beverages or fructose-sweetened beverages. The beverages provided 30% of total kilocalories. Blood samples were collected over 24 h.Main Outcome Measures: Area under the curve (24 h AUC) for glucose, lactate, insulin, leptin, ghrelin, uric acid, triglycerides (TGs), and free fatty acids was measured.Results: Compared with glucose-sweetened beverages, fructose consumption was associated with lower AUCs for insulin (1052.6 ± 135.1 vs. 549.2 ± 79.7 μU/ml per 23 h, P P P P Conclusions: In obese subjects, consumption of fructose-sweetened beverages with meals was associated with less insulin secretion, blunted diurnal leptin profiles, and increased postprandial TG concentrations compared with glucose consumption. Increases of TGs were augmented in obese subjects with insulin resistance, suggesting that fructose consumption may exacerbate an already adverse metabolic profile present in many obese subjects. Compared to glucose-sweetened beverages, consumption of fructose-sweetened beverages increases postprandial triglycerides in obese subjects, potentially exacerbating the known adverse metabolic profile associated with obesity.

Metabolic and endocrine profiles in response to systemic infusion of fructose and glucose in rhesus macaques.

Adams, S. H., Stanhope, K. L., Grant, R. W., Cummings, B. P. & Havel, P. J. (2008). Endocrinology, 149(6), 3002-3008. Link to Article Read Abstract Diurnal patterns of circulating leptin concentrations are attenuated after consumption of fructose-sweetened beverages compared with glucose-sweetened beverages, likely a result of limited postprandial glucose and insulin excursions after fructose. Differences in postprandial exposure of adipose tissue to peripheral circulating fructose and glucose or in adipocyte metabolism of the two sugars may also be involved. Thus, we compared plasma leptin concentrations after 6-h iv infusions of saline, glucose, or fructose (15 mg/kg•min) in overnight-fasted adult rhesus monkeys (n = 9). Despite increases of plasma fructose from undetectable levels to about 2 mm during fructose infusion, plasma leptin concentrations did not increase, and the change of insulin was only about 10% of that seen during glucose infusion. During glucose infusion, plasma leptin was significantly increased above baseline concentrations by 240 min and increased steadily until the final 480-min time point (change in leptin = +2.5 ± 0.9 ng/ml, P vs. saline; percent change in leptin = +55 ± 16%; P vs. saline). Substantial anaerobic metabolism of fructose was suggested by a large increase of steady-state plasma lactate (change in lactate = 1.64 ± 0.15 mm from baseline), which was significantly greater than that during glucose (+0.53 ± 0.14 mm) or saline (−0.51 ± 0.14 mm) infusions (P i.e. limited post-fructose insulin excursions and/or hexose-specific differences in adipocyte metabolism) are likely to underlie disparate effects of fructose and glucose to increase circulating leptin concentrations.

β-Fructofuranosidase and sucrose phosphorylase of rumen bacterium Pseudobutyrivibrio ruminis strain 3.

Kasperowicz, A., Stan-Glasek, K., Guczynska, W., Pristas, P., Javorsky, P., Vandzurova, A. & Michalowski, T. (2012). World Journal of Microbiology and Biotechnology, 28(3), 1271-1279. Link to Article Read Abstract The subject of this study was the fructan and sucrose degrading enzymes of bacterium Pseudobutyrivibrio ruminis strain 3. It was stated that cell extract from bacteria growing on inulin contained β-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7), while the bacteria maintained on sucrose showed only phosphorylase. Partially purified β-fructofuranosidase digested inulooligosaccharides and sucrose to fructose or fructose and glucose, respectively, but was unable to degrade the long chain polymers of commercial inulin and Timothy grass fructan. Digestion rate of inulooligosaccharides fit Michaelis–Menten kinetics with V_max 5.64 μM/mg/min and K_m 1.274%, respectively, while that of sucrose was linear. Partially purified sucrose phosphorylase digested only sucrose. The digestion products were fructose, glucose-1P and free glucose. The reaction was in agreement with Michaelis–Menten kinetics. The V_max were 0.599 and 0.584 μM/mg/min, while K_m were 0.190 and 0.202% for fructose release and glucose-1P formation, respectively, when bacteria grew on inulin. The V_max were, however, 1.37 and 1.023 μM/mg/min, while K_m were 0.264 and 0.156%, if bacteria were grown on sucrose. The free glucose was hardly detectable for the enzyme originated from inulin grown bacteria, but glucose levels ranged from 0.05 to 0.25 μM/mg/min, when cell extract from bacteria grown on sucrose was used. Release of free glucose was observed when no inorganic phosphate was present in reaction mixture.

Comparative effects of fructose and glucose on lipogenic gene expression and intermediary metabolism in HepG2 liver cells.

