An SPRI beads-based DNA purification strategy for flexibility and cost-effectiveness.

BACKGROUND: Current solid-phase reversible immobilization (SPRI) beads technology is widely used in molecular biology due to its convenience for DNA manipulation. However, the high performance commercial SPRI beads have no price advantage over our method. Furthermore, the use of commercially available SPRI beads standards does not provide the flexibility required for a number of specific nucleic acid handling scenarios. RESULTS: We report an efficient DNA purification strategy by combining home-made beads-suspension buffer with SPRI beads. The method tests the critical concentrations of polyethylene glycol (PEG) 8000 and beads to maximise recovery. And the composition of the SPRI beads DNA purification system (SDPS) was determined at 20% PEG 8000, 2 M NaCl and 16.3 mM MgCl2, and 1.25 mg/ml beads (1/8th original concentration). Then, we tested the DNA recovery of the SDPS, and the result showed that it was comparable to the control (AMPure XP beads). In the study, we have also developed an adjustment SPRI beads DNA purification system (ASDPS), the volume of ASDPS per reaction is 0.6× reaction volume (beads/samples). The performance of ASDPS is similar to SDPS and the control. But the cost of our methods is only about 1/24th of the control. To further assess its performance, we prepare the DNA-seq libraries to evaluate the yield, library quality, capture efficiency and consistency. We have compared all these results with the performance of the control and confirmed its efficiency. CONCLUSION: We have proposed an alternative DNA purification approach with great flexibility, allowing researchers to manipulate DNA in different conditions. And ultimately, its application will benefit molecular biology research in the future.

Development of a high-yield, high-quality purification process for adeno-associated virus vectors that can be used in vivo without ultracentrifugation: Application to a lung endothelial cell-targeted adeno-associated virus.

Recombinant adeno-associated viruses (rAAVs) are useful vectors for expressing genes of interest in vivo because of their low immunogenicity and long-term gene expression. Various mutations have been introduced in recent years and have enabled high-efficacy, stabilized, and organ-oriented transduction. Our purpose for using rAAV is to express our target gene in the mouse lung to investigate pulmonary artery hypertension. We constructed a self-complementary AAV having mutant capsids with the ESGHGYF insert, which directs the vectors to lung endothelial cells. However, when this mutant virus was purified from the producing cells by the conventional method using an ultracentrifuge, it resulted in a low yield. In addition, the purification method using an ultracentrifuge is tedious and labor-intensive. Therefore, we aimed to develop a simple, high-quality method for obtaining enough lung-targeted rAAV. First, we modified amino acids (T491V and Y730F) of the capsid to stabilize the rAAV from degradation, and we optimized culture conditions. Next, we noticed that many rAAVs were released from the cells into the culture medium. We, therefore, improved our purification method by purifying from the culture medium without the ultracentrifugation step. Purification without ultracentrifugation had the problem that impurities were mixed in, causing inflammation. However, by performing PEG precipitation and chloroform extraction twice, we were able to purify rAAV that caused only as little inflammation as that obtained by the ultracentrifuge method. Sufficient rAAV was obtained and can now be administered to a rat as well as mice from a single dish: 1.50 × 1013 ± 3.58 × 1012 vector genome from one φ150 mm dish (mean ± SEM).

Tissue-specific isolation of Arabidopsis/plant mitochondria - IMTACT (isolation of mitochondria tagged in specific cell types).

