Mixture Of Lactose Research Articles

Model-based estimation of selectivities of the β-galactosidase of Aspergillus oryzae in the production of lactulose and fructosyl-galactooligosaccharides

There has been recent interest in using the β-galactosidase of Aspergillus oryzae to produce lactulose and fructosyl-galactooligosaccharides, for use as prebiotics. The success of the enzymatic process depends on the selectivities of the enzyme for the various transgalactosylation and hydrolysis reactions that occur in these systems, but the methods that have been used to date to express these selectivities are not adequate. In the current work, we demonstrate a method for determining the selectivity of the β-galactosidase of A. oryzae in two case studies done with literature data: (1) the production of lactulose from a mixture of lactose and fructose and (2) the production of fructosyl-galactooligosaccharides (fGOS) from lactulose. In the first case study, we demonstrate that the enzyme has a 4- to 5-fold preference for producing GOS over lactulose when an equimolar mixture of fructose to lactose is used, but that the selectivity for producing lactulose increases as the initial fructose to lactose molar ratio increases. In the second case study, we show that the enzyme has about a 1.5-fold preference for producing fGOS4 and fGOS5 over the initial transgalactosylation product, fGOS3. The selectivities that we determine in our work will be important parameters for time-based models used to guide the development and optimization of processes for the production of lactulose and fructosyl-galactooligosaccharides as prebiotics.

Nanoscale colocalized thermal and chemical mapping of pharmaceutical powder aerosols

Inhalation of pharmaceutical aerosol formulations is widely used to treat respiratory diseases. Spatially resolved thermal characterization offers promise for better understanding drug release rates from particles; however, this has been an analytical challenge due to the small particle size (from a few micrometers down to nanometers) and the complex composition of the formulations. Here, we employ nano-thermal analysis (nanoTA) to probe the nanothermal domain of a pharmaceutical aerosol formulation containing a mixture of fluticasone propionate (FP), salmeterol xinafoate (SX), and excipient lactose, which is widely used to treat asthma and chronic obstructive pulmonary disease (COPD). Furthermore, atomic force microscopy-infrared spectroscopy (AFM-IR) and AFM force measurements are performed to provide nanochemical and nanomechanical information to complement the nanothermal data. The colocalized thermal and chemical mapping clearly reveals the surface heterogeneity of the drugs in the aerosol particles and demonstrates the contribution of the surface chemical composition to the variation in the thermal properties of the particles. We present a powerful analytical approach for in-depth characterization of thermal/chemical/morphological properties of dry powder inhaler particles at micro- and nanometer scales. This approach can be used to facilitate the comparison between generics and reference inhalation products and further the development of high-performance pharmaceutical formulations.