Hirahatake, K. M., Meissen, J. K., Fiehn, O. & Adams, S. H. (2011). PloS one, 6(11), e26583. Link to Article Read Abstract Consumption of large amounts of fructose or sucrose increases lipogenesis and circulating triglycerides in humans. Although the underlying molecular mechanisms responsible for this effect are not completely understood, it is possible that as reported for rodents, high fructose exposure increases expression of the lipogenic enzymes fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC-1) in human liver. Since activation of the hexosamine biosynthesis pathway (HBP) is associated with increases in the expression of FAS and ACC-1, it raises the possibility that HBP-related metabolites would contribute to any increase in hepatic expression of these enzymes following fructose exposure. Thus, we compared lipogenic gene expression in human-derived HepG2 cells after incubation in culture medium containing glucose alone or glucose plus 5 mM fructose, using the HBP precursor 10 mM glucosamine (GlcN) as a positive control. Cellular metabolite profiling was conducted to analyze differences between glucose and fructose metabolism. Despite evidence for the active uptake and metabolism of fructose by HepG2 cells, expression of FAS or ACC-1 did not increase in these cells compared with those incubated with glucose alone. Levels of UDP-N-acetylglucosamine (UDP-GlcNAc), the end-product of the HBP, did not differ significantly between the glucose and fructose conditions. Exposure to 10 mM GlcN for 10 minutes to 24 hours resulted in 8-fold elevated levels of intracellular UDP-GlcNAc (PPPin vivo, it would suggest that high fructose consumption promotes triglyceride synthesis primarily through its action to provide lipid precursor carbon and not by activating lipogenic gene expression.

Measurement of glucose and fructose in clinical samples using gas chromatography/mass spectrometry.

Wahjudi, P. N., Patterson, M. E., Lim, S., Yee, J. K., Mao, C. S. & Lee, W. N. P. (2010). Clinical Biochemistry, 43(1-2), 198-207. Link to Article Read Abstract Objective: The impact of increased fructose consumption on carbohydrate metabolism is a topic of current interest, but determination of serum level has been hindered due to low concentration and interference from serum glucose. We are reporting a method for the quantification of glucose and fructose in clinical samples using gas chromatography/mass spectrometry (GC/MS). The accuracy and precision of GC/MS and an enzymatic assay were compared.Design and methods: Mass spectrometry fragmentation patterns of methyloxime peracetate derivatized aldose and ketose were determined. Unique fragments for glucose and fructose were used for quantitative analysis using isotope labeled recovery standards.Results: Methyloxime peracetate derivatives of glucose and fructose showed characteristic loss of acetate (M-60) or ketene (M-42) under chemical ionization (CI). Under electron impact (EI) ionization, a unique C1–C2 fragment of glucose was formed, while a C1–C3 fragment was formed from keto-hexoses. These unique fragments were used in the quantitative assay of glucose and fructose in clinical samples. In clinical samples, the GC/MS assay has a lower limit of detection than that of the enzymatic assay. In plasma samples from patients evaluated for diabetes the average serum glucose and fructose were 6.19 ± 2.72 mM and 46 ± 25.22 μM. Fructose concentrations in many of these samples were below the limit of detection of the enzymatic method.Conclusion: Derivatization of aldose and ketose monosaccharides to their respective O-methyloxime acetates for GC/MS analysis is a facile method for determination of serum/plasma glucose and fructose samples.

Physiological studies of Leuconostoc mesenteroides strain NRRL B-1149 during cultivation on glucose and fructose media.

Bivolarski, V., Vasileva, T., Shukla, R., Goyal, A. & Iliev, I. (2012). Journal of BioScience & Biotechnology, 1(3), 235-240. Link to Article Read Abstract Glycosyltransferases are extracellular and cell-associated sucrase enzymes produced mainly by lactic acid bacteria Leuconostoc mesenteroides, oral Streptococcus species and also Lactobacillus species. According to the synthesized polymer (glucan or fructan) in the presence of sucrose, these enzymes are divided into two groups: glucosyltransferases (GTFs) and fructosyltransferases (FTFs). Only Streptococcus, Lactobacillus and Leuconostoc strains are known as producers of both GTFs and FTFs. The enzymes from Lactobacillus and Leuconostoc spp. are implicated in the synthesis of polymers and oligosaccharides (OS) important for human health because of their prebiotic properties and immunomodulating activity. In the present work, we studied the production of extracellular and cell-associated glycosyltransferases by Leuconostoc mesenteroides strain NRRL B-1149 during its growth on media containing glucose or fructose as a main carbon source. The enzyme activities, pH and biomass formation were measured and compared during the cultivation. We have shown that glucose and fructose have not an equal role for enzyme production. The highest extracellular activity was detected at the 4^th hour during the cultivation of the strain in medium with fructose - 5.45 U/mg. When the strain was cultivated in medium with glucose, the maximum of extracellular enzyme activity was detected at the 5^th hour of the cultivation but the measured activity was about 9 times lower compared to these, obtained after cultivation in fructose medium. The studied strain produced mainly extracellular glycosyltransferases in glucose or fructose medium, which were 92.4% and 97.1% of the total enzyme activity, respectively. In order to characterize the produced enzymes, cell-associated and extracellular enzymes were determined using SDS-PAGE and in situ Periodic Acid Schiffs staining after incubation with 10% sucrose. When the investigated strain was grown in media with sucrose, glucose or fructose, several types of glycosyltransferases were detected - dextransucrase with molecular weight 180 kDa and two fructosyltransferases, corresponding to 120 kDa and 86 kDa molecular weights.