Plant cells contain numerous subcompartments with clearly delineated metabolic functions. Mitochondria represent a very small fraction of the total cell volume and yet are the site of respiration and thus crucial for cells throughout all developmental stages of a plant's life. As such, their isolation from the rest of the cellular components is a basic requirement for numerous biochemical and physiological experiments. Although procedures exist to isolate plant mitochondria from different organs (i.e. leaves, roots, tubers, etc.), they are often tedious and do not provide resolution at the tissue level (i.e. phloem, mesophyll or pollen). Here, we present a novel method called IMTACT (isolation of mitochondria tagged in specific cell types), developed in Arabidopsis thaliana (Arabidopsis) that involves biotinylation of mitochondria in a tissue-specific manner using transgenic lines expressing a synthetic version of the OM64 (Outer Membrane 64) gene combined with BLRP and the BirA biotin ligase gene. Tissue specificity is achieved with cell-specific promoters (e.g. CAB3 and SUC2). Labeled mitochondria from crude extracts are retained by magnetic beads, allowing the simple and rapid isolation of highly pure and intact organelles from organs or specific tissues. For example, we could show that the mitochondrial population from mesophyll cells was significantly larger in size than the mitochondrial population isolated from leaf companion cells. To facilitate the applicability of this method in both wild-type and mutant Arabidopsis plants we generated a set of OM64-BLRP one-shot constructs with different selection markers and tissue-specific promoters.

Comparison of different methods for the isolation and purification of rat nucleus pulposus-derived mesenchymal stem cells.

PURPOSE: Recently, nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have been identified and have shown good prospects for the repair of degenerative intervertebral discs. However, there is no consensus about the methods for the isolation and purification of NPMSCs. Therefore, a reliable and efficient isolation and purification method is potentially needed. We aimed to compare different methods and to identify an optimal method for isolating and purifying NPMSCs. METHODS: NPMSCs were isolated and purified using two common methods (a low-density culture (LD) method and a mesenchymal stem cell complete medium culture (MSC-CM) method) and two novel methods (a cloning cylinder (CC) method and a combination of the CC and MSC-CM methods (MSC-CM+CC)). The morphology, MSC-specific surface markers (CD44, CD73, CD90, CD105, CD34 and HLA-DR), multiple-lineage differentiation potential, colony formation ability, and stemness gene (Oct4, Nanog, and Sox2) expression were evaluated and compared. RESULTS: NPMSCs isolated from nucleus pulposus (NP) tissues via the four methods met the criteria stated by the International Society of Cell Therapy (ISCT) for MSCs, including adherent growth ability, MSC-specific surface antigen expression, and multi-lineage differentiation potential. In particular, the MSC-CM+CC method yielded a relatively higher quality of NPMSCs in terms of cell surface markers, multiple-lineage differentiation potential, colony formation ability, and stemness gene expression. CONCLUSIONS: Our results indicated that NPMSCs can be obtained via all four methods and that the MSC-CM+CC method is more reliable and efficient than the other three methods. The findings from this study provide an alternative option for isolating and purifying NPMSCs.

A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli.

To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6 × His-TF-CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF-CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase-human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25 mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.

Purification and Immunoprotection Evaluation of AaHIV from Complex Venom Metalloproteinases of Deinagkistrodon acutus.

The aim of this study was to investigate the immunoprotective effects of AaHIV in mice. After purification, a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Bicinchoninic acid was used to determine the molecular weight and concentration of AaHIV. AaHIV, venom complex (VC), and phosphate buffered saline (PBS) were subsequently used to immunize the mice three times, and the blood was sampled 1 week after the third immunization to determine the serum immunoglobulin G (IgG) antibody titer. A skin-bleeding inhibition assay and toxin-eliminating assay were performed on the immunized mice. The purity and concentration of AaHIV were 86.6% and 1.20 mg/mL, respectively. The AaHIV group exhibited higher antibody titers than the VC group. The survival rate of the AaHIV group (7/10) was significantly higher than that of the PBS group (0/10) (P = 0.0031). The high titer of antibodies induced by AaHIV partially neutralized the bleeding activity of the Deinagkistrodon acutus venom complex.

Highly efficient and easy protease-mediated protein purification.

As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate.

PA tag: a versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin.

Peptide-based epitope tagging technology is universally used in nearly all kind of research projects that involve biochemical characterization of a target protein, but not many systems are fully compatible with purification purpose. By utilizing an anti-human podoplanin antibody NZ-1, we constructed a novel epitope tag system. NZ-1 possesses exceptionally high affinity toward a dodecapeptide dubbed "PA tag", with a characteristic slow dissociation kinetics. Because of its high affinity, PA-tagged proteins in a dilute sample can be captured by immobilized NZ-1 resin in a near complete fashion and eluted by a solution of free PA peptide. This enabled efficient one-step purification of various proteins including soluble (an ectodomain fragment of neuropilin-1) and membrane (epidermal growth factor receptor) proteins expressed in mammalian cells. Mild regeneration condition of the peptide-bound antibody ensures repeated use of the antibody resin, indicating a cost-efficient nature of the system. Together with its outstanding performance in the immunodetection experiments (i.e., Western blotting and flow cytometry), PA tag/NZ-1 system will offer a great chance to facilitate protein production in many biomedical research projects.

[Opening experiments: synthesis, purification, and characterization of polyethyleneimine-modified carbon dots].

Open experiments are an effective means of cultivating top-notch innovative talents. Based on student interests, research hotspots and our laboratory conditions, an experimental scheme was designed. In this experiment, polyethyleneimine modified carbon dots (PEI-CDs) were prepared via a one-step hydrothermal method using citric acid (CA) as the carbon source and PEI as the surface passivator. First, CA and PEI were completely dissolved in 0.1 mol/L HCl and transferred into an autoclave. The autoclave was heated to 130 ℃ for 2 h. PEI-CDs solution was obtained. After cooling to room temperature, the solution was concentrated to 2 mL by rotary evaporation. Finally, the PEI-CDs were precipitated, washed with ethanol, and dried under vacuum at 70 ℃ for 12 h. The obtained PEI-CDs were characterized by fluorescence spectrophotometry, absorption spectrophotometry, infrared spectrometry, and transmission electron microscopy. The results indicated that anhydrous-ethanol precipitation is a simple, rapid, economical, and green purification method. The as-prepared PEI-CDs had unique properties, such as good water solubility, high luminescence, uniform particle sizes, and good stability. Through this open experiment, students can not only master the operation of large-scale instruments but also enhance their interest in scientific research.

A unified purification method for actin-binding proteins using a TEV-cleavable His-Strep-tag.

The actin cytoskeleton governs the dynamic functions of cells, ranging from motility to phagocytosis and cell division. To elucidate the molecular mechanism, in vitro reconstructions of the actin cytoskeleton and its force generation process have played essential roles, highlighting the importance of efficient purification methods for actin-binding proteins. In this study, we introduce a unified purification method for actin-binding proteins, including capping protein (CP), cofilin, ADF, profilin, fascin, and VASP, key regulators in force generation of the actin cytoskeleton. Exploiting a His-Strep-tag combined with a TEV protease cleavage site, we purified these diverse actin-binding proteins through a simple two-column purification process: initial purification through a Strep-Tactin column and subsequent tag removal through the reverse purification by a Ni-NTA column. Biochemical and microscopic assays validated the functionality of the purified proteins, demonstrating the versatility of the approach. Our methods not only delineate critical steps for the efficient preparation of actin-binding proteins but also hold the potential to advance investigations of mutants, isoforms, various source species, and engineered proteins involved in actin cytoskeletal dynamics.•Unified purification method for various actin-binding proteins.•His-Strep-tag and TEV protease cleavage for efficient purification.•Functional validation through biochemical and microscopic assays.

Development of RNA/DNA automated extraction and purification device for infectious disease diagnosis.

