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L-asparaginase (ASNase) is an aminohydrolase enzyme widely used in the pharmaceutical and food industries. Although currently its main applications are focused on the treatment of lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and acrylamide reduction in starch-rich foods cooked at temperatures above 100 °C, its use as a biosensor in the detection and monitoring of L-asparagine levels is of high relevance. ASNase-based biosensors are a promising and innovative technology, mostly based on colorimetric detection since the mechanism of action of ASNase is the catalysis of the L-asparagine hydrolysis, which releases L-aspartic acid and ammonium ions, promoting a medium pH value change followed by color variation. ASNase biosensing systems prove their potential for L-asparagine monitoring in ALL patients, along with L-asparagine concentration analysis in foods, due to their simplicity and fast response.

Made available in DSpace on 2022-04-28T19:47:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-07 Despite being a vital asset for global sustenance and economy, ocean aquatic ecosystems are in danger due to the effects of incorrect management of their resources, pollution, and climate change. Considering the seafood industry discards half of its fish-product mass in the ocean, a proper valorization of its residues would decrease not only the ocean contamination but also improve the management of marine resources and increase the sector competitiveness. With these goals in mind, ocean-based industries are adopting new sustainable production models, similar to biorefineries, which are effective for waste valorization, namely, converting low-value biomass into commercially relevant by-products. Based on a deeper knowledge of aquatic feedstocks, the development and implementation of a marine biorefinery can be fundamental to consolidate a greenersocioeconomic development, similar to that observed in green chemistry. However, biorefineries are sophisticated multi-step systems with numerous feedstocks and commodities. Therefore, their implementation requires expertise in all stages of manufacturing, in addition to a clear vision of all raw materials, residues, and products. In this sense, with this perspective, we provide an initial overview of the current state-of-the-art on marine biorefineries and the sources and applications of their by-products. Afterward, we suggest how to integrate green chemistry and blue economy principles into ocean-based industries, aiming to support a more sustainable, profitable, and conscious ocean economy. This journal is School of Pharmaceutical Sciences São Paulo University (USP) Cidade de Universitária, Av. Prof. Lineu Prestes, no. 580, B16 Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01 Univ Coimbra CIEPQPF Department of Chemical Engineering, Rua Sílvio Lima, Pólo II-Pinhal de Marrocos Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01

Ionic liquids (ILs) and deep eutectic solvents (DESs) debuted with a promise of a superior sustainability footprint due to their low vapor pressure. However, their toxicity and high cost compromise this footprint, impeding their real-world applications. Fortunately, their property tunability through a rational selection of precursors, including bioderived ones, provides a strategy to ameliorate toxicity, lower cost, and endow new functions. This Review discusses whether ILs and DESs are sustainable solvents and how they contribute to sustainable chemical processes.

Made available in DSpace on 2022-04-28T19:44:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Lipase is an important group of biocatalysts, which combines versatility and specificity, and can catalyze several reactions when applied in a high amount of industrial processes. In this study, the lipase produced by Aspergillus japonicus under submerged cultivation, was immobilized by physical adsorption, using clay supports, namely, diatomite, vermiculite, montmorillonite KSF (MKSF) and kaolinite. Besides, the immobilized and free enzyme was characterized, regarding pH, temperature and kinetic parameters. The most promising clay support was MKSF that pre-sented 69.47% immobilization yield and hydrolytic activity higher than the other conditions studied (270.7 U g−1). The derivative produced with MKSF showed high stability at pH and temperature, keeping 100% of its activity throughout 12 h of incubation in the pH ranges between 4.0 and 9.0 and at a temperature from 30 to 50◦ C. In addition, the immobilized lipase on MKSF support showed an improvement in the catalytic performance. The study shows the potential of using clays as support to immobilized lipolytic enzymes by adsorption method, which is a simple and cost-effective process. Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) Department of Pharmaceutical-Biochemical Technology School of Pharmaceutical Sciences University of São Paulo Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) CAPES: 2017/11482-7 FAPESP: 2017/11482-7 CAPES: 2018/06908-8 FAPESP: 2018/06908-8 CAPES: 2019/15493-9 FAPESP: 2019/15493-9 CAPES: 2020/08655-0 FAPESP: 2020/08655-0 CAPES: 2020/09592-1 FAPESP: 2020/09592-1

Made available in DSpace on 2021-06-25T10:25:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Selective recovery and purification of carotenoids and lipids from oleaginous red yeast Rhodotorula glutinis require a complex integration of different downstream operations. Envisioning a future industrial application in the bioeconomy framework, an experimental planning was used to design ternary mixtures of bio-based solvents (biosolvents) for simultaneous recovery and selective separation of carotenoids (i.e., β-carotene and torularhodin) and fatty acids (i.e., margaric acid, stearic acid and pentadecylic acid) from untreated (fresh wet) and freeze/thaw pre-treated (wet and dry) biomass of Rhodotorula glutinis CCT-2186. Two ternary systems of biosolvents, (i) ethanol:ethyl lactate:water and ii) ethanol:ethyl acetate:water, were investigated in solid–liquid extraction procedures to permeabilize/disintegrate the yeast cells and to enhance the selective recovery of β-carotene, torularhodin and fatty acids from the intracellular environment. Optimization of the ternary mixture composition for recovery of carotenoids and fatty acids from the three types of biomass allowed to obtain yields higher than 75% (w/w) from pre-treated dry biomass using mixture of ethanol:ethyl acetate:water (67:33:00% w/w/w). The best systems were then integrated with a liquid–liquid extraction for the selective separation of carotenoids and fatty acids and solvent recycling. The mixtures of biosolvents were reused up to three consecutive extraction cycles, maintaining high extraction efficiency yields of both carotenoids (>45% w/w) and lipids (>30% w/w). At the end, the biological activity of the carotenoids extracts was confirmed by determining the antioxidant activity using Electron Paramagnetic Resonance (EPR) and the stable free radical 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH·). The highest antioxidant activity (93%) was obtained with torularhodin (250 μg/mL). This study confirms that extraction platforms using mixed biosolvents are simple, efficient and sustainable solutions for the selective recovery and separation of carotenoids and fatty acids from microbial biomass. Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) CICECO-Aveiro Institute of Materials Department of Chemistry University of Aveiro Univ Coimbra CIEPQPF Department of Chemical Engineering Rua Sílvio Lima Pólo II – Pinhal de Marrocos Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP)

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application

Made available in DSpace on 2022-04-29T08:34:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-03-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Laboratório Associado para a Química Verde Centro de Investigação em Materiais Cerâmicos e Compósitos Currently, the ability to produce several kinds of water-soluble red natural colorants makes the genus Talaromyces particularly important to the dye industry, which can be an alternative to the use of harmful synthetic colorants. In this study, colored compounds produced by Talaromyces amestolkiae were extracted, characterized chemically and the color stability of the fermented broth without any extraction procedure was further evaluated over pH variation. Five azaphilones compounds were detected by Ultrahigh Performance Liquid Chromatography-Mass Spectrometry system, all being complexes of the fatty acid amino-hexanedioic acid and azaphilone Monascus colorants. The color of the fermented broth was stable at a wide range of pH (3–9). Furthermore, T. amestolkiae colorants precipitated through hydrolysis of key chemical groups at extremely acidic (pH 1) and lose red color in extremely basic (pH 13) medium, showing negative halochromism. Nevertheless, these findings enhance the industrial relevance of azaphilone colorants produced by biotechnological process. Department of Engineering Bioprocess and Biotechnology School of Pharmaceutical Sciences São Paulo State University - UNESP CICECO – Aveiro Institute of Materials Chemistry Department University of Aveiro, Campus Universitário de Santiago LAQV - REQUIMTE Department of Chemistry and University of Aveiro Department of Drugs and Medicines School of Pharmaceutical Sciences São Paulo State University - UNESP Laboratory of Mass Spectrometry Applied to Natural Products Chemistry Department of Chemistry Faculty of Philosophy Sciences and Letters of Ribeirão Preto (FFCLRP) University of São Paulo (USP) Department of Engineering Bioprocess and Biotechnology School of Pharmaceutical Sciences São Paulo State University - UNESP Department of Drugs and Medicines School of Pharmaceutical Sciences São Paulo State University - UNESP FAPESP: 2014/01580-3 FAPESP: 2018/06908-8 FAPESP: 2019/15493-9 Laboratório Associado para a Química Verde: UIDB/50006/2020 Centro de Investigação em Materiais Cerâmicos e Compósitos: UIDB/50011/2020 Centro de Investigação em Materiais Cerâmicos e Compósitos: UIDP/50011/2020

Made available in DSpace on 2020-12-12T00:55:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-07 Rhodotorula glutinis (R. glutinis) yeasts are natural sources of intracellular carotenoids such as β-carotene, torularhodin, and torulene. Since these yeasts are constituted by a rigid cell-wall structure, the use of energy-saving and high-efficiency cell disruption procedures is critical for carotenoids recovery. A new technology using protic ionic liquids (PILs) was here evaluated as an alternative platform to permeabilize the R. glutinis cells and to improve the extraction of β-carotene, torularhodin, and torulene. The cell disruption ability of 12 highly concentrated aqueous solutions of ammonium-based PILs was determined, evaluating the influence of the relative ion hydrophobicity, solid-liquid ratio, water content, and temperature. Carotenoid extraction yields increased with the hydrophobicity of the PILs (i.e., increase of alkyl chain length of the anion or cation), temperature (from 25 to 65 °C), and PIL concentration (from 75 to 90% v/v). Additionally, to demonstrate the potential of PILs in carotenoids recovery, solvent recycling and carotenoids polishing were carried out using a three-phase partitioning system. The results demonstrate that the use of PILs as cell-disrupting agents can be a simple, efficient, sustainable, and feasible method to recover intracellular carotenoids from microbial biomass. Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences Sao Paulo State University (UNESP), Rodovia Araraquara Jaú, Km 01, Campos Ville Departamento de Ingeniería Química Industrial y Del Medio Ambiente ETS Ingenieros Industriales Universidad Politécnica de Madrid, C/José Gutiérrez Abascal 2 CICECO-Aveiro Institute of Materials Department of Chemistry University of Aveiro Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences Sao Paulo State University (UNESP), Rodovia Araraquara Jaú, Km 01, Campos Ville

Made available in DSpace on 2019-10-06T15:48:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-16 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The replacement of synthetic colors in food products by natural alternatives has been boosted by consumers willing to pay more for healthier products. However, the success of microbial colorants depends not only on its acceptability on the market but also its production costs. Talaromyces species can produce water-soluble red colorants induced by glucose and monosodium glutamate (MSG). In this study, the influence of several conditions was evaluated to produce natural red colorants by submerged culture of Talaromyces amestolkiae. Under optimal conditions (g/L: glucose 10, MSG 25, MgSO4 0.012, FeSO4 0.01, CaCl2 0.015; and initial pH of 5.0), a 30-fold increase in the production was achieved, reaching a red colorant production of 13.44 UA500nm. Depending on the initial pH, colorants with different hues and chroma values were obtained. Deep yellow colorants were derived from neutral and basic pH, while deep red colors were derived from acidic pH. The fluorescence spectrum of culture broth obtained before and after complexation with salts presented red colorants with yellow fluorescence spectra. The information generated in this study would be useful for the formulation of industrial media for large-scale cultivation of T. amestolkiae, which have the potential to produce Talaromyces fermented colorants for use in health foods and pharmaceutics. Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences Campus (Araraquara) Universidade Estadual Paulista – UNESP, Rodovia Araraquara-Jaú/ Km 01, Campos Ville - Araraquara/SP Culture Collection DPUA/UFAM Universidade Federal do Amazonas Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences Campus (Araraquara) Universidade Estadual Paulista – UNESP, Rodovia Araraquara-Jaú/ Km 01, Campos Ville - Araraquara/SP CAPES: 001 CNPq: 155317/2016-4 FAPESP: 2014/01580-3

Made available in DSpace on 2021-06-25T10:15:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-15 The green fluorescent protein (GFP) is a biomolecule used in many biological applications such as biomarkers and biosensors, which require high purity levels. It is usually obtained from recombinant Escherichia coli strains, which also produces other endogenous proteins, demanding multiple purification steps, and consequently, increasing the overall costs to obtain pure GFP. Simpler and cheaper purification methods like Aqueous Biphasic Systems (ABS) were already successfully applied to purify GFP at lab scale. Therefore, the development of automatized industrially compatible purification platforms, such as countercurrent chromatography using ABS, can potentially improve the GFP production. This work studied the continuous purification of the variant enhanced GFP (EGFP) by applying ABS composed of polyethylene glycol (PEG 8000), sodium polyacrylate (NaPA 8000) and sodium sulfate (Na2SO4) as electrolyte. An initial screening was carried by changing the electrolyte content in the ABS. The increase of this condition has demonstrated an increase on the EGFP partition for the PEG-rich phase. The most efficient ABS and, at the same time, with the most appropriate conditions, namely the system composed of 15 wt% PEG 8000 + 4.5 wt% NaPA 8000 + 2.5 wt% Na2SO4 was chosen and applied on the fast centrifugal partition chromatography (FCPC). After optimization, the best operational conditions were identified, i.e. a flow rate of 2.5 mL.min−1 and rotation speed of 2000 rpm at ascending mode, and the best results obtained, meaning a purification of 89.93% and a recovery yield of 82.3%, confirming the potential of FCPC to the continuous purification of EGFP. CICECO – Aveiro Institute of Materials Department of Chemistry University of Aveiro Department of Biochemical and Pharmaceutical Technology University of São Paulo Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) Univ Coimbra CIEPQPF Department of Chemical Engineering, Rua Sílvio Lima, Pólo II – Pinhal de Marrocos Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP)

Astaxanthin (AXT) is a natural xanthophyll with strong antioxidant, anticancer and antimicrobial activities, widely used in the food, feed, pharmaceutical and nutraceutical industries. So far, 95% of the AXT global market is produced by chemical synthesis, but growing customer preferences for natural products are currently changing the market for natural AXT, highlighting the production from microbially-based sources such as the yeast Phaffia rhodozyma. The AXT production by P. rhodozyma has been studied for a long time at a laboratory scale, but its use in industrial-scale processes is still very scarce. The optimization of growing conditions as well as an effective integration of upstream-downstream operations into P. rhodozyma-based AXT processes has not yet been fully achieved. With this critical review, we scrutinized the main approaches for producing AXT using P. rhodozyma strains, highlighting the impact of using conventional and non-conventional procedures for the extraction of AXT from yeast cells. In addition, we also pinpointed research directions, for example, the use of low-cost residues to improve the economic and environmental sustainability of the bioprocess, the use of environmentally/friendly and low-energetic integrative operations for the extraction and purification of AXT, as well as the need of further human clinical trials using yeast-based AXT.

Made available in DSpace on 2021-06-25T10:18:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-07 Natural bioactive compounds have been attracting growing interest from the industries as a greeneralternative to synthetic raw materials/products. Rhodotorula glutinis yeast naturally synthesizes added value compounds such as lipids and carotenoids, commonly used for cosmetic, pharmaceutical, and food applications. R. glutinis constitutes a rigid cell-wall structure, requiring energy-saving and efficient cell disruption methods for a sustainable recovery of the intracellular compounds. A simple and ecofriendly technology using mixed bio-based solvents (biosolvents) was evaluated here as an alternative platform to permeabilize yeast cells and to improve the selective recovery of β-carotene, torularhodin, torulene and lipids. The extraction ability of pure and solvent mixtures (methanol, ethanol, ethyl acetate, isopropanol, cyclohexane and 2-methyl tetrahydrofuran) was initially screened, demonstrating the clear impact of using mixtures to improve the extraction yields (up to three-fold increase). After identifying ethyl acetate/ethanol/water as the solvent mixture with a greater capacity to extract carotenoids and lipids, the selective recovery of carotenoids and lipids was enhanced by optimizing the solvent mixture composition ratio. Envisioning the industrial application, an integrated biosolvent-based downstream platform was designed. Two different strategies were investigated to further isolate carotenoids and lipids from R. glutinis biomass and to recycle the ethyl acetate/ethanol/water mixture: (i) precipitation using cold acetone; (ii) sequential liquid-liquid extraction. The integrated process for each strategy was compared with a conventional extraction procedure in terms of recovery efficiencies and its environmental impact. Regardless of the strategy, it is shown that the mixture of ethyl acetate, ethanol and water (15/27/58% w/w) can be reused up to three consecutive extractive cycles, ensuring high extraction efficiency yields, while decreasing the process carbon footprint by about 75% compared to the conventional method. Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences Saõ Paulo State University (UNESP) CICECO Aveiro Institute of Materials Department of Chemistry University of Aveiro Department of Marine Faculty of Fisheries and Marine Universitas Airlangga Jalan, Mulyorejo Kampus C CESAM Centre for Environmental and Marine Studies Department of Environment and Planning University of Aveiro Univ Coimbra CIEPQPF Department of Chemical Engineering, Rua Sílvio Lima Pólo II-Pinhal de Marrocos Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences Saõ Paulo State University (UNESP)

Made available in DSpace on 2021-06-25T10:15:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Natural colorants from microbial fermentation have gained significant attention in the market to replace the synthetic ones. Talaromyces spp. produce yellow-orange-red colorants, appearing as a potential microorganism to be used for this purpose. In this work, the production of natural colorants by T. amestolkiae in a stirred-tank bioreactor is studied, followed by its application as additives in bio-based films. The effect of the pH-shift control strategy from 4.5 to 8.0 after 96 h of cultivation is evaluated at 500 rpm, resulting in an improvement of natural colorant production, with this increase being more significant for the orange and red ones, both close to 4-fold. Next, the fermented broth containing the colorants is applied to the preparation of cassava starch-based films in order to incorporate functional activity in biodegradable films for food packaging. The presence of fermented broth did not affect the water activity and total solids of biodegradable films as compared with the standard one. In the end, the films are used to pack butter samples (for 45 days) showing excellent results regarding antioxidant activity. It is demonstrated that the presence of natural colorants is obtained by a biotechnology process, which can provide protection against oxidative action, as well as be a functional food additive in food packing biomaterials. Department of Engineering Bioprocess and Biotechnology School of Pharmaceutical Sciences Universidade Estadual Paulista—UNESP Faculty of Pharmaceutical Sciences University of Campinas—FCF/UNICAMP Laboratory of Biotechnology Applied to Infectious and Parasitic Diseases Biological Science Institute University of Pernambuco-ICB/UPE Department of Bromatological Analysis Faculty of Pharmacy Postgraduate Program in Science of Food Federal University of Bahia Department of Biochemical and Pharmaceutical Technology University of São Paulo Department of Engineering Bioprocess and Biotechnology School of Pharmaceutical Sciences Universidade Estadual Paulista—UNESP

l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.

Made available in DSpace on 2020-12-12T01:35:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fibrinolytic enzymes have been considered promising for treatment and protection of healthy circulation due its ability to dissolve the fibrin in blood clots. Extractive fermentation is a not explored and efficient downstream process which segregates the desired product simultaneously in a fermentation process fast and economically. Extraction of fibrinolytic enzymes by Bacillus stearothermophilus DPUA 1729 employing conventional aqueous two-phase systems (ATPS) and extractive fermentation with ATPS was evaluated. The results of both systems were compared using a factorial design with PEG molar mass, PEG and salt concentrations as independent variables and extraction parameters as a response. In all conditions evaluated it was observed a similar partitioning of fibrinolytic enzymes through the phases, both in conventional ATPS and extractive fermentation. Salt concentration and interaction among PEG and salt concentration influenced in the partition coefficient. The fibrinolytic activity was determined by hydrolysis of fibrin in plate using the extract of one condition from extractive fermentation. The zone degradation presented a diameter of 7.03 ± 0.94 mm. In conclusion, there was no significant difference among the results obtained using conventional ATPS and extractive fermentation, however, the second one presents more advantages and can integrate production and extraction in one single step, reducing the costs. Culture Collection DPUA/UFAM Federal University of Amazonas Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences UNESP-University Estadual Paulista Laboratory of Biotechnology Applied to Infectious and Parasitic Diseases Biological Science Institute University of Pernambuco Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences UNESP-University Estadual Paulista

This book chapter provides an overview on the use of nanoparticles in the biomedical field. Nanomaterials have been a topic of research due to their unique properties, which have shown to be of crucial relevance towards the development of innovative technologies within the biomedical field. Nanoparticles (NPs) can be prepared from a wide diversity of materials and modified with a plethora of functional groups or ligands, allowing them to display specific properties and to be applied in a range of applications. In this book chapter, both the methods to functionalize nanoparticles and their applications in the biomedical field are overviewed. Metal-based, silica and carbon NPs, and their use in membranes, are described. Since drug delivery systems are the main application of functionalized nanoparticles, a specific section devoted to this topic is also presented. published

Made available in DSpace on 2021-06-25T10:17:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Lipases are enzymes employed in several industrial process and their applicability can be increased if these biocatalysts are in the immobilize form. The objective of this work was to study the immobilization of lipase produced by submerged cultivation of Aspergillus sp. by hydrophobic interaction, evaluating its stability and reuse capacity. The immobilization process on octyl-sepharose (C8) and octadecyl-sepabeads (C18) carriers was possible after the removal of oil excess presented in the fermented broth. The results showed that the enzyme was isolated and concentrated in octyl-sepharose with 22% of the initial activity. To increase the amount of enzyme adsorbed on the carrier, 4 immobilization cycles were performed in a row, on the same carrier, with a final immobilization yield of 151.32% and an increase in the specific activity of 136%. The activity test with immobilized lipase showed that the immobilized enzyme maintained 75% of the initial activity after 20 cycles. Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) Department of Biocatalysis Institute of Catalysis (ICP-CSIC) Department of Pharmacy School of Pharmaceutical Sciences University of São Paulo (USP) Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP)

Made available in DSpace on 2018-12-11T16:48:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-08 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Over the last few years, synthetic colorants have been increasingly replaced by natural alternatives that cause fewer adverse health effects and can show biological activity. These biocompounds can be produced by fermentative processes using microorganisms such as bacteria and filamentous fungi. However, the biocompounds must be extracted from the fermented broth before industrial application. Thus, the development of effective techniques for the extraction and purification of biocolorants with high recovery is of great interest. The main goal of this study was to evaluate the recovery of natural colorants produced by Talaromyces amestolkiae using aqueous micellar two-phase system (AMTPS) composed of Triton X-114 and Ionic Liquids (ILs) from the imidazolium family as co-surfactants. Preliminary experiments were performed to determine the binodal curves in both presence and absence of fermented broth containing the red colorants. Subsequently, a set of partitioning experiments was performed using the developed systems. Under all conditions, the red colorants were partitioned mainly into the micelle-rich phase. The systems with low concentrations of Triton X-114 and ILs showed high partition coefficients. The system (0.1 wt% [C10mim]Cl + 3 wt% Triton X-114) with the highest partition coefficient (KRC = 6.12) was used to study the effect of temperature on partitioning. The partitioning of the red colorants was strongly influenced by the temperature, and KRC increased to 24.71 at 45 °C. The results of this study demonstrate that the partitioning of biomolecules can be controlled by the presence of ILs, mainly by electrostatic interactions, or by the proper adjustment of the partitioning temperature. Universidade Estadual Paulista (UNESP) School of Pharmaceutical Sciences Campus (Araraquara) Department of Bioprocess and Biotechnology Universidade Estadual Paulista (UNESP) School of Pharmaceutical Sciences Campus (Araraquara) Department of Bioprocess and Biotechnology FAPESP: 2014/01580-3 FAPESP: 2014/16424-7 FAPESP: 2015/04751-6 FAPESP: 2016/01076-9 FAPESP: 443984/2014-0

Made available in DSpace on 2018-12-11T16:50:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-01 The partitioning of bacteriocin, a promising alternative to chemical preservatives, in a novel inexpensive and stable aqueous two-phase system (ATPS) comprising poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The ATPS was generated by mixing both polymers with Na2SO4 or choline chloride ([Ch]Cl) and a bacteriocin extract from the fermented broth of Lactobacillus plantarum ST16Pa. Bacteriocin showed stability at different pH values (3.0–8.0) and temperatures (50–80 °C), as well as in the presence of ATPS components. Hydrophobic and electrostatic interactions were found to be the major driving forces for bacteriocin partitioning. The peptide partitioned preferentially to the PEG-rich phase (partition coefficient, KBact > 1). However, the highest partition coefficient was achieved in the polymeric-based ATPS using [Ch]Cl as additive, as follows: 8 wt% PEG 10,000 g/mol/8 wt% NaPA 8,000 g/mol/0.5 M [Ch]Cl, resulting in a KBact equal to 32. Moreover, these conditions promoted high selectivity (S = 62.7), since the greater part of total proteins partitioned into the NaPA-rich phase (Kprot = 0.51). In conclusion, ATPS composed of PEG/NaPA using choline-based salts as additive can be considered mainly as a first step for bacteriocin extraction from fermented broth by applying low polymer content and mild conditions. Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical Sciences University of São Paulo – FCF/USP Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences State University of São Paulo – FCFar/UNESP Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences State University of São Paulo – FCFar/UNESP

Biopharmaceuticals are one of the most important groups of biotechnological products, representing one-quarter of all pharmaceutical sales and providing suitable and efficient medical care for many previously untreatable diseases. However, their production and purification usually require complex processes, resulting in extremely expensive final products. Thus, the development of newer, simpler and cheaper methods for biosynthesis and purification of these biocompounds, as well as proper integration between these stages, are crucial to reduce their commercial costs and to increase the scale of biopharmaceuticals' medical use. One solution for this concern relies on the proper integration between upstream and downstream processing applying liquid–liquid extraction systems as an intermediate stage. Recently, several works reported that a proper choice of liquid–liquid systems such as aqueous biphasic systems (ABS) can be a suitable platform to simplify, reduce the number of the downstream stages, improve the recovery and purification yields and, consequently, decrease biopharmaceuticals' manufacturing costs. This review will explain the general concept of biopharmaceuticals, their main production and purification methods, particularly, exploring the use of ABS as effective and integrative platforms for their recovery and purification, and providing further insights into the future trends and prospects in the field. © 2017 Society of Chemical Industry

Made available in DSpace on 2018-12-11T16:48:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-25 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Federación Española de Enfermedades Raras Clavulanic acid (CA) is an important pharmaceutical compound produced by batch fermentation of Streptomyces clavuligerus. Since, CA is chemically unstable, its downstream processing should be studied to develop more efficient and resolute techniques. Herein, the use of aqueous two-phase systems (ATPS) composed of cholinium chloride, [Ch]Cl, was proposed as a novel platform for the recovery and purification of CA. Thus, the stability of CA in presence of different [Ch]Cl concentrations was initially studied, and the high biocompatibility of this salt was demonstrated by the low CA degradation levels. Then, the partitioning of CA using two types of polymeric ATPS has been investigated. Two ATPS formed by the combination of [Ch]Cl and two polyethylene glycol (PEG) polymers, PEG 600 g mol−1 (PEG-600) and polyethylene glycol methyl ether 550 g mol−1 (PEG-500-OMe), were used to assess the influence of the PEG nature, in addition to the concentration of the phase forming agents on the CA partitioning. It has shown that CA is almost equally distributed between the two-phases in equilibrium (0.6 < KCA < 1.6). Nevertheless, the selective extraction of CA for the [Ch]Cl-rich or PEG-rich phase by the proper adjustment of ATPS composition was attained. In the search for higher extraction efficiencies [EE (%)] and partition coefficients (KCA), a second polymeric ATPS platform composed of PEG-600 and sodium polyacrylate 8000 g mol−1 (NaPA-8000), applying [Ch]Cl as adjuvant was tested. The main results suggest the recovery of CA towards the PEG-rich phase (KCA ≥ 5.6 ± 0.6 and EE ≥ 85.5± 1.4%). The higher migration levels of CA have mainly resulted from the electronegative repulsion of NaPA-8000 over CA molecules. The ATPS with best performance for the CA extraction were selected for the recovery of CA directly from fermented broth of Streptomyces clavuligerus. In this set of experiments, the highest values of CA recovery yield and purification factor (respectively, 64.91± 1.99% and 22.70 ± 0.87) were attained for the systems PEG-600/NaPA-8000 and PEG-600/[Ch]Cl, respectively. Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences UNESP – Univ Estadual Paulista, Rodovia Araraquara-Jaú/01, Campos Ville Institute of Botany Leibniz Universität Hannover, Herrenhäuser Str. 2 CICECO – Aveiro Institute of Materials Department of Chemistry University of Aveiro Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences UNESP – Univ Estadual Paulista, Rodovia Araraquara-Jaú/01, Campos Ville FAPESP: 2014/01580-3 Federación Española de Enfermedades Raras: PT2020

Made available in DSpace on 2019-10-06T15:40:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-07-12 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This work studied the effect of the cation alkyl chain length of 1-alkyl-n-methylimidazolium chloride ([Cnmim]Cl)-based ILs on the activity of Aspergillus niger lipase. First, the lipase activity in the presence of different ILs concentration over time was determined. ILs with shorter cation alkyl side chain length, namely [C4mim]Cl and [C6mim]Cl, promoted an increase of lipase activity; while, [C8mim]Cl, depending on its concentration, maintained or decreased the enzyme activity. In the presence of ILs with longer cation alkyl chain length, i.e., [C10mim]Cl and [C12mim]Cl, the lipase relative activity was reduced with 0.1 (%v/v) and until suppressed ([C12mim]Cl at 0.3 (%v/v)) as a result of irreversible changes in its secondary structure. Fluorescence and circular dichroism spectroscopy analysis confirmed the results achieved. These findings show that [Cnmim]Cl-based ILs can exert different behavior on the lipase’ activity (enhance, maintain or even inhibit) and structural conformation, depending on the cation alkyl chain length and their relative concentration. Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/km 01 Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/km 01 CAPES: 001 CNPq: 140331/2016-6 FAPESP: 2014/01580-3 FAPESP: 2014/16424-7

Lipases are well-known biocatalysts used in several industrial processes/applications. Thus, as with other enzymes, changes in their surrounding environment and/or their thermodynamic parameters can induce structural changes that can increase, decrease, or even inhibit their catalytic activity. The use of ionic compounds as solvents or additives is a common approach for adjusting reaction conditions and, consequently, for controlling the biocatalytic activity of enzymes. Herein, to elucidate the effects of ionic compounds on the structure of lipase, the stability and enzymatic activity of lipase from Aspergillus niger in aqueous solutions (at 0.05, 0.10, 0.50, and 1.00 M) of six cholinium-based ionic liquids (cholinium chloride [Ch]Cl; cholinium acetate ([Ch][Ac]); cholinium propanoate ([Ch][Prop]); cholinium butanoate ([Ch][But]); cholinium pentanoate ([Ch][Pent]); and cholinium hexanoate ([Ch][Hex])) were evaluated over 24 hr. The enzymatic activity of lipase was maintained or enhanced in the lower concentrations of all the [Ch]+ -ILs (below 0.1 M). [Ch][Ac] maintained the biocatalytic behavior of lipase, independent of the IL concentration and incubation time. However, above 0.1 M, [Ch][Pent] and [Ch][Hex] caused complete inhibition of the catalytic activity of the enzyme, demonstrