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BACKGROUND: To perform fast, reproducible, and absolute quantitative measurements in an automated manner has become of paramount importance when monitoring industrial processes, including fermentations. Due to its numerous advantages - including its inherent quantitative nature - Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy provides an ideal tool for the time-resolved monitoring of fermentations. However, analytical conditions, including non-automated sample preparation and long relaxation times (T1) of some metabolites, can significantly lengthen the experimental time and make implementation in an industrial set up unfeasible. RESULTS: We present a high throughput method based on Standard Operating Procedures (SOPs) and 1H NMR, which lays the foundation for what we call Fermentation Analytical Technology (FAT). Our method was developed for the accurate absolute quantification of metabolites produced during Escherichia coli industrial fermentations. The method includes: (1) a stopped flow system for non-invasive sample collection followed by sample quenching, (2) automatic robot-assisted sample preparation, (3) fast 1H NMR measurements, (4) metabolites quantification using multivariate curve resolution (MCR), and (5) metabolites absolute quantitation using a novel correction factor (k) to compensate for the short recycle delay (D1) employed in the 1H NMR measurements. The quantification performance was tested using two sample types: buffer solutions of chemical standards and real fermentation samples. Five metabolites - glucose, acetate, alanine, phenylalanine and betaine - were quantified. Absolute quantitation ranged between 0.64 and 3.40 mM in pure buffer, and 0.71-7.76 mM in real samples. SIGNIFICANCE: The proposed method is generic and can be straight forward implemented to other types of fermentations, such as lactic acid, ethanol and acetic acid fermentations. It provides a high throughput automated solution for monitoring fermentation processes and for quality control through absolute quantification of key metabolites in fermentation broth. It can be easily implemented in an at-line industrial setting, facilitating the optimization of the manufacturing process towards higher yields and more efficient and sustainable use of resources.
Assuntos
Escherichia coli , Fermentação , Espectroscopia de Prótons por Ressonância Magnética , Escherichia coli/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/métodosRESUMO
Lactose crystallization during storage deteriorates reconstitution performance of milk powders, but the relationship between lactose crystallization and reconstitution is inexplicit. The objective of this study is to characterize crystalline lactose in the context of formulation and elucidate the complex relationship between lactose crystallization and powder functionality. Lactose in Skim Milk Powder (SMP), Whole Milk Powder (WMP) and Fat-Filled Milk Powder (FFMP) stored under 23 %, 53 % and 75 % Relative Humidity (RH) at 25 â for four months was compared. Lactose, surface chemistry and microstructure of FFMP stored at 25 â and 40 â at 23 % to 75 % RH for four months were also analyzed and interpreted. At the same RH, FFMP crystallized in the same pattern as WMP. At 53 % RH, FFMP and WMP differentiated from SMP in terms of lactose morphology as well as the ratio between anhydrous α-lactose and anhydrous ß-lactose. Lactose remained amorphous at 23 % RH, crystallized predominantly to α/ß-lactose (1:4) at 40 to 58 % RH and to α-lactose monohydrate at 75 % RH. The crystallinity index was similar for all powders containing crystalline lactose. The estimated crystallite size increased from approx. 0.1 to 20 µm with increasing RH and temperature. When amorphous lactose crystallized into crystals below approx. 0.1 µm at 25 °C and 43 % RH, the microstructure and surface lipid were comparable to that of the reference powder. This powder reconstituted into a stable suspension system comparable to that of reference (well performing) powders. These results demonstrate that crystallite size is the key property linking lactose crystallization and reconstitution. Our finding thus indicates limiting crystallite size is important for maintaining desired product quality.
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Lactose , Leite , Animais , Cristalização/métodos , Leite/química , Lactose/química , Pós/química , Difração de Raios XRESUMO
To understand shearing on cheese curds during high shear extrusion, the controlable parameters of a twin-screw extruder were related with measured and calculated parameters that characterise the extrusion process effects on product properties. Curd properties were correlated with specific mechanical energy SME (23-390 kJ·kg-1), Texit (22-54 °C) and residence time RT (36-507 s); the wide experimental range studied provided new insights regarding extrusion of cheese curds. Longer and finer fibers were produced at low SME (23-27 kJ·kg-1), high Texit (50-54 °C) and short RT (55-60 s). Whereas extruded curds produced at high SME (166-390 kJ·kg-1), low Texit (22-23 °C) and long RT (371-396 s) tend to form a compact structure with less fiber formation. Temperature in the heating section, Th, and temperature of the cooling die, Tc, were found to determine critical curd phase transitions during extrusion, from viscoelastic solid to viscoelastic liquid and vice versa, that are important for the creation of fibrous cheese curd structures. Tc was the most important factor influencing SME, indicating the considerable contribution of the cooling process in increasing the shear forces. Curd composition and textural properties were significantly influenced by Th and Tc, showing that a higher Th enhances curd elasticity and reduces melt strength while a higher Tc induces lower water content and increases melt strength. We concluded that a variety of structured mozzarella products with customized properties can be produced by controlling the extrusion parameters.
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Queijo , Queijo/análise , Culinária , Transição de Fase , Temperatura , ÁguaRESUMO
Cyclodextrins (CDs) are cyclic oligosaccharides that have found widespread application in numerous fields. CDs have revealed a number of various health benefits, making them potentially useful food supplements and nutraceuticals. In this study, the impact of α-, ß-, and γ-CD at different concentrations (up to 8% of the flour weight) on the wheat dough and bread properties were investigated. The impact on dough properties was assessed by alveograph analysis, and it was found that especially ß-CD affected the viscoelastic properties. This behavior correlates well with a direct interaction of the CDs with the proteins of the gluten network. The impact on bread volume and bread staling was also assessed. The bread volume was in general not significantly affected by the addition of up to 4% CD, except for 4% α-CD, which slightly increased the bread volume. Larger concentrations of CDs lead to decreasing bread volumes. Bread staling was investigated by texture analysis and low field nuclear magnetic resonance spectroscopy (LF-NMR) measurements, and no effect of the addition of CDs on the staling was observed. Up to 4% CD can, therefore, be added to wheat bread with only minor effects on the dough and bread properties.
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Pão , Triticum , gama-Ciclodextrinas/química , Ciclodextrinas/química , Espectroscopia de Ressonância MagnéticaRESUMO
Bone char catalyzed dechlorination of trichloroethylene (TCE) by green rust (iron(II)-iron(III) hydroxide, GR) has introduced a promising new reaction platform for degradation of chlorinated solvents. This study aimed to reveal whether a broader class of biochars are catalytically active for the dechlorination reaction and to identify which biochar properties are the most important for the catalytic activity. Biochars produced by pyrolysis of animal, plant, and sewage waste substrates at 950 °C were prepared for catalytic dechlorination of TCE by GR tested in batch experiments with 0.15 g L-1 biochar, 3.2 g L-1 GR, and ~ 20 µM TCE. The results showed that the biochar substrate significantly affects its catalytic activity, with the highest TCE reduction rate observed for bone and shrimp-based biochars (k ≥ 0.18 h-1), whereas no reactivity was seen for graphite and activated carbon references. Multivariate regression indicated that the biochar catalytic activity is controlled by multiple biochar properties - biochar surface area, TCE sorption, abundance of C-O groups, and pore size are the properties that impact the catalytic activity most. Derivation of biochar reactivity relationship for a broad spectrum of biochars provides a new approach for identifying proper biochar catalysts for pollutant degradation.
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Carvão Vegetal , Tricloroetileno , Animais , Catálise , Compostos FérricosRESUMO
Biochars function as electron transfer mediators and thus catalyze redox transformations of environmental pollutants. A previous study has shown that bone char (BC) has high catalytic activity for reduction of chlorinated ethylenes using layered Fe(II)-Fe(III) hydroxide (green rust) as reductant. In the present study, we studied the rate of trichloroethylene (TCE) reduction by green rust in the presence of BCs obtained at pyrolysis temperatures (PTs) from 450 to 1050 °C. The reactivity increased with PT, yielding a maximum pseudo-first-order rate constant (k) of 2.0 h-1 in the presence of BC pyrolyzed at 950 °C, while no reaction was seen for BC pyrolyzed at 450 °C. TCE sorption, specific surface area, extent of graphitization, carbon content, and aromaticity of the BCs also increased with PT. The electron-accepting capacity (EAC) of BC peaked at PT of 850 °C, and EAC was linearly correlated with the sum of concentrations of quinoid, quaternary N, and pyridine-N-oxide groups measured by XPS. Moreover, no TCE reduction was seen with graphene nanoparticles and graphitized carbon black, which have high degrees of graphitization but low EAC values. Further analyses showed that TCE reduction rates are well correlated with the EAC and the C/H ratio (proxy of electrical conductivity) of the BCs, strongly indicating that both electron-accepting functional groups and electron-conducting domains are crucial for the BC catalytic reactivity. The present study delineates conditions for designing redox-reactive biochars to be used for remediation of sites contaminated with chlorinated solvents.
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Tricloroetileno , Poluentes Químicos da Água , Cloro , Ferro , OxirreduçãoRESUMO
Fluorescence spectroscopy is a sensitive and selective technique, which can be of great value in bioprocesses to provide online, real-time measures of chemical compounds. Although fluorescence spectroscopy is a widely studied method, not much attention has been given to issues concerning intensity variations in the fluorescence landscapes due to pH fluctuations. This study elucidates how pH fluctuations cause intensity changes in fluorescence measurements and thereby decreases the quality of the subsequent quantification. A photo-degradation process of riboflavin was investigated by fluorescence spectroscopy and used as a model system. A two-step modeling approach, combining weighted PARAllel FACtor analysis (PARAFAC) with weighted non-linear regression of the known reaction kinetics, is suggested as a way of handling the fluorescence intensity shifts caused by the pH changes. The suggested strategy makes it possible to compensate for uncertainties in the shifted data and thereby obtain more reliable concentration profiles for the chemical compounds and kinetic parameters of the reaction.
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BACKGROUND: A number of human diseases such as obesity and diabetes are associated with changes or imbalances in the gut microbiota (GM). Laboratory mice are commonly used as experimental models for such disorders. The introduction and dynamic development of next generation sequencing techniques have enabled detailed mapping of the GM of both humans and animal models. Nevertheless there is still a significant knowledge gap regarding the human and mouse common GM core and thus the applicability of the latter as an animal model. The aim of the present study was to identify inter- and intra-individual differences and similarities between the GM composition of particular mouse strains and humans. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1509428 high quality tag-encoded partial 16S rRNA gene sequences determined using 454/FLX Titanium (Roche) pyro-sequencing reflecting the GM composition of 32 human samples from 16 individuals and 88 mouse samples from three laboratory mouse strains commonly used in diabetes research were analyzed using Principal Coordinate Analysis (PCoA), nonparametric multivariate analysis of similarity (ANOSIM) and alpha diversity measures. A reliable cutoff threshold for low abundant taxa estimated on the basis of the present study is recommended for similar trials. CONCLUSIONS/SIGNIFICANCE: Distinctive quantitative differences in the relative abundance of most taxonomic groups between the examined categories were found. All investigated mouse strains clustered separately, but with a range of shared features when compared to the human GM. However, both mouse fecal, caecal and human fecal samples shared to a large extent not only representatives of the same phyla, but also a substantial fraction of common genera, where the number of shared genera increased with sequencing depth. In conclusion, the GM of mice and humans is quantitatively different (in terms of abundance of specific phyla and species) but share a large qualitatively similar core.
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Bactérias/genética , Microbiota/genética , Animais , Fezes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Análise Multivariada , Análise de Componente Principal , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de RNA , Estatísticas não ParamétricasRESUMO
The aim of the current study was to elucidate whether cows and young bulls require different combinations of heating temperature and heating time to reduce toughness of the meat. The combined effect of heating temperature and time on toughness of semitendinosus muscle from the two categories of beef was investigated and the relationship to properties of connective tissue was examined. Measurements of toughness, collagen solubility, cathepsin activity and protein denaturation of beef semitendinosus heated at temperatures between 53°C and 63°C for up to 19 1/2 h were conducted. The results revealed that slightly higher temperatures and prolonged heating times were required to reduce toughness of semitendinosus from cows to the same level as in young bulls. Reduced toughness of semitendinosus as a result of low temperature for prolonged time is suggested to result from weakening of the connective tissue, caused partly by denaturation or conformational changes of the proteins and/or by solubilization of collagen.
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Colágeno/química , Tecido Conjuntivo/química , Manipulação de Alimentos/métodos , Temperatura Alta , Carne/análise , Desnaturação Proteica , Estresse Mecânico , Animais , Bovinos , Dieta , Feminino , Humanos , Masculino , Proteínas Musculares/química , Solubilidade , TemperaturaRESUMO
BACKGROUND: Recent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut. The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control. METHODS AND FINDINGS: The study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2. The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N = 20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene. The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P = 0.03). Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C. coccoides-E. rectale group correlated positively and significantly with plasma glucose concentration (P = 0.04) but not with BMIs. Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P = 0.02) and positively correlated with plasma glucose (P = 0.04). CONCLUSIONS: The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota. The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota.
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Bactérias/isolamento & purificação , Diabetes Mellitus Tipo 2/fisiopatologia , Intestinos/microbiologia , Adulto , Idoso , Bactérias/classificação , Bactérias/genética , Índice de Massa Corporal , Contagem de Colônia Microbiana , DNA Bacteriano/química , DNA Bacteriano/genética , Fezes/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
In this paper, the effect of process analyzer selection and positioning on plant-wide process monitoring is investigated. A fundamental problem in process analytical chemistry is the incomparability of different instrument characteristics. A fast but imprecise instrument is incomparable to a slow but precise instrument. Theory is developed to overcome this problem by using an abstract definition of a process analyzer. This definition allows us to put all instrument characteristics for a particular monitoring task on an equal footing. This results in a measurability factor M that expresses monitoring performance of any process measurement by combining instrument characteristics such as precision, sampling rate, grab size, response correlation, and delay time. Both the choice of location and the performance characteristics of different process analyzers can be evaluated using the measurability factor. The unifying nature of the measurability factor allows for a rational decision between completely different process analyzers and locations (Smilde et al., in this issue). The theory is illustrated and validated with an experiment. A tubular reactor for free-radical bulk polymerization of styrene is monitored by in-line short-wave near-infrared spectroscopy at different positions. Alternatively, product samples are collected for at-line near-infrared analysis. Both analyzers measure styrene monomer concentration. The analysis results are used to predict conversion as well as number and weight average molecular mass of the polystyrene reactor product. The theoretical measurability factors for this case study correspond well with the experimental findings.