Measurement of total starch in cereal products by amyloglucosidase-alpha-amylase method: collaborative study.
McCleary, B. V., Gibson, T. S. & Mugford, D. C. (1997). Journal of AOAC International, 80, 571-579.
An American Association of Cereal Chemists/AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measurement of total starch in a range of cereal grains and products. The flour sample is incubated at 95 degrees C with thermostable alpha-amylase to catalyze the hydrolysis of starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze the dextrins to glucose. Glucose is measured with glucose oxidase-peroxidase reagent. Thirty-two collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included chicken feed pellets, white bread, green peas, high-amylose maize starch, white wheat flour, wheat starch, oat bran, and spaghetti. All samples were analyzed by the standard procedure as detailed above; 4 samples (high-amylose maize starch and wheat starch) were also analyzed by a method that requires the samples to be cooked first in dimethyl sulfoxide (DMSO). Relative standard deviations for repeatability (RSD(r)) ranged from 2.1 to 3.9%, and relative standard deviations for reproducibility (RSD(R)) ranged from 2.9 to 5.7%. The RSD(R) value for high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%; the value was reduced to 2.9% when the DMSO procedure was used, and the determined starch values increased from 86.9 to 97.2%.
Grape and wine analysis: Oenologists to exploit advanced test kits.
Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.
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.
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.
Measurement of carbohydrates in grain, feed and food.
McCleary, B. V., Charnock, S. J., Rossiter, P. C., O’Shea, M. F., Power, A. M. & Lloyd, R. M. (2006). Journal of the Science of Food and Agriculture, 86(11), 1648-1661.
Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successfully subjected to interlaboratory evaluation. All methods are based on the use of enzymes either purified by conventional chromatography or produced using molecular biology techniques. Such methods allow specific, accurate and reliable quantification of a particular component. Problems in calculating the actual weight of galactosyl-sucrose oligosaccharides in test samples are discussed in detail.
Measurement of Starch: Critical evaluation of current methodology.
B. V. McCleary, L. M. J. Charmier & V. A. McKie. (2018). Starch‐Stärke, In Press.
Most commonly used methods for the measurement of starch in food, feeds and ingredients employ the combined action of α‐amylase and amyloglucosidase to hydrolyse the starch to glucose, followed by glucose determination with a glucose oxidase/peroxidase reagent. Recently, a number of questions have been raised concerning possible complications in starch analytical methods. In this paper, each of these concerns, including starch hydrolysis, isomerisation of maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase, enzyme purity and hydrolysis of sucrose and β‐glucans have been studied in detailed. Results obtained for a range of starch containing samples using AOAC Methods 996.11 and 2014 .10 are compared and a new simpler format for starch measurement is introduced. With this method that employs a thermostable α‐amylase (as distinct from a heat stable α‐amylase) which is both stable and active at 100°C and pH 5.0, 10 samples can be analysed within 2 h, as compared to the 6 h required with AOAC Method 2014.10.
Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant.
Dato, L., Branduardi, P., Passolunghi, S., Cattaneo, D., Riboldi, L., Frascotti, G., Valli, M. & Porro, D. (2010). FEMS Yeast Research, 10(7), 894-908.
The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2μm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application.
Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524.
Mikkelsen, D., Flanagan, B. M., Dykes, G. A. & Gidley, M. J. (2009). Journal of Applied Microbiology, 107(2), 576-583.
Aims: To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced. Methods and Results: Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga. xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3•83 and 3•75 g l-1 respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2•5 g l-1. Solid state 13C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles. Conclusions: The production rate of bacterial cellulose by Ga. xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features. Significance and Impact of the Study: Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga. xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.
Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus.
Centeno, M. S., Guerreiro, C. I. P. D., Dias, F. M.V., Morland, C., Tailford, L. E., Goyal, A., Prates, J. A. M., Ferreira, L. M. A., Caldeira, R. M. H., Mongodin, E. F., Nelson, K. E., Gilbert, H. J. & Fontes, C. M. (2006). FEMS Microbiology Letters, 261(1), 123-132.
Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and α-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 α-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.
Sensor combination and chemometric variable selection for online monitoring of Streptomyces coelicolor fed-batch cultivations.
Ödman, P., Johansen, C. L., Olsson, L., Gernaey, K. V. & Lantz, A. E. (2010). Applied Microbiology and Biotechnology, 86(6), 1745-1759.
Fed-batch cultivations of Streptomyces coelicolor, producing the antibiotic actinorhodin, were monitored online by multiwavelength fluorescence spectroscopy and off-gas analysis. Partial least squares (PLS), locally weighted regression, and multilinear PLS (N-PLS) models were built for prediction of biomass and substrate (casamino acids) concentrations, respectively. The effect of combination of fluorescence and gas analyzer data as well as of different variable selection methods was investigated. Improved prediction models were obtained by combination of data from the two sensors and by variable selection using a genetic algorithm, interval PLS, and the principal variables method, respectively. A stepwise variable elimination method was applied to the three-way fluorescence data, resulting in simpler and more accurate N-PLS models. The prediction models were validated using leave-one-batch-out cross-validation, and the best models had root mean square error of cross-validation values of 1.02 g l-1 biomass and 0.8 g l-1 total amino acids, respectively. The fluorescence data were also explored by parallel factor analysis. The analysis revealed four spectral profiles present in the fluorescence data, three of which were identified as pyridoxine, NAD(P)H, and flavin nucleotides, respectively.
Sealing ability of a new polydimethylsiloxane-based root canal filling material.
Özok, A. R., van der Sluis, L. W. M., Wu, M. K. & Wesselink, P. R. (2008). Journal of Endodontics, 34(2), 204-207.
We tested the null hypothesis that there is no difference in the sealing ability of GuttaFlow, RoekoSeal, and AH26 in root canals. Sixty extracted mandibular premolars were filled with AH26 (lateral compaction), RoekoSeal, or GuttaFlow (modified single-cone). The sealing ability of the root canal fillings was measured weekly (4 weeks) by using a glucose penetration model. Kruskal-Wallis test revealed significant differences in glucose penetration between the experimental groups at weeks 1, 2, 3, and 4. Whereas GuttaFlow showed the highest amount of leakage at all times, AH26 showed the lowest. There was no significant difference between RoekoSeal-filled and AH26-filled root canals throughout the experimental period. AH26 showed better sealing ability in root canals than GuttaFlow.
Adipose triglyceride lipase plays a key role in the supply of the working muscle with fatty acids.
Schoiswohl, G., Schweiger, M., Schreiber, R., Gorkiewicz, G., Preiss-Landl, K., Taschler, U., Zierler, K. A., Radner, F. P. W., Eichmann, T. O., Kienesberger, P. C., Eder, S., Lass, A., Haemmerle, G., Alsted, T. J., Kiens, B., Hoefler, G., Zechner, R. & Zimmermann, R. (2010). Journal of Lipid Research, 51(3), 490-499.
FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice deficient for adipose triglyceride lipase (ATGL-ko) exhibit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL deficiency affects energy availability and substrate utilization in working muscle. In this study, we investigated the effect of moderate treadmill exercise on blood energy metabolites and liver glycogen stores in mice lacking ATGL. Because ATGL-ko mice exhibit massive accumulation of TG in the heart and cardiomyopathy, we also investigated a mouse model lacking ATGL in all tissues except cardiac muscle (ATGL-ko/CM). In contrast to ATGL-ko mice, these mice did not accumulate TG in the heart and had normal life expectancy. Exercise experiments revealed that ATGL-ko and ATGL-ko/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. Together, our studies suggest that ATGL-ko mice cannot adjust circulating FA levels to the increased energy requirements of the working muscle, resulting in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.
Experimental evidence for a significant contribution of cellulose to indoor aerosol mass concentration.
Cerqueira, M., Marques, D., Caseiro, A. & Pio, C. (2010). Atmospheric Environment, 44(6), 867-871.
An apartment bedroom located in a residential area of Aveiro (Portugal) was selected with the aim of characterizing the cellulose content of indoor aerosol particles. Two sets of samples were taken: (1) PM10 collected simultaneously in indoor and outdoor air; (2) PM10 and PM2.5 collected simultaneously in indoor air. The aerosol particles were concentrated on quartz fibre filters with low-volume samplers equipped with size selective inlets. The filters were weighed and then extracted for cellulose analysis by an enzymatic method. The average indoor cellulose concentration was 1.01 ± 0.24 μg m-3, whereas the average outdoor cellulose concentration was 0.078 ± 0.047 μg m-3, accounting for 4.0% and 0.4%, respectively, of the PM10 mass. The corresponding average ratio between indoor and outdoor cellulose concentrations was 11.1 ± 4.9, indicating that cellulose particles were generated indoors, most likely due to the handling of cotton-made textiles as a result of routine daily activities in the bedroom. Indoor cellulose concentrations averaged 1.22 ± 0.53 μg m-3 in the aerosol coarse fraction (determined from the difference between PM10 and PM2.5 concentrations) and averaged 0.38 ± 0.13 μg m-3 in the aerosol fine fraction. The average ratio between the coarse and fine fractions of cellulose concentrations in the indoor air was 3.6 ± 2.1. This ratio is in line with the primary origin of this biopolymer. Results from this study provide the first experimental evidence in support of a significant contribution of cellulose to the mass of suspended particles in indoor air.
Effect of HXT1 and HXT7 hexose transporter overexpression on wild-type and lactic acid producing Saccharomyces cerevisiae cells.