Aldose reductase is implicated in high glucose‐induced oxidative stress in mouse embryonic neural stem cells.

Fu, J., Tay, S. S. W., Ling, E. A. & Dheen, S. T. (2007). Journal of Neurochemistry, 103(4), 1654-1665. Link to Article Read Abstract Oxidative stress caused by hyperglycemia is one of the key factors responsible for maternal diabetes-induced congenital malformations, including neural tube defects in embryos. However, mechanisms by which maternal diabetes induces oxidative stress during neurulation are not clear. The present study was aimed to investigate whether high glucose induces oxidative stress in neural stem cells (NSCs), which compose the neural tube during development. We also investigated the mechanism by which high glucose disturbs the growth and survival of NSCs in vitro. NSCs were exposed to physiological D-glucose concentration (PG, 5 mmol/L), PG with L-glucose (25 mmol/L), or high D-glucose concentration (HG, 30 or 45 mmol/l). HG induced reactive oxygen species production and mRNA expression of aldose reductase (AR), which catalyzes the glucose reduction through polyol pathway, in NSCs. Expression of glucose transporter 1 (Glut1) mRNA and protein which regulates glucose uptake in NSCs was increased at early stage (24 h) and became down-regulated at late stage (72 h) of exposure to HG. Inhibition of AR by fidarestat, an AR inhibitor, decreased the oxidative stress, restored the cell viability and proliferation, and reduced apoptotic cell death in NSCs exposed to HG. Moreover, inhibition of AR attenuated the down-regulation of Glut1 expression in NSCs exposed to HG for 72 h. These results suggest that the activation of polyol pathway plays a role in the induction of oxidative stress which alters Glut1 expression and cell cycle in NSCs exposed to HG, thereby resulting in abnormal patterning of the neural tube in embryos of diabetic pregnancy.

Immobilised yeast grape must deacidification in a recycle fixed bed reactor.

Portugal, I., Ribeiro, S. C., Xavier, A. M. R. B., Centeno, F. & Strehaiano, P. (2011). International Journal of Food Science & Technology, 46(2), 284-289. Link to Article Read Abstract Maloalcoholic fermentation (MAF) of grape must by Schizosaccharomyces pombe immobilised in calcium-alginate double-layer beads (ProMalic®) was studied in Erlenmeyer flasks and in a total recycle fixed-bed reactor operating in batch mode. The reaction is pseudo-first order with respect to L-malic acid and under similar conditions deacidification is faster in the recycle reactor. This was attributed to mass transfer limitations which were confirmed in the recycle reactor by studying the influence of yeast load on the rate of MAF. Mass transfer limitations are also responsible for the lower activation energy of fermentation with the immobilised yeast (67 ± 9 kJ mol^-1) in comparison with the free cells (126 ± 19 kJ mol^-1). Alcoholic fermentation and MAF were performed simultaneously, both in the recycle reactor and in the industrial trials, confirming the efficacy of immobilised S. pombe to reduce grape must acidity without interfering with the main fermentation. Altogether, the present results are useful for the scale-up of a recycle reactor to process large volumes of grape must.

UV-method for the determination of D-Fructose and D-Glucose in foodstuffs, beverages and other materialsPrinciple: (hexokinase)(1) D-Glucose + ATP → G-6-P + ADP (hexokinase)(2) D-Fructose + ATP → F-6-P + ADP (glucose-6-phosphate dehydrogenase)(3) G-6-P + NADP⁺ → gluconate-6-phosphate + NADPH + H⁺ (phosphoglucose isomerase)(4) F-6-P ↔ G-6-P

Kit size: *110 assays (manual) / 1100 (microplate) / 1100 (auto-analyser)

*The number of manual tests per kit can be doubled if all volumes are halved.This can be readily accommodated using the MegaQuant^TMWaveSpectrophotometer (D-MQWAVE).

Method: Spectrophotometric at 340 nmReaction time: ~ 13 minDetection limit: 0.66 mg/LApplication examples: Wine, beer, fruit juices, soft drinks, milk, jam, honey, dietetic foods, bread, bakery products, candies, desserts, confectionery, ice-cream, fruit and vegetables, condiments, tobacco, cosmetics, pharmaceuticals, paper and other materials (e.g. biological cultures, samples, etc.)Method recognition: Methods based on this principle have been accepted by AOAC, EN,NEN, NF, DIN, GOST, OIV, IFU, AIJN, MEBAK and IOCCC

Advantages

• PVP incorporated to prevent tannin inhibition
• Validated by the University of Wine, Suze la Rousse, France
• Very competitive price (cost per test)
• All reagents stable for > 2 years after preparation (manual analysis applications)
• Rapid reaction at either 25 or 37°C
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing
• Standard included
• Extended cofactors stability
• Suitable for manual, microplate and auto-analyser formats