Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Tea saponins as natural stabilizers for the production of hesperidin nanosuspensions.

Tea saponins (TS), a novel multifunctional stabilizer, were explored to stabilize the nanosuspensions. The purpose of this study was to investigate the effect of TS on the stability and redispersibility of nanosuspensions. In present work, hesperidin (HDN), a poorly soluble drug, was used as a model drug. HDN nanosuspensions (HDN-NS) with particle size of 250-270 nm were prepared by high-speed shearing and high-pressure homogenization. The zeta potential of HDN-NS was -23.16 ± 1.12 mV. Compared with traditional stabilizers, TS were superior in stabilization efficiency at low concentrations. Nanosuspensions freeze-dried powder using TS and lactose as cryoprotectants had good redispersibility, and the average particle size was 266.5 ± 9.0 nm after reconstitution. TS and lactose can effectively prevent the irreversible agglomeration of HDN-NS during freeze-drying. The dissolution was enhanced owing to particle size reduction. Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) results showed that HDN nanocrystals were irregularly lumpy. The chemical structure and crystal state of HDN had not significantly changed during production. In conclusion, TS have the potential to stabilize and disperse nanosuspensions and provide a promising strategy for the development of poorly soluble drugs.

Pinus residue/pectin-based composite hydrogels for the immobilization of ß-D-galactosidase.

The aim of this work was to synthesize pinus residue/pectin-based composite hydrogels for the immobilization of ß-D-galactosidase. These hydrogels were synthesized via chemical crosslinking, and characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy, thermal analysis, mechanical assays, X-ray diffraction, and swelling kinetics. The water absorption mechanism in the hydrogel networks occurs by non-Fickian transport. The ß-D-galactosidase immobilization capacities of the hydrogels containing 0, 5 and 10% of pinus residue were respectively 242.08 ± 0.36, 181.27 ± 0.50 and 182.71 ± 0.36 mg enzyme per g dried hydrogel, at pH 4.0 and after 600 min. These values were 182.99 ± 0.41, 219.99 ± 0.47 and 218.56 ± 0.39 mg g-1, respectively, at pH 5.6. Pectin-based hydrogels demonstrated to be excellent solid supports for the immobilization of enzymes. ß-D-Galactosidase immobilized in pectin-based hydrogels could be applied in the hydrolysis of lactose contained in either dairy foods or lactose-intolerant individuals.

Improving oxidative stability and release behavior of docosahexaenoic acid algae oil by microencapsulation.

BACKGROUND: Spray-dried docosahexaenoic acid algae oil (DHA AO) microcapsules were prepared using whey protein isolate and glucose syrup (WPI + GS), or sodium starch octenylsuccinate and glucose syrup (SSOS + GS), or whey protein isolate and lactose (WPI + L). The effect of the formulations on encapsulation properties, oxidative protection and in vitro oil release pattern of the resulting microencapsulates was investigated. RESULTS: A high encapsulation efficiency of over 98% of DHA AO was obtained for microcapsules with all three wall materials. Among the wall materials, SSOS + GS exhibited a better micro-particulation ability reflected by more uniform size and smoother surface of the formed microcapsules and no agglomerates. DHA AO microcapsules with all the wall materials showed good protection of the oil from oxidation during storage with an increasing order of WPI + GS, SSOS + GS and WPI + L. Moreover, microencapsulation significantly increased the release of DHA AO in the intestinal phase of the in vitro digestion process with an increasing order of SSOS + GS, WPI + GS and WPI + L, indicating the increased stability of the oil in the highly acidic gastric environment and the enhanced lipid digestibility in the small intestine. CONCLUSIONS: The results suggest that it is possible to transform a highly oxidizable liquid functional food ingredient such as DHA AO into a stable and easy-to-handle solid powder through spray drying with properly selected wall materials. © 2020 Society of Chemical Industry.

Impact of Fenugreek on Milk Production in Rodent Models of Lactation Challenge.

Fenugreek, a herbal remedy, has long been used as galactologue to help mothers likely to stop breastfeeding because of perceived insufficient milk production. However, few studies highlight the efficacy of fenugreek in enhancing milk production. The aims of our study were to determine whether fenugreek increased milk yield in rodent models of lactation challenge and if so, to verify the lack of adverse effects on dam and offspring metabolism. Two lactation challenges were tested: increased litter size to 12 pups in dams fed a 20% protein diet and perinatal restriction to an 8% protein diet with eight pups' litter, with or without 1 g.kg-1.day-1 dietary supplementation of fenugreek, compared to control dams fed 20% protein diet with eight pups' litters. Milk flow was measured by the deuterium oxide enrichment method, and milk composition was assessed. Lipid and glucose metabolism parameters were assessed in dam and offspring plasmas. Fenugreek increased milk production by 16% in the litter size increase challenge, resulting in an 11% increase in pup growth without deleterious effect on dam-litter metabolism. Fenugreek had no effect in the maternal protein restriction challenge. These results suggest a galactologue effect of fenugreek when mothers have no physiological difficulties in producing milk.