Rossi, G., Sauer, M., Porro, D. & Branduardi, P. (2010). Microbial Cell Factories, 9(1), 15.
Background: Since about three decades, Saccharomyces cerevisiae can be engineered to efficiently produce proteins and metabolites. Even recognizing that in baker's yeast one determining step for the glucose consumption rate is the sugar uptake, this fact has never been conceived to improve the metabolite(s) productivity. In this work we compared the ethanol and/or the lactic acid production from wild type and metabolically engineered S. cerevisiae cells expressing an additional copy of one hexose transporter.
Results: Different S. cerevisiae strains (wild type and metabolically engineered for lactic acid production) were transformed with the HXT1 or the HXT7 gene encoding for hexose transporters. Data obtained suggest that the overexpression of an Hxt transporter may lead to an increase in glucose uptake that could result in an increased ethanol and/or lactic acid productivities. As a consequence of the increased productivity and of the reduced process timing, a higher production was measured.
Conclusions: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is a well established concept. A high production relies on a multi-factorial system. We showed that by modulating the first step of the pathway leading to lactic acid accumulation an improvement of about 15% in lactic acid production can be obtained in a yeast strain already developed for industrial application.
Construction and characterization of three lactate dehydrogenase-negative Enterococcus faecalis V583 mutants.
Jönsson, M., Saleihan, Z., Nes, I. F. & Holo, H. (2009). Applied and Environmental Microbiology, 75(14), 4901-4903.
The roles of the two ldh genes of Enterococcus faecalis were studied using knockout mutants. Deletion of ldh-1 causes a metabolic shift from homolactic fermentation to ethanol, formate, and acetoin production, with a high level of formate production even under aerobic conditions. Ldh-2 plays only a minor role in lactate production.
Preloaded Tissues for Descemet Membrane Endothelial Keratoplasty.
Parekh, M., Ruzza, A., Ferrari, S., Busin, M. & Ponzin, D. (2016). American Journal of Ophthalmology, 166, 120-125.
INTRODUCTION: To determine the feasibility of preloading endothelial tissues for Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Laboratory investigation. METHODS: setting: Institutional. PARTICIPANTS: Twenty human donor corneas unsuitable for transplantation with endothelial cell density in a range of 1600-2700 cells/mm(2). INTERVENTION: The endothelium was punched, stripped (8.5 mm diameter) and manually tri-folded with the endothelial side inward. The excised membranes were gently moved in a 2.2 intraocular lens (IOL) cartridge and pulled further in the funnel using 25 G end-grasping forceps. The cartridge was filled with transport media (TM) (sealed at its funnel and back entrance with a stopper) and the tissue was preserved for 4 days at room temperature in the bottles containing TM. MAIN OUTCOME MEASURES: Success rate of preparation, processing time, endothelial cell loss (ECL), and active metabolism. RESULTS: The tissues were peeled and loaded successfully in all cases. Average stripping and loading time was 20 and 4.5 minutes, respectively. ECL after preservation was 4.35% with 3.55% (± 5.89%) mortality and 7.80% (± 14.12%) uncovered areas. A total of 0.55 (± 0.26) mg/mL of glucose was consumed by the cells showing active metabolism. CONCLUSIONS: Tri-folded (endothelium-in) DMEK grafts can be preloaded using TM in an IOL cartridge and stored up to 4 days with limited endothelial damage. Direct injection of TM should be avoided because of the presence of bovine serum, but the tissue can be washed using balanced salt solution and gently injected. Alternatively, the graft can be easily delivered using a bimanual pull-through technique. Preloading DMEK grafts will simplify the surgery with reproducibility, reduced surgical time, and reduced tissue wastage, cost, and logistical requirements.
Characterization of the isolated active layer of a NF membrane by electrochemical impedance spectroscopy.
Efligenir, A., Fievet, P., Déon, S. & Salut, R. (2015). Journal of Membrane Science, 477, 172-182.
The dielectric and electric properties of the isolated active layer of an NF membrane were determined by electrochemical impedance spectroscopy (EIS). To our knowledge, this was the first attempt aimed at assessing the above properties from measurements performed directly on the active layer alone. Experiments were conducted with Desal DK membrane and various electrolyte solutions (KCl, KI, LiCl, NH4 Cl, CaCl2 and BaCl2 at 10-2 mol m-3. The comparison of impedance spectra obtained for the whole membrane and active layer alone revealed very low contribution of the active layer to the overall membrane impedance. The dielectric constant (εp) and conductivity (κca) inside the pores of the active layer were evaluated from capacity and resistance measurements. The dielectric constant inside the pores was found to be smaller than its bulk value, irrespective of the electrolyte used. The reduction in εp was attributed only to confinement effect. It was also shown that pore conductivity exceeded the bulk conductivity. This increased conduction inside the pores could have been due to the excess of counter-ions inside the pores resulting from fixed charges on the pore walls. The present study shows that the dielectric constant inside the pores of the active layer of NF membranes can be determined independently from nano-filtration measurements and without any ambiguity, provided that EIS measurements are performed directly on the isolated active layer.
Interaction mechanism between green tea extract and human α-amylase for reducing starch digestion.
Miao, M., Jiang, B., Jiang, H., Zhang, T. & Li, X. (2015). Food Chemistry, 186, 20-25.
This study evaluated the inhibitory effects of the green tea extract on human pancreatic α-amylase activity and its molecular mechanism. The green tea extract was composed of epicatechin (59.2%), epigallocatechin gallate (14.6%) and epicatechin gallate (26.2%) as determined by HPLC analysis. Enzyme activity measurement showed that % inhibition and IC50 of the green tea extract (10%, based on starch) were 63.5% and 2.07 mg/ml, respectively. The Michaelis–Menten constant remained unchanged but the maximal velocity decreased from 0.43 (control) to 0.07 mg/(ml × min) (4 mg/ml of the green tea extract), indicating that the green tea extract was an effective inhibitor against α-amylase with a non-competitive mode. The fluorescence data revealed that the green tea extract bound with α-amylase to form a new complex with static quenching mechanism. Docking study showed the epicatechin gallate in the green tea extract presented stronger affinity than epigallocatechin gallate, with more number of amino acid residues involved in amylase binding with hydrogen bonds and Van der Waals forces. Thus, the green tea extract could be used to manipulate starch digestion for potential health benefits.
Spatial reorganization of telomeres in long-lived quiescent cells.
Guidi, M., Ruault, M., Marbouty, M., Loïodice, I., Cournac, A., Billaudeau, C., Hocher, A., Mozziconacci, J., Koszul, R.. & Taddei, A. (2015). Genome Biology, 16(1), 206.
Background: The spatiotemporal behavior of chromatin is an important control mechanism of genomic function. Studies in Saccharomyces cerevisiae have broadly contributed to demonstrate the functional importance of nuclear organization. Although in the wild yeast survival depends on their ability to withstand adverse conditions, most of these studies were conducted on cells undergoing exponential growth. In these conditions, as in most eukaryotic cells, silent chromatin that is mainly found at the 32 telomeres accumulates at the nuclear envelope, forming three to five foci. Results: Here, combining live microscopy, DNA FISH and chromosome conformation capture (HiC) techniques, we report that chromosomes adopt distinct organizations according to the metabolic status of the cell. In particular, following carbon source exhaustion the genome of long-lived quiescent cells undergoes a major spatial re-organization driven by the grouping of telomeres into a unique focus or hypercluster localized in the center of the nucleus. This change in genome conformation is specific to quiescent cells able to sustain long-term viability. We further show that reactive oxygen species produced by mitochondrial activity during respiration commit the cell to form a hypercluster upon starvation. Importantly, deleting the gene encoding telomere associated silencing factor SIR3 abolishes telomere grouping and decreases longevity, a defect that is rescued by expressing a silencing defective SIR3 allele competent for hypercluster formation. Conclusions: Our data show that mitochondrial activity primes cells to group their telomeres into a hypercluster upon starvation, reshaping the genome architecture into a conformation that may contribute to maintain longevity of quiescent cells.
Human Corneal Endothelial Cell Cultivation From Old Donor Corneas With Forced Attachment.
Parekh, M., Ahmad, S., Ruzza, A. & Ferrari, S. (2017). Scientific Reports, 7.
Human corneal endothelial cells (HCEnCs) are responsible for maintaining the transparency of the cornea. Damaged or diseased HCEnCs may cause blindness. Replacement of the diseased cells with a healthy donor endothelium is the only currently available treatment. Tissue-engineering can serve as an alternative to conventional donor corneal transplantation. Due to the global shortage of donor corneas, a wide interest in the development of cultured graft substitutes and artificial corneas has increased. Availability of the old donor corneas is higher especially for research. Although it can be proposed as a valuable source for cell culture, its less proliferative capability emerges a challenge for the researchers. This article describes the use of hyaluronic acid (HA) in combination with Rho-kinase inhibitor (ROCK) Y-27632 for the cultivation of HCEnCs from older donor corneas (age > 60 years). Four conditions including and excluding HA + ROCK and its effect on early attachment rates and proliferation was studied on forty-eight corneas. It was observed that HCEnCs reach confluence within 10–15 days when cultured with HA + ROCK. This approach improves the efficiency of cell adhesion due to force attachment. HCEnCs from old donor corneas can be cultured using this method which may further lead to cell-based therapy for treating corneal endothelial dysfunction.