Genetic tests using RNA/DNA are the most accurate for diagnosing infectious diseases and assessing disease susceptibility, including COVID-19. However, manual specimen handling and the risk of secondary infections by medical staff highlight the need for automated equipment. Automation methods, such as bead purification, have limitations with high-viscosity specimens, while column purification requires complex equipment. This study aimed to develop an automated device using the column purification method for safe and reliable infectious disease diagnosis. We compared the yield and purification of three nucleic acid extraction methods (centrifugation, pressurization, and depressurization) and examined the adaptation of the extraction methods to automated device. Furthermore, we examined the feasibility of extracting SARS-CoV-2 RNA from COVID-19 patients and using qPCR analysis to determine whether the extraction method could be used as a clinical analyzer. Results varied with different columns and reagents, but pressurization method was selected for the automated device's RNA/DNA extraction. Using an automated device equipped with a pressurization method, RNA extracted from pharyngeal fluids from COVID-19 patients who had already been diagnosed with SARS-CoV-2 by qRT-PCR again tested positive. These findings demonstrate the device's effectiveness for nucleic acid extraction and virus-targeted diagnostics. Moreover, it holds potential for genetic testing in fields like food and environmental measurements. The automated device addresses specimen handling challenges and provides a reliable tool for infectious disease diagnosis.

Vesicles as antibiotic carrier: State of art.

Antimicrobial resistance (AMR) has become a global health problem. Bacteria are able to adapt to different environments, with the presence or absence of a host, forming colonies and biofilms. In fact, biofilm formation confers chemical protection to the microbial cells, thus making most of the conventional antibiotics ineffective. Prevention and destruction of biofilms is a challenging task that should be addressed by a multidisciplinary approach from different research fields. One of the medical strategies used against biofilms is the therapy with drug delivery systems. Lipidic nanovesicles are a good choice for encapsulating drugs, increasing their pharmacodynamics and reducing side effects. These soft nanovesicles show significant advantages for their high biocompatibility, physical and chemistry properties, good affinity with drugs, and easy route of administration. This review summarizes the current knowledge on different types of vesicles which may be used as antibiotic carriers. The main preparation and purification methods for the synthesis of these vesicles are also presented. The advantages of drug encapsulation are critically reviewed. In addition, recent works on endolysin formulations as novel, "greener" and efficient antibiofilm solution are included. This paper can provide useful background for the design of novel efficient formulations and synergistic nanomaterials and could be also useful at the pharmaceutical industry to develop wastewater treatments and reduce the antibiotics in the environmental waters.

Development of an adverse drug event network to predict drug toxicity.

Despite of their therapeutic effects, drug's exposure may have negative effects on human health such as adverse drug reaction (ADR) and side effects (SE). Adverse drug events (ADEs), that correspond to an event occurring during the drug treatment (i.e. ADR and SE), is not necessarily caused by the drug itself, as this is the case with medical errors and social factors. Due to the complexity of the biological systems, not all ADEs are known for marketed drugs. Therefore, new and effective methods are needed to determine potential risks, including the development of computational strategies. We present an ADE association network based on 90,827 drug-ADE associations between 930 unique drug and 6221 unique ADE, on which we implemented a scoring system based on a pull-down approach for prediction of drug-ADE combination. Based on our network, ADEs proposed for three drugs, safinamide, sonidegib, rufinamide are further discussed. The model was able to identify, already known drug-ADE associations that are supported by the literature and FDA reports, and also to predict uncharacterized associations such as dopamine dysregulation syndrome, or nicotinic acid deficiency for the drugs safinamide and sonidegib respectively, illustrating the power of such integrative toxicological approach.

Production and characterization of no-carrier-added 161Tb as an alternative to the clinically-applied 177Lu for radionuclide therapy.

BACKGROUND: 161Tb is an interesting radionuclide for cancer treatment, showing similar decay characteristics and chemical behavior to clinically-employed 177Lu. The therapeutic effect of 161Tb, however, may be enhanced due to the co-emission of a larger number of conversion and Auger electrons as compared to 177Lu. The aim of this study was to produce 161Tb from enriched 160Gd targets in quantity and quality sufficient for first application in patients. METHODS: No-carrier-added 161Tb was produced by neutron irradiation of enriched 160Gd targets at nuclear research reactors. The 161Tb purification method was developed with the use of cation exchange (Sykam resin) and extraction chromatography (LN3 resin), respectively. The resultant product (161TbCl3) was characterized and the 161Tb purity compared with commercial 177LuCl3. The purity of the final product (161TbCl3) was analyzed by means of γ-ray spectrometry (radionuclidic purity) and radio TLC (radiochemical purity). The radiolabeling yield of 161Tb-DOTA was assessed over a two-week period post processing in order to observe the quality change of the obtained 161Tb towards future clinical application. To understand how the possible drug products (peptides radiolabeled with 161Tb) vary with time, stability of the clinically-applied somatostatin analogue DOTATOC, radiolabeled with 161Tb, was investigated over a 24-h period. The radiolytic stability experiments were compared to those performed with 177Lu-DOTATOC in order to investigate the possible influence of conversion and Auger electrons of 161Tb on peptide disintegration. RESULTS: Irradiations of enriched 160Gd targets yielded 6-20 GBq 161Tb. The final product was obtained at an activity concentration of 11-21 MBq/µL with ≥99% radionuclidic and radiochemical purity. The DOTA chelator was radiolabeled with 161Tb or 177Lu at the molar activity deemed useful for clinical application, even at the two-week time point after end of chemical separation. DOTATOC, radiolabeled with either 161Tb or 177Lu, was stable over 24 h in the presence of a stabilizer. CONCLUSIONS: In this study, it was shown that 161Tb can be produced in high activities using different irradiation facilities. The developed method for 161Tb separation from the target material yielded 161TbCl3 in quality suitable for high-specific radiolabeling, relevant for future clinical application.

Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells Can Be Enriched by a Combination of Mitochondrial Content and Activated Leukocyte Cell Adhesion Molecule.

INTRODUCTION: Non-genetic purification methods for pluripotent stem cell-derived hepatocyte-like cells are useful for liver regenerative therapy and pharmaceutical applications. METHODS: Fluorescent activated cell sorting (FACS) was used to separate cells by combining two parameters: cellular mitochondrial content evaluated by the mitochondrial membrane potential-dependent fluorescent probe (TMRM) and immunocytochemical detection of activated leukocyte cell adhesion molecule (ALCAM). This method was applied to murine fetal, human embryonic stem cell (ESC)-derived, and human induced pluripotent stem cell (iPSC)-derived cell-mixtures. Separately sorted cell fractions were evaluated by quantitative PCR, immunohistochemistry, and cytochemistry for HNF4a, AFP, and albumin mRNA and/or protein expression. RESULTS: Hepatocyte-like cells were segregated into the high TMRM signal and ALCAM-positive population. The purity of hepatocyte-like cells derived from human iPSCs was 97 ± 0.38% (n = 5). CONCLUSIONS: This hepatocyte-like cell purification method may be applicable to the quality control of cells for liver regenerative cell therapy and pharmaceutical development.

A Facile and Low-Cost Method to Produce Ultrapure 99.99999% Gallium.

As one of the critical raw materials, very pure gallium is important for the semiconductor and photoelectric industry. Unfortunately, refining gallium to obtain a purity that exceeds 99.99999% is very difficult. In this paper, a new, facile and efficient continuous partial recrystallization method to prepare gallium of high purity is investigated. Impurity concentrations, segregation coefficients, and the purification effect were measured. The results indicated that the contaminating elements accumulated in the liquid phase along the crystal direction. The order of the removal ratio was Cu > Mg > Pb > Cr > Zn > Fe. This corresponded to the order of the experimentally obtained segregation coefficients for each impurity: Cu < Mg < Pb < Cr < Zn < Fe. The segregation coefficient of the impurities depended strongly on the crystallization rate. All observed impurity concentrations were substantially reduced, and the purity of the gallium obtained after our refinement exceeded 99.99999%.

A simple cleanup method for the removal of humic substances from soil protein extracts using aluminum coagulation.

Soil proteomics, the large-scale characterization of the entire protein complement in soils, provides a promising approach for deciphering the role of microbial functioning in terrestrial ecosystems. However, the extraction of soil proteins in sufficient quantities and of adequate purity remains a challenging task mainly due to the co-extraction of interfering humic substances. Up to now, the treatment of soil extracts with liquid phenol has been the "gold standard" for reducing humics, while the NoviPure cleanup kit was recently launched as a non-toxic approach. The present study describes an alternative method for delivering high-purity proteins based on humic coagulation with trivalent aluminum ions (Al3+). Various experimental parameters were optimized individually in order to maximize protein yield and diminish co-extracted humics. The optimized method was applied on a set of soil samples with diverse physicochemical characteristics and a comparison with the other two techniques was conducted. The amount of residual humics resulting from Al3+-based method was 26 and 35% higher than that from phenol treatment and NoviPure Kit, respectively, but these differences were of marginal statistical significance. With regard to extracted proteins, the average yields of the three methods were comparable, without showing any statistically significant differences. Overall, humic coagulation with Al3+ offers comparable cleanup performance in terms of protein yield and purity, but it is less toxic and less complex than the phenol-partitioning method, whereas it is far less expensive than the NoviPure Kit. The new technique is expected to facilitate the implementation of proteomic studies in soils.

Optimización de la extracción alcohólica para la obtención de soluciones concentradas de propóleos / Optimization of the alcoholic extraction to obtain concentrated solutions of propolis

Introducción: existen distintos métodos para la obtención de soluciones concentradas de propóleos, utilizados por diferentes autores tanto en el ámbito nacional como internacional. En este trabajo se propone una variante del método Pichansky que consiste en una secuencia de pasos de extracción/filtración, para optimizar el proceso de obtención de soluciones alcohólicas concentradas de propóleos en el Centro de Inmunología y Biopreparados de Holguín. Objetivo: optimizar el proceso de obtención de las soluciones alcohólicas concentradas de propóleos que contribuya a eliminar contaminantes disueltos y elevar la calidad del producto final. Materiales y métodos: para la obtención de las soluciones se emplearon tres lotes experimentales y se realizaron tres extracciones alcohólicas sucesivas con filtraciones intermedias a temperatura controlada. Los parámetros de calidad se determinaron según procedimientos del Sistema de Gestión implantado, realizándose una comparación entre los lotes experimentales y los lotes elaborados posteriormente en el proceso de producción utilizando la misma variante del método de extracción. La información se procesó estadísticamente mediante un análisis de medias con el software Medcal. Resultados: en los resultados se obtuvieron valores óptimos de concentración de sólidos totales superior al 30% para todos los lotes ensayados de las soluciones concentradas, las propiedades organolépticas se encuentran dentro de los estándares de calidad establecidos. Conclusiones: se logró optimizar el proceso de obtención de la solución concentrada de propóleos manteniendo la conformidad del producto, lográndose un mejor agotamiento de la materia prima, teniendo en cuenta la incorporación de un tercer paso en el proceso de elaboración y la filtración a temperatura controlada.

Introduction: There are different methods to obtain concentrated propolis solution that have used by different national and international authors. In this document a variant of Pichansky method is proposed to optimize the process for obtaining concentrated propolis in the Immunology and Blood by Product Center. Objective: To optimize the process of obtaining concentrated propolis alcoholic solutions through a sequence of steps of extraction/filtration to remove dissolved contaminants and increase the quality of the final product. Materials and methods: To obtain solutions, three experimental plots were performed. This process was executed with three successive extractions using alcohol intermediate to temperature controlled. The quality parameters were determined according to procedures implemented of management system. A comparison was performed between experimental batches and batches subsequently produced with the change. The information was processed statistically by means of (means t-test) with Medcal software. Results: The optimal values of volume of total solids concentration above 30% were obtained for all tested batches of concentrated solutions, the organoleptic properties were found according to the standard quality. Conclusions: It was possible to optimize the process of obtaining the propolis concentrated solution of maintaining product compliance, achieving a better recovery of the first product due to considering the addition of a third step in the process with a controlled temperature.