Empirical investigation of preparations produced according to the European Pharmacopoeia monograph 1038.

According to the European Pharmacopoeia monograph 1038 (Praeparationes homoeopathicae), homeopathic preparations are produced by successive dilution and succussion steps. Dilution levels beyond Avogadro's limit, however, render specific effects implausible according to standard scientific knowledge. Accordingly, we were interested in a critical empirical investigation of preparations produced according to this monograph. Within a precursor study we developed a bioassay based on a fingerprint metabolomic analysis of Lepidium sativum seeds germinated in vitro in either homeopathic preparations or controls in a blinded and randomized assignment. Results of the precursor study were not consistent with the hypothesis that the effects of a Stannum metallicum 30x preparation are identical to placebo. In the present study we investigated the reproducibility of these effects after scrutinizing and optimizing experimental procedures. Ten independent experiments were performed in a blinded and randomized assignment in two independent laboratories. Additionally, 10 systematic negative water control experiments were performed in both laboratories to critically assess the stability of the experimental set-up. The effects of the Stannum metallicum 30x treatment were reproduced. The systematic negative control experiments did not yield false-positive results, indicating a stable experimental set-up. We thus repeatedly observed biological effects conflicting with the assumption that Stannum metallicum 30x is identical to placebo. We therefore wish to discuss whether these findings are to be considered a scientific anomaly or whether they might stimulate further investigations to clarify whether application of the European Pharmacopoeia monograph 1038 may result in pharmaceutical preparations with specific effects.

Effects of intra-articular hyaluronic acid associated to Chitlac (arty-duo®) in a rat knee osteoarthritis model.

Among conventional osteoarthritis (OA) treatments, intra-articular (i.a) viscosupplementation with hyaluronic acid (HA) is used to restore joint viscoelasticity. However, the rapid clearance and elimination of HA may limit its application. The aim of this study was to verify the improved efficacy of HA within the joint, using a lactose-modified chitosan (chitlac) as a potentially chondroprotective additive. Four weeks after induction of experimental OA by destabilization of the medial meniscus (DMM), 12-week-old Sprague Dawley male rats (n = 30), received once a week, for three weeks, i.a injections of: (i) HA associated to chitlac (ARTY-DUO®), (ii) HA; and (iii) sodium chloride (NaCl). Five animals for each group were euthanized 4 weeks after the first i.a injection, while the remaining five were euthanized 8 weeks after the first i.a injection. The restoration of physiological joint microenvironment was tested by histology, histomorphometry, immunohistochemistry, and microtomography (micro-CT). At 4 and even more at 8 weeks, histological analysis showed a significant decrease in OARSI and Mankin scores, with weaker matrix metalloproteinase (MMP)-3, MMP-13, and Galectin-3 in ARTY-DUO® group versus NaCl and HA groups. A reduction in Galectin-1 and a stronger Collagen II staining was seen in both ARTY-DUO® and HA versus NaCl. A reduction in Kreen-modified score, for synovium inflammation, was observed in the ARTY-DUO® group. Micro-CT measurements did not shown significant differences between the groups. The present results show that i.a ARTY-DUO® injections produce a significant improvement in knee articular cartilage degeneration and synovium inflammation in a rat model of DMM-induced OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

The physical features -size and granularity- of solid materials are strongly affected by trituration in lactose, before turning them into homeopathic solutions.

Trituration is a mechanical process (a form of comminution) for reducing the particle size of a substance. In this manuscript, six different Raw Solid Materials (RSM) which are used in Homeopathy after successive grindings are studied before they are turned into homeopathic solutions. The impact of trituration, with the presence of α­lactose monohydrate (milk sugar) seems to be quite great and interesting because of the variety of grain size which largely differentiate the properties of the materials. The grain sizes obtained triturations by hand according to C. Herring's suggestion leads, finally, measurement scale dimensions. The obtained results can be useful information for all the pharmacy industries, as well as for preparing any kind of powder.

Kinetics of lactose fermentation in milk with kombucha starter.

The aim of this research was to investigate the effect of new, non-conventional starter culture on the kinetics of the lactose transformation during milk fermentation by kombucha, at pH 5.8; 5.4; 5.1; 4.8; and 4.6, at two different temperatures 37 °C and 42 °C. Milk fermentation at 42 °C lasted significantly shorter (about 5 h, 30 min) compared to the fermentation at 37 °C. Changes of lactose concentration at the both temperatures are consisting of two retaining stages and very steep decline in-between. The analysis of the rate curves showed that the reaction rate passes through the maximum after 9 h, 30 min at 37 °C and after 4 h at 42 °C. The sigmoidal saturation curve indicates a complex kinetics of lactose fermentation by kombucha starter.

Application of a commercial digestive supplement formulated with enzymes and probiotics in lactase non-persistence management.

Strategies to avoid lactose malabsorption, which affects 70% of the world's population, are focused on the restriction of milk and dairy products or the use of non-human ß-galactosidases or probiotics endowed with ß-galactosidase activity added at mealtime. Our evaluation of a commercial blend of probiotics and enzymes (protease, lactase, lipase and amylase) and its potential application in lactase non-persistence management is described in this work. Recommended amounts (460-1000 mg) of the commercial probiotics-enzyme blend were shown to be adequate for performing in vitro lactose hydrolysis in standard solutions (0.25-5%) and commercial dairy products, namely milks (5% lactose) and yogurts (3% lactose), reaching hydrolysis values between 44 and 96%. According to these percentages, the use of the enzymatic preparation would guarantee the intake of less than 12 g, the recommendation of the EFSA for lactose intolerance. Furthermore, formation of prebiotic galactooligosaccharides was also detected, increasing the potential benefits of the enzymatic preparation in the gastrointestinal system.

A new therapeutic avenue for bronchiectasis: Dry powder inhaler of ciprofloxacin nanoplex exhibits superior ex vivo mucus permeability and antibacterial efficacy to its native ciprofloxacin counterpart.

Non-cystic fibrosis bronchiectasis (NCFB) characterized by permanent bronchial dilatation and recurrent infections has been clinically managed by long-term intermittent inhaled antibiotic therapy among other treatments. Herein we investigated dry powder inhaler (DPI) formulation of ciprofloxacin (CIP) nanoplex with mannitol/lactose as the excipient for NCFB therapy. The DPI of CIP nanoplex was evaluated against DPI of native CIP in terms of their (1) dissolution characteristics in artificial sputum medium, (2) ex vivo mucus permeability in sputum from NCFB and healthy individuals, (3) antibacterial efficacy in the presence of sputum against clinical Pseudomonas aeruginosa strains (planktonic and biofilm), and (4) cytotoxicity towards human lung epithelial cells. Despite their similarly fast dissolution rates in sputum, the DPI of CIP nanoplex exhibited superior mucus permeability to the native CIP (5-7 times higher) attributed to its built-in ability to generate highly supersaturated CIP concentration in the sputum. The superior mucus permeability led to the CIP nanoplex's higher antibacterial efficacy (>3 log10 CFU/mL). The DPI of CIP nanoplex exhibited similar cytotoxicity towards the lung epithelial cells as the native CIP indicating its low risk of toxicity. These results established the promising potential of DPI of CIP nanoplex as a new therapeutic avenue for NCFB.

Maillard-Reaction-Functionalized Egg Ovalbumin Stabilizes Oil Nanoemulsions.

Egg white proteins are an excellent source of nutrition, with high biological and technological values. However, their limited functional properties prevent their widespread industrial applications. In this study, the ovalbumin functionality was improved via glycation by Maillard reaction with d-lactose. The free amino groups and sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile were determined, confirming that glycation occurred between ovalbumin and lactose. The emulsification of the conjugate was 2.69-fold higher than that of ovalbumin at pH 7.0 after glycation. The thermal stability also improved remarkably. The glycated protein products were used to form an oil-water nanoemulsion for polymethoxyflavone-rich aged orange peel oil. The resulting nanoemulsion showed good pH, thermal, and storage stabilities.

Limitations of high dose carrier based formulations.

PURPOSE: This study was performed to investigate how increasing the active pharmaceutical ingredient (API) content within a formulation affects the dispersion of particles and the aerosol performance efficiency of a carrier based dry powder inhalable (DPI) formulation, using a custom dry powder inhaler (DPI) development rig. METHODS: Five formulations with varying concentrations of API beclomethasone dipropionate (BDP) between 1% and 30% (w/w) were formulated as a multi-component carrier system containing coarse lactose and fine lactose with magnesium stearate. The morphology of the formulation and each component were investigated using scanning electron micrographs while the particle size was measured by laser diffraction. The aerosol performance, in terms of aerodynamic diameter, was assessed using the British pharmacopeia Apparatus E cascade impactor (Next generation impactor). Chemical analysis of the API was observed by high performance liquid chromatography (HPLC). RESULTS: Increasing the concentration of BDP in the blend resulted in increasing numbers and size of individual agglomerates and densely packed BDP multi-layers on the surface of the lactose carrier. BDP present within the multi-layer did not disperse as individual primary particles but as dense agglomerates, which led to a decrease in aerosol performance and increased percentage of BDP deposition within the Apparatus E induction port and pre-separator. CONCLUSION: As the BDP concentration in the blends increases, aerosol performance of the formulation decreases, in an inversely proportional manner. Concurrently, the percentage of API deposition in the induction port and pre-separator could also be linked to the amount of micronized particles (BDP and Micronized composite carrier) present in the formulation. The effect of such dose increase on the behaviour of aerosol dispersion was investigated to gain greater insight in the development and optimisation of higher dosed carrier-based formulations.

Comparison of anticancer activity of biocompatible ZnO nanoparticles prepared by solution combustion synthesis using aqueous leaf extracts of Abutilon indicum, Melia azedarach and Indigofera tinctoria as biofuels.

Recently, there has been an upsurge in the use of naturally available fuels for solution combustion synthesis (SCS) of nanoparticles. Although many reports suggest that these biofuels pose less harm to the environment, their strategic advantages and reliability for making NPs has not been discussed. In the present work, we try to address this issue using plant extracts as biofuels for the SCS of zinc oxide nanoparticles as a model system. In the present work, combustion synthesis of ZnO NPs using lactose and aqueous leaf extracts of Abutilon indicum, Melia azedarach, Indigofera tinctoria as biofuels has been carried out. A comparative analysis of the obtained powders has been conducted to understand the strategic advantages of using plant extracts over a chemical as combustion fuel for the synthesis of zinc oxide nanoparticles. The X-ray diffractograms of the samples revealed the presence of Wurtzite hexagonal structure with varying crystallite sizes. Morphological studies indicated that samples prepared using biofuels had smaller diameter than those prepared using lactose as fuel. Surface characteristics of the samples were measured by X-ray photoelectron spectroscopy. Qualitative phytochemical screening of aqueous leaf extracts revealed the presence of many phytochemicals in them, which might be responsible for combustion. Gas chromatography mass spectrum was carried out to detect the phytochemicals present in the aqueous extracts of the leaves. Further, anticancer evaluation carried out against DU-145 and Calu-6 cancer cells indicated higher anticancer activity of zinc oxide nanoparticles prepared using biofuels. The results of blood haemolysis revealed the biocompatibility of zinc oxide nanoparticles at lower concentrations. In conclusion, we propose that multiple other studies would be required in order to vindicate the potential advantages of using naturally available fuels in SCS.

Green Tea Polyphenols Decrease Strecker Aldehydes and Bind to Proteins in Lactose-Hydrolyzed UHT Milk.

The effect of epigallocatechin gallate enriched green tea extract (GTE) on flavor, Maillard reactions and protein modifications in lactose-hydrolyzed (LH) ultrahigh temperature (UHT) processed milk was examined during storage at 40 °C for up to 42 days. Addition of GTE inhibited the formation of Strecker aldehydes by up to 95% compared to control milk, and the effect was similar when GTE was added either before or after UHT treatment. Release of free amino acids, caused by proteolysis, during storage was also decreased in GTE-added milk either before or after UHT treatment compared to control milk. Binding of polyphenols to milk proteins was observed in both fresh and stored milk samples. The inhibition of Strecker aldehyde formation by GTE may be explained by two different mechanisms; inhibition of proteolysis during storage by GTE or binding of amino acids and proteins to the GTE polyphenols.

Atomic scale surface engineering of micro- to nano-sized pharmaceutical particles for drug delivery applications.

Atomic layer deposition on pharmaceutical particles for drug delivery applications is demonstrated using assisted fluidized bed dry powder processing. Complete and conformal layering is achieved on particle sizes from the lower micron to upper nanometer range under near ambient conditions. As few as 2-14 atomic alumina layers alter particle properties: dissolution, dispersibility and heat transfer.

Hydroxyurea-Lactose Interaction Study: In Silico and In Vitro Evaluation.

