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To prevent neural tube defects and other cardiovascular diseases in newborns, folic acid (FA) is recommended in pregnant women. A daily dose of 600 µg FA consumption is widely prescribed for women during pregnancy and 400 µg for women with childbearing potential. FA is a class IV compound according to the Biopharmaceutics Classification System (BCS) due to its low permeability (1.7 × 10-6 cm/s) and low solubility (1.6 mg/L); therefore, it must be administered via a formulation that enhances its solubility. Studies reported in the literature have proved that co-amorphization and salt formation of a poorly soluble drug with amino acids (AA) can significantly increase its solubility. Although arginine has been used with FA as a supplement, there is no information on the effect of basic AA (arginine and lysine) on the physical and chemical properties of FA-AA binary formulations. The present study implemented a conductimetric titration methodology to find the effective molar ratio to maximize FA solubility. The results showed that a 1:2.5 FA:AA molar ratio maximized solubility for arginine and lysine. Binary formulations were prepared using different methods, which led to an amorphous system confirmed by the presence of a glass transition, broad FTIR bands, and the absence of an X-ray diffraction pattern. Results of FA:AA (1:2.5) solubility increased in the range of 5500-6000 times compared with pure FA. In addition to solubility enhancement, the binary systems presented morphological properties that depend on the preparation method and whose consideration could be strategic for scaling purposes.

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Almost half of orally administered active pharmaceutical ingredients (APIs) have low solubility, which affects their bioavailability. In the last two decades, several alternatives have been proposed to modify the crystalline structure of APIs to improve their solubility; these strategies consist of inducing supramolecular structural changes in the active pharmaceutical ingredients, such as the amorphization and preparation of co-crystals or polymorphs. Since many APIs are thermosensitive, non-thermal emerging alternative techniques, such as mechanical activation by milling, have become increasingly common as a preparation method for drug formulations. This review summarizes the recent research in preparing pharmaceutical formulations (co-amorphous, co-crystals, and polymorphs) through ball milling to enhance the physicochemical properties of active pharmaceutical ingredients. This report includes detailed experimental milling conditions (instrumentation, temperature, time, solvent, etc.), as well as solubility, bioavailability, structural, and thermal stability data. The results and description of characterization techniques to determine the structural modifications resulting from transforming a pure crystalline API into a co-crystal, polymorph, or co-amorphous system are presented. Additionally, the characterization methodologies and results of intermolecular interactions induced by mechanical activation are discussed to explain the properties of the pharmaceutical formulations obtained after the ball milling process.

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BACKGROUND: Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose. RESULTS: The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h-1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h-1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h-1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L. CONCLUSION: The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

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Phenolic compounds from fruits and vegetables have shown antioxidant, anticancer, anti-inflammatory, among other beneficial properties for human health. All these benefits have motivated multiple studies about preserving, extracting, and even increasing the concentration of these compounds in foods. A diverse group of vegetable products treated with High Hydrostatic Pressure (HHP) at different pressure and time have shown higher phenolic content than their untreated counterparts. The increments have been associated with an improvement in their extraction from cellular tissues and even with the activation of the biosynthetic pathway for their production. The application of HHP from 500 to 600 MPa, has been shown to cause cell wall disruption facilitating the release of phenolic compounds from cell compartments. HPP treatments ranging from 15 to 100 MPa during 10-20 min at room temperature have produced changes in phenolic biosynthesis with increments up to 155%. This review analyzes the use of HHP as a method to increase the phenolic content in vegetable systems. Phenolic content changes are associated with either an immediate stress response, with a consequent improvement in their extraction from cellular tissues, or a late stress response that activates the biosynthetic pathways of phenolics in plants.

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Since conventional thermal processing can have detrimental consequences on aroma compounds, non-thermal technologies such as high hydrostatic pressure (HHP) have been explored. HHP may alter the weak chemical bonds of enzymes. These changes can modify the secondary, tertiary, and quaternary structures of key enzymes in the production of aroma compounds. This can result in either an increase or decrease in their content, along with reactions or physical processes associated with a reduction of molecular volume. This article provides a comprehensive review of HHP treatment's effects on the content of lipid-derived aroma compounds, aldehydes, alcohols, ketones, esters, lactones, terpenes, and phenols, on various food matrices of vegetable and animal origin. The content of aldehydes and ketones in food samples increased when subjected to HHP, while the content of alcohols and phenols decreased, probably due to oxidative processes. Both ester and lactone concentrations appeared to decline due to hydrolysis reactions. There is no clear tendency regarding terpenes concentration when subjected to HHP treatments. Because of the various effects of HHP on aroma compounds, an area of opportunity arises to carry out future studies that allow optimizing and controlling the effect.