A comprehensive compatibility study of ganciclovir with some common excipients

Objectives: The aim of the present study is to illustrate compatibility testing of ganciclovir (GCV) with some common excipients that would be used to manufacture solid oral dosage forms. Different spectroscopy techniques were utilized to see the interaction of GCV with excipients such as lactose, microcrystalline cellulose (MCC), magnesium stearate, and talc, and dicalcium phosphate. Further, a molecular docking study was also done to know the interaction of GCV with excipients. In vitro study of a physical mixture of GCV with excipients was performed to get the release of drug. Material and Methods: A number of analytical techniques (differential scanning calorimetry [DSC] using DSC-Q20, TA instruments, Fourier-transform infrared spectroscopy [FTIR] spectroscopy using Spectrum RX 1, nuclear magnetic resonance [NMR] using Bruker Advance Neo 500 MHz NMR spectrometer, etc.) have been used to explore the drug-excipient compatibility. Further, a suspected interaction was evaluated by thin-layer chromatography (TLC). In vitro dissolution studies in different sets of experiments were accomplished to determine the influence of hydrophobic and hydrophilic attributes of excipients (MCC, lactose, dicalcium phosphate, and talc) on the dissolution profile of GCV using USP1-type dissolution apparatus. Furthermore, in silico molecular docking studies were also performed to evaluate any probable molecular interactions among drugs and excipients using Auto Dock VINA 1.2.0 software and GROMACS 5.0 software. Results: Comparing FTIR and 1H NMR spectra of GCV and physical mixtures of GCV and excipients, no significant deviation of characteristic peaks in infrared spectroscopy and 1H NMR signals was observed. The DSC of GCV showed two sharp endothermic peaks at 238.82°C and 255°C. The endothermic peak of GCV in DSC thermogram of physical mixtures was observed in nearly the same position except with lactose and dicalcium phosphate. A slightly deviated peak of GCV with a physical mixture of drug and lactose and dicalcium phosphate indicated that there were suspected interactions between the drug with lactose and dicalcium phosphate. These interactions were evaluated by thin-layer chromatography (TLC) and it confirmed that there was no interaction between drugs and excipients. In vitro dissolution studies determined the influence of hydrophobic and hydrophilic attributes of excipients on the dissolution profile of GCV. The physical mixture of GCV with MCC displayed a maximum amount (66.48%) of drug release in 10 min. On the other hand, a physical mixture of GCV with talc showed a minimum amount (12.08%) of drug release in 10 min. Docking study predicted that the number of interactions were more between GCV and lactose (four nos.) in comparison to GCV and MCC (two nos.). This interaction supported the in vitro drug release of a physical mixture of GCV with MCC which was higher than a mixture of GCV with lactose. Conclusion: Compatibility testing of GCV with used excipients by analytical techniques confirmed that GCV should be compatible with used excipients. Drug dissolution of GCV and physical mixture of MCC exhibited the maximum amount of drug release whereas a mixture of GCV with talc released the minimum amount of drug for both short (10 min.) and long (60 min.) periods. Docking studies disclosed that the lactose complex showed less deviation with less root mean square deviation value in comparison to the microcrystalline complex. Thus, the lactose complex has more hydrogen bonds and it was more stable as compared with the MCC complex. GCV indicates that the total energy of the MCC complex is less than that of the lactose complex. This indicates that GCV is more soluble when combined with the microcrystalline complex. Therefore, GCV and used excipients could be used for solid dosage formulations.

Emergent Lag Phase in Flux-Regulation Models of Bacterial Growth

Lag phase is observed in bacterial growth during a sudden change in conditions: growth is inhibited whilst cells adapt to the environment. Bi-phasic, or diauxic growth is commonly exhibited by many species. In the presence of two sugars, cells initially grow by consuming the preferred sugar then undergo a lag phase before resuming growth on the second. Biomass increase is characterised by a diauxic growth curve: exponential growth followed by a period of no growth before a second exponential growth. Recent literature lacks a complete dynamic description, artificially modelling lag phase and employing non-physical representations of precursor pools. Here, we formulate a rational mechanistic model based on flux-regulation/proteome partitioning with a finite precursor pool that reveals core mechanisms in a compact form. Unlike earlier systems, the characteristic dynamics emerge as part of the solution, including the lag phase. Focussing on growth of Escherichia coli on a glucose–lactose mixture we show results accurately reproduce experiments. We show that for a single strain of E. coli, diauxic growth leads to optimised biomass yields. However, intriguingly, for two competing strains diauxic growth is not always the best strategy. Our description can be generalised to model multiple different microorganisms and investigate competition between species/strains.

Effect of Diluents on Active Pharmaceutical Ingredient Loss Until Patients Ingest Powder Formulation.

In powder formulations, it is a problem that the required therapeutic dose is not obtained because of loss of the active pharmaceutical ingredient (API). In this study, we investigated three types of lactose diluents, which are widely used as pharmaceutical excipients, for dispensing prednisolone powder. Extra-fine crystalline lactose, commonly used as a diluent in compounding powder formulations, was used as a comparison. The effect of lactose on the API loss rate was examined by analyzing the amount of prednisolone in the powder formulation taken out of a single-dose package after dispensing. The results showed that Dilactose-F had the lowest API loss rate (22%), followed by powder lactose (37.8%), extra-fine crystalline lactose (45.9%), and crystal form lactose (48.6%), indicating that the use of Dilactose-F as a diluent significantly improved API loss when compounding the powder formulation. Because each mixture of commercial prednisolone powder and lactose was within acceptable uniformity and loss rate before packaging, we considered that API loss occurred when the powder was taken out of the single-dose package before patients ingested them. Then, the physical properties of these lactose types affecting the API loss rate were examined. Strong correlation was not found between flowability and the API loss rate, but particle size distribution and bulk density were strongly correlated with the API loss rate. Furthermore, Dilactose-F, which showed the lowest API loss rate, did not show an exothermic peak due to epimerization to anhydrous β -lactose in differential scanning calorimetry and showed a peak specific to β -lactose in powder X-ray diffractometer. These results suggested that in powder compounding where the API content is low, the physical properties of lactose, such as particle size distribution, bulk density, and crystalline form, are intricately related to API loss.

P AOX1 expression in mixed-substrate continuous cultures of Komagataella phaffii (Pichia pastoris) is completely determined by methanol consumption regardless of the secondary carbon source.

The expression of recombinant proteins by the AOX1 promoter of Komagataella phaffii is typically induced by adding methanol to the cultivation medium. Since growth on methanol imposes a high oxygen demand, the medium is often supplemented with an additional secondary carbon source which serves to reduce the consumption of methanol, and hence, oxygen. Early research recommended the use of glycerol as the secondary carbon source, but more recent studies recommend the use of sorbitol because glycerol represses P AOX1 expression. To assess the validity of this recommendation, we measured the steady state concentrations of biomass, residual methanol, and LacZ expressed from P AOX1 over a wide range of dilution rates (0.02-0.20h-1) in continuous cultures of the Mut+ strain fed with methanol + glycerol (repressing) and methanol + sorbitol (non-repressing). We find that under these conditions, the specific P AOX1 expression rate (measured as either specific LacZ productivity or specific AOX productivity) is completely determined by the specific methanol consumption rate regardless of the type (repressing/non-repressing) of the secondary carbon source. In both cultures, the specific P AOX1 expression rate is proportional to the specific methanol consumption rate, provided that the latter is below 0.15g/(gdw-h); beyond this threshold consumption rate, the specific P AOX1 expression rate of both cultures saturates to the same value. Analysis of the data in the literature shows that the same phenomenon also occurs in continuous cultures of Escherichia coli fed with mixtures of lactose plus repressing/non-repressing carbon sources. The specific P lac expression rate is completely determined by the specific lactose consumption rate, regardless of the type of secondary carbon source, glycerol or glucose.

Characterization of microbial communities in anaerobic acidification reactors fed with casein and/or lactose

Protein-rich agro-industrial waste streams are high in organic load and represent a major environmental problem. Anaerobic digestion is an established technology to treat these streams; however, retardation of protein degradation is frequently observed when carbohydrates are present. This study investigated the mechanism of the retardation by manipulating the carbon source fed to a complex anaerobic microbiota and linking the reactor performance to the variation of the microbial community. Two anaerobic acidification reactors were first acclimated either to casein (CAS reactor) or lactose (LAC reactor), and then fed with mixtures of casein and lactose. Results showed that when lactose was present, the microbial community acclimated to casein shifted from mainly Chloroflexi to Proteobacteria and Firmicutes, the degree of deamination in the CAS reactor decreased from 77 to 15%, and the VFA production decreased from 75 to 34% of the effluent COD. A decrease of 75% in protease activity and 90% in deamination activity of the microbiota was also observed. The microorganisms that can ferment both proteins and carbohydrates were predominant in the microbial community, and from a thermodynamical point of view, they consumed carbohydrates prior to proteins. The frequently observed negative effect of carbohydrates on protein degradation can be mainly attributed to the substrate preference of these populations.Keypoints• The presence of lactose shifted the microbial community and retarded anaerobic protein degradation.• Facultative genera were dominant in the presence and absence of lactose.• Substrate-preference caused retardation of anaerobic protein degradation.

A highly selective fluorescent sensor for manganese(II) ion detection based on N,S-doped carbon dots triggered by manganese oxide

A highly selective, carbon dots (CDs)-based fluorescent sensor for manganese(II) ions (Mn2+) was proposed. The synthesis of N,S-doped CDs (N,S-CDs) involved simple pyrolysis of a mixture of lactose and l-cysteine in a hot NaOH solution. The as-synthesized codoped CDs exhibited a weak fluorescent emission peak at 510 nm when excited at 430 nm. In the presence of Mn2+, the intensity of the fluorescence signals was effectively increased with a small redshift in the spectrum. Thus, by tracing the degree of fluorescence enhancement, the material could be used as a selective probe for Mn2+ detection. The possible sensing mechanism was based on the oxidation of the N,S-CDs by MnO2, which was generated from mixing Mn2+ with NaOH in the N,S-CDs-containing medium. A linear relationship was established between the extent of the increase in fluorescence and the increasing Mn2+ concentration from 0 to 5.0 μM with a limit of detection (LOD) of 0.12 μM. Remarkably, even in the presence of other cations, the developed sensor remained highly selective toward Mn2+. The feasibility of our developed sensor in real applications was confirmed with environmental samples, showing excellent accuracy and precision.

Physicochemical Properties of Zinc and Lactose in Solid Mixtures: Influence of Trituration Process.

Recent experimental results supporting the dynamization process show modification in the characteristics of solid mixtures. The present work aims to evaluate the physicochemical properties of metallic zinc and lactose, evidencing the interactions between all chemical components presented in dynamized solid mixtures by analytical techniques. Mixtures of zinc and lactose (1:9 w/w) were successively triturated at the same proportion according to the Brazilian Homeopathic Pharmacopoeia, receiving the designation of 10-1 - 10-6 (1dH - 6dH). All samples were submitted to the following characterization techniques: Atomic Absorption Spectrometry (AAS), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Differential Scanning Calorimetry (DSC), Thermogravimetry (TG), and Raman Spectroscopy (RS). AAS results detected 97.0% of zinc in the raw material, and the triturated zinc lactose system (ZnMet) presented mean values similar to those expected for the physical mixtures: i.e., 9.94%, 1.23%, and 0.11% in the three first proportions (10-1, 10-2, 10-3), respectively. SEM images showed particle size reduction due to the trituration process. The XRD assays of ZnMet 10-3 and 10-6 indicated peak changes at 12.3° and 43.26°, probably associated with modifications of inter-atomic crystalline spacing. The thermal analysis results of dynamized samples suggest modifications in the chemical interaction between zinc and lactose induced by the physical forces applied. RS experiments showed variation in vibration frequencies due to the dynamization procedure, in which marked ZnMet 10-6 spectral modifications were detected at 357, 477, 1086 and 1142 cm-1, and in the wavelength range 860-920 cm-1. These results highlight the importance of applying suitable characterization methods to improve our understanding of the properties of homeopathic solid mixtures, whereas the uses of sensitive tools evidence the influence of trituration on the crystalline properties and in the enthalpy variation of dynamized samples.

Increasing Lovastatin Production by Re-routing the Precursors Flow of Aspergillus terreus via Metabolic Engineering.

Lovastatin is an anti-cholesterol medicine that is commonly prescribed to manage cholesterol levels, and minimise the risk of suffering from heart-related diseases. Aspergillus terreus (ATCC 20542) supplied with carbohydrates or sugar alcohols can produce lovastatin. The present work explored the application of metabolic engineering in A. terreus to re-route the precursor flow towards the lovastatin biosynthetic pathway by simultaneously overexpressing the gene for acetyl-CoA carboxylase (acc) to increase the precursor flux, and eliminate ( +)-geodin biosynthesis (a competing secondary metabolite) by removing the gene for emodin anthrone polyketide synthase (gedC). Alterations to metabolic flux in the double mutant (gedCΔ*accox) strain and the effects of using two different substrate formulations were examined. The gedCΔ*accox strain, when cultivated with a mixture of glycerol and lactose, significantly (p < 0.05) increased the levels of metabolic precursors malonyl-CoA (48%) and acetyl-CoA (420%), completely inhibited the (+)-geodin biosynthesis, and increased the level of lovastatin [152mg/L; 143% higher than the wild-type (WT) strain]. The present work demonstrated how the manipulation of A. terreus metabolic pathways could increase the efficiency of carbon flux towards lovastatin, thus elevating its overall production and enabling the use of glycerol as a substrate source. As such, the present work also provides a framework model for other medically or industrially important fungi to synthesise valuable compounds using sustainable carbon sources.

Multicenter project Brazil-Italy: physicochemical analysis of Zincum metallicum and lactose in dynamized mixtures

Background: The use of sensitive techniques can be useful to increase the understanding of homeopathic systems behavior. &#x0D; Aim: To analyze physicochemical properties of Zincum metallicum and lactose mixtures prepared according to Brazilian Homeopathic Pharmacopeia.&#x0D; Methodology: Zincum metallicum was prepared using lactose as vehicle, from 1 to 3 cH, and from 1 to 6dH. All samples including respective controls (dynamized lactose) were analyzed by Atomic Absorption Spectroscopy (AAS), X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM) with X-Ray Energy Dispersive Spectroscopy (EDX), Scanning Electron Microscopy (SEM), and Differential Scanning Calorimetry (DSC).&#x0D; Results: AAS analysis showed the dynamization process decreases zinc concentration in a proportional manner to the increase of homeopathic potencies. Quantities of 4ppm and 132 ppm of zinc were detected in 6dH and 3cH, respectively. XRD analysis showed crystalline form of lactose was not modified by dynamization. Amorphous lactose spheres were observed by TEM in all samples, with mean size ranging from 800 to 200 nm. EDX obtained in the TEM confirmed zinc presence throughout the amorphous matter and the absence of individualized zinc crystalline particles. These results are in accordance with XRD which showed absence of zinc diffraction peaks, in all lactose samples containing zinc suggesting this metal could be modified from crystalline to amorphous state, and consequently adsorbed into lactose molecules. SEM structural analysis of lactose and zinc (1cH) samples using BSE detector showed no zinc metal grains confirming XRD and TEM results. DSC analyses presented statistical significance differences (p

Evaluation of Binders in Twin-Screw Wet Granulation.

The binders povidone (Kollidon 30), copovidone (Kollidon VA64), hypromellose (Pharmacoat 606), and three types of hyprolose (HPC SSL-SFP, HPC SSL, and HPC SL-FP) were evaluated regarding their suitability in twin-screw wet granulation. Six mixtures of lactose and binder as well as lactose without binder were twin-screw granulated with demineralized water at different barrel fill levels and subsequently tableted. A screening run with HPC SSL determined the amount of water as an influential parameter for oversized agglomerates. Subsequent examination of different binders, especially Kollidon 30 and Kollidon VA64 resulted in large granules. All binders, except Pharmacoat 606, led to a reduction of fines compared to granulation without a binder. The molecular weight of applied hyproloses did not appear as influential. Tableting required an upstream sieving step to remove overlarge granules. Tableting was possible for all formulations at sufficient compression pressure. Most binders resulted in comparable tensile strengths, while Pharmacoat 606 led to lower and lactose without a binder to the lowest tensile strength. Tablets without a binder disintegrated easily, whereas binder containing tablets of sufficient tensile strength often nearly failed or failed the disintegration test. Especially tablets containing Pharmacoat 606 and HPC SL-FP disintegrated too slowly.

Loss on Drying Using Halogen Moisture Analyzer: An Orthogonal Technique for Monitoring Volatile Content for In-Process Control Samples during Pharmaceutical Manufacturing

Organic solvents and water are routinely used during the synthetic pathway of an active pharmaceutical ingredient (API) and extraction work up, respectively. Residual amounts of solvents and water tend to be strictly controlled at each step of a process as carryover can often negatively affect crystallization or the reactivity of catalysts and/or activating agents. A rapid, accurate, and precise measurement of these attributes is an important part of this overall control strategy. Here, we have developed a loss on drying (LOD) method for measuring total volatile matter for in-process control liquid and solid samples during a small molecule API manufacturing process, which relies on a halogen moisture analyzer with an integrated mass balance. Mixtures of lactose and microcrystalline cellulose with varying water content were prepared and used for evaluating the precision of the LOD technique with varying sample sizes. We have successfully demonstrated here that not only are the moisture measurements using this LOD method comparable to the traditional methods like Karl Fischer (KF) titration and/or gas chromatography (GC) for volatile solvents but also the LOD measurement process is much faster and requires little to no sample preparation. Further, we present a case study of using LOD and KF together to obtain the ratio of water and DMA (a high boiling point solvent), enabling a more efficient manufacturing process as it obviates the need for specialized HPLC or GC methods needed for measuring high boiling solvents such as DMA.

Flavor release from lactose/protein matrix during storage: Effects of lactose crystallization and powder microstructure

Glass-forming carbohydrates are widely used to encapsulate volatile flavor substances to avoid excessive flavor release and increase storage stability. The objective of this study was to investigate the effects of the crystallization of lactose on the microstructures of powders, as well as subsequent influences on flavor release from lactose-containing matrices. Mixtures of lactose and whey protein isolate were used to constitute the wall materials in ratios of 0:1, 1:4, 1:1 and 4:1 (w/w). Ethyl acetate (EA) was used as a model core material and microparticles were prepared by freeze-drying. Microparticles with higher lactose contents had lower glass transition temperatures and exhibited increased water adsorption behaviors at various water activities (0.33, 0.54 and 0.75 aw). A rapid decrease in flavor retention was found in matrices with high lactose contents, especially during storage at 0.75 aw. X-ray diffraction and crystallinity analysis proved that the degrees of crystallization of lactose in the encapsulation systems differed, which was induced by differences in water activities. Images of microstructures showed that the formation of crystals caused structural modifications of the powder. Consequently, EA particles were prone to accumulate and migrate to the surface of the powder, which resulted in considerable release of EA flavor.

Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30

BackgroundThe degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30.ResultsExperimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the β-glucosidase regulation and iii) a negative regulation of the development process and growth.ConclusionsThis work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the β-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.

UTILIZATION OF NEMBA STEM AS AN ANTITUBERCULOSIS COMPARED WITH RIFAMPICIN AND ETAMBUTOL

Indonesia ranks third in the number of tuberculosis, after India and China. Treatment of tuberculosis takes a long time, and the lack of discipline in taking medication has caused the Mycobacterium tuberculosis to be resistant to many synthetic drugs that have been used so far. In contrast, the discovery of new synthetic drugs is very slow. Traditionally, neem bark (Azadirachta indica JUSS.) has been used to treat coughing up blood and phlegm, and the results of previous studies show that ethanol extract of neem bark in vitro can inhibit the growth of Mycobacterium tuberculosis. This study was conducted to determine the toxicity of neem bark extract, formulation of neem bark ethanol extract into tablet preparations, and the potential of this tablet preparation as anti-tuberculosis in vivo in animals. Neem bark extract was made by percolation using an ethanol extractor. Toxicity test was carried out in the form of an acute toxicity test on mice to calculate the LD50 value and histopathological observations of the liver, lungs, and intestines. The tablets were made by wet granulation. The potential test of tablet preparations as anti-tuberculosis was performed in vivo on animals infected with Mycobacterium tuberculosis H37Rv using a nebulizer. The results showed LD 50 = 11.85 ± 0.571, including mild toxic. The ethanolic extract of neem bark can be formulated into tablets using a 6% gelatin binder, Manihot starch and primojel as a binder, and a mixture of starch and lactose as a filler. Neem bark extract tablets can cure tuberculosis of experimental animals that have been infected with Mycobacterium tuberculosis H37RV, which is given a dose of 3 times a day two tablets (50 mg/tablet) for six weeks seen tuberculosis bacteria from +3 to negative, and a dose of 3 times a day once a day. Tablet looks from +3 to +1.

Enhanced 2′-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.

Meloxicam Carrier Systems Having Enhanced Release and Aqueous Wettability Prepared Using Micro-suspensions in Different Liquid Media.

One of the conventional methods of alleviating the problem of poor drug solubility is the particle size reduction. The efficiency of this approach depends on successful formulation suppressing the drug agglomeration. The aim of this study was to circumvent the dissolution problems of model hydrophobic meloxicam drug (MLX) by using liquid media of different wetting capacity to comminute and formulate a rapidly dissolving carrier system without the use of surfactants. Micro-suspensions of MLX were prepared by ball milling, using water or n-Heptane as a liquid medium. The suspensions were used as granulation liquids to formulate granulate from microcrystalline cellulose and lactose mixture. The release kinetics from prepared granulates were studied using the USP-4 dissolution apparatus. Micro-suspensions prepared via wet milling in non-water liquid media exhibited a massive improvement of release rate compared with source meloxicam and they outperformed their water-milled counterparts. The release rates from those formulations, despite not comprising any surfactant, were comparable to those obtained by different authors using surfactant stabilized nanosuspension formulations. Thus, they can present an interesting formulation alternative for hydrophobic drugs that are dissolution limited.

Continuous Manufacturing of Homogeneous Ultralow-Dose Granules by Twin-Screw Wet Granulation

Homogeneous ultralow-dose (30 mg) tablets were prepared from perfectly free-flowing granules manufactured by continuous Twin-Screw Wet Granulation. The gravimetrically fed mixture of lactose and potato starch of low particle size was successfully agglomerated and the size enlargement technology proved to be very robust. Since the incorporated drug was dissolved in ethanol-based granulation liquid, the resulting homogeneity largely depended on the dosing of the applied liquid administering units.A peristaltic pump generated higher deviation of the drug content in tablets (Relative Standard Deviation (RSD): 7.7 %) compared to a syringe pump (RSD: 2.3 %) or a piston pump (RSD: 4.6 %). This is due to the pulsation of the liquid flow generated by the peristaltic pump according to the real-time measured mass of the fed liquid. A good correlation was found between the RSD of the liquid mass flow (calculated from the recorded masses) and the RSD of the drug content. Based on the results, the goodness of Content Uniformity, as the most relevant critical quality attribute of low-dose products, was determined by the characteristics of the applied dosing units. The feeding characteristic of the different pumps could be easily measured by the introduced balance-based method and therefore, the applicability of the pumps could be evaluated.

Ultrasensitive detection of saccharides using terahertz sensor based on metallic nano-slits

Unambiguous identification of trace amounts of biochemical molecules in a complex background using terahertz spectroscopy is extremely challenging owing to the extremely small absorption cross sections of these molecules in the terahertz regime. Herein, we numerically propose a terahertz nonresonant nano-slits structure that serves as a powerful sensor. The structure exhibits strongly enhanced electric field in the slits (five orders of magnitude), as well as high transmittance over an extra-wide frequency range that covers the characteristic frequencies of most molecules. Fingerprint features of lactose and maltose are clearly detected using this slits structure, indicating that this structure can be used to identify different saccharides without changing its geometrical parameters. The absorption signal strengths of lactose and maltose with a thickness of 200 nm are strongly enhanced by factors of 52.5 and 33.4, respectively. This structure is very sensitive to thin thickness and is suitable for the detection of trace sample, and the lactose thickness can be predicted on the basis of absorption signal strength when the thickness is less than 250 nm. The detection of a mixture of lactose and maltose indicates that this structure can also achieve multi-sensing which is very difficult to realize by using the resonant structures.