The Maillard reaction between hydroxyurea (a primary amine-containing drug) and lactose (used as an excipient) was explored. The adduct of these compounds was synthesized by heating hydroxyurea with lactose monohydrate at 60 °C in borate buffer (pH 9.2) for 12 h. Synthesis of the adduct was confirmed using UV-visible spectroscopy and Fourier transform infrared, differential scanning calorimetry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry studies. An in silico investigation of how the adduct formation affected the interactions of hydroxyurea with its biological target oxyhemoglobin, to which it binds to generate nitric oxide and regulates fetal hemoglobin synthesis, was carried out. The in silico evaluations were complemented by an in vitro assay of the anti-sickling activity. Co-incubation of hydroxyurea with deoxygenated blood samples reduced the percentage of sickled cells from 38% to 12 ± 1.6%, whereas the percentage of sickled cells in samples treated with the adduct was 17 ± 1.2%. This indicated loss of anti-sickling activity in the case of the adduct. This study confirmed that hydroxyurea can participate in a Maillard reaction if lactose is used as a diluent. Although an extended study at environmentally feasible temperatures was not carried out in the present investigation, the partial loss of the anti-sickling activity of hydroxyurea was investigated along with the in silico drug-target interactions. The results indicated that the use of lactose in hydroxyurea formulations needs urgent reconsideration and that lactose must be replaced by other diluents that do not form Maillard adducts.

Co-milled API-lactose systems for inhalation therapy: impact of magnesium stearate on physico-chemical stability and aerosolization performance.

CONTEXT: Particle micronization for inhalation can impart surface disorder (amorphism) of crystalline structures. This can lead to stability issues upon storage at elevated humidity from recrystallization of the amorphous state, which can subsequently affect the aerosol performance of the dry powder formulation. OBJECTIVE: The aim of this study was to investigate the impact of an additive, magnesium stearate (MGST), on the stability and aerosol performance of co-milled active pharmaceutical ingredient (API) with lactose. METHODS: Blends of API-lactose with/without MGST were prepared and co-milled by the jet-mill apparatus. Samples were stored at 50% relative humidity (RH) and 75% RH for 1, 5, and 15 d. Analysis of changes in particle size, agglomerate structure/strength, moisture sorption, and aerosol performance were analyzed by laser diffraction, scanning electron microscopy (SEM), dynamic vapor sorption (DVS), and in-vitro aerodynamic size assessment by impaction. RESULTS: Co-milled formulation with MGST (5% w/w) led to a reduction in agglomerate size and strength after storage at elevated humidity compared with co-milled formulation without MGST, as observed from SEM and laser diffraction. Hysteresis in the sorption/desorption isotherm was observed in the co-milled sample without MGST, which was likely due to the recrystallization of the amorphous regions of micronized lactose. Deterioration in aerosol performance after storage at elevated humidity was greater for the co-milled samples without MGST, compared with co-milled with MGST. CONCLUSION: MGST has been shown to have a significant impact on co-milled dry powder stability after storage at elevated humidity in terms of physico-chemical properties and aerosol performance.

Investigation into the Manufacture and Properties of Inhalable High-Dose Dry Powders Produced by Comilling API and Lactose with Magnesium Stearate.

The aim of the study was to understand the impact of different concentrations of the additive material, magnesium stearate (MGST), and the active pharmaceutical ingredient (API), respectively, on the physicochemical properties and aerosol performance of comilled formulations for high-dose delivery. Initially, blends of API/lactose with different concentrations of MGST (1-7.5% w/w) were prepared and comilled by the jet-mill apparatus. The optimal concentration of MGST in comilled formulations was investigated, specifically for agglomerate structure and strength, particle size, uniformity of content, surface coverage, and aerosol performance. Secondly, comilled formulations with different API (1-40% w/w) concentrations were prepared and similarly analyzed. Comilled 5% MGST (w/w) formulation resulted in a significant improvement in in vitro aerosol performance due to the reduction in agglomerate size and strength compared to the formulation comilled without MGST. Higher concentrations of MGST (7.5% w/w) led to reduction in aerosol performance likely due to excessive surface coverage of the micronized particles by MGST, which led to failure in uniformity of content and an increase in agglomerate strength and size. Generally, comilled formulations with higher concentrations of API increased the agglomerate strength and size, which subsequently caused a reduction in aerosol performance. High-dose delivery was achieved at API concentration of >20% (w/w). The study provided a platform for the investigation of aerosol performance and physicochemical properties of other API and additive materials in comilled formulations for the emerging field of high-dose delivery by dry powder inhalation.