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The substitution of wheat gluten in the food industry is a relevant research area because the only known treatment for celiac disease is abstinence from this protein complex. The use of gluten-free cereals in dough systems has demonstrated that the viscoelastic properties of gluten cannot be achieved without the modification of the protein fraction. The quality of the final product is determined by the ability of the modification to form a matrix similar to that of gluten and to reach this, different methods have been proposed and tested. These procedures can be classified into four main types: chemical, enzymatic, physical, and genetic. This article provides a comprehensive review of the most recent research done in protein modification of cereal and pseudocereals for gluten substitution. The reported effects and methodologies for studying the changes made with each type of modification are described; also, some opportunity areas for future works regarding the study of the effect of protein modifications on gluten-free products are presented.

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The melanins constitute a diverse group of natural products found in most organisms, having functions related to protection against chemical and physical stresses. These products originate from the enzyme-catalyzed oxidation of phenolic and indolic substrates that polymerize to yield melanins, which include eumelanin, pheomelanin, pyomelanin, and the allomelanins. The enzymes involved in melanin formation belong mainly to the tyrosinase and laccase protein families. The melanins are polymeric materials having applications in the pharmaceutical, cosmetic, optical, and electronic industries. The biotechnological production of these polymers is an attractive alternative to obtaining them by extraction from plant or animal material, where they are present at low concentrations. Several species of microorganisms have been identified as having a natural melanogenic capacity. The development and optimization of culture conditions with these organisms has resulted in processes for generating melanins. These processes are based on the conversion of melanin precursors present in the culture medium to the corresponding polymers. With the application of genetic engineering techniques, it has become possible to overexpress genes encoding enzymes involved in melanin formation, mostly tyrosinases, leading to an improvement in the productivity of melanogenic organisms, as well as allowing the generation of novel recombinant microbial strains that can produce diverse types of melanins. Furthermore, the metabolic engineering of microbial hosts by modifying pathways related to the supply of melanogenic precursors has resulted in strains with the capacity of performing the total synthesis of melanins from simple carbon sources in the scale of grams. In this review, the latest advances toward the generation of recombinant melanin production strains and production processes are summarized and discussed.

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One-third of the population of the USA suffers from metabolic syndrome (MetS). Treatment of patients with MetS regularly includes drugs prescribed simultaneously to treat diabetes and cardiovascular diseases. Therefore, the development of novel multidrug formulations is recommended. However, the main problem with these drugs is their low solubility. The use of binary co-amorphous systems emerges as a promising strategy to increase drug solubility. In the present study, irbesartan (IBS) and glimepiride (GMP), class II active pharmaceutical ingredients (API), widely used in the treatment of arterial hypertension and diabetes, were selected to develop a novel binary co-amorphous system with remarkable enhancement in the dissolution of both APIs. The phase diagram of IBS-GMP was constructed and co-amorphous systems were prepared by melt-quench, in a wide range of compositions. Dissolution profile (studied at pH 1.2 and 37°C for mole fractions 0.01, 0.1, and 0.5) demonstrated that the xGMP = 0.01 formulation presents the highest enhancement in its dissolution. GMP went from being practically insoluble to reach 3.9 ± 0.9 µg/mL, and IBS showed a 12-fold increment with respect to the dissolution of its crystalline form. Infrared studies showed that the increase in the dissolution profile is related to the intermolecular interactions (hydrogen bonds), which were dependent of composition. Results of structural and thermal characterization performed by XRD and DSC showed that samples have remained in amorphous state for more than 10 months of storage. This work contributes to the development of a highly soluble co-amorphous drugs with potential used in the treatment of MetS.

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The high index of simultaneous incidence of hypertension and hypercholesterolemia in the population of many countries demands the preparation of more efficient drugs. Therefore, there is a significant area of opportunity to provide as many alternatives as possible to treat these illnesses. Taking advantage of the solubility enhancement that can be achieved when an active pharmaceutical ingredient (API) is obtained and stabilized in its amorphous state, in the present work, new drug-drug co-amorphous formulations (Simvastatin SIM- Nifedipine NIF) with enhanced solubility and stability were prepared and characterized. Results show that the co-amorphous system (molar ratio 1:1) is more soluble than the pure commercial APIs studied separately. Aqueous dissolution profiles showed increments of solubility of 3.7 and 1.7 times for SIM and NIF, correspondingly, in the co-amorphous system. The new co-amorphous formulations, monitored in time, (molar fractions 0.3, 0.5 and 0.7 of SIM) remained stable in the amorphous state for more than one year when stored at room temperature and did not show any signs of crystallization when re-heating. Inspection on the remainder of a sample after six hours of dissolution showed no recrystallization, confirming the stability of co-amorphous system. The enhanced solubility of the co-amorphous formulations makes them promising for simultaneously targeting of hypertension and hypercholesterolemia through combination therapy.

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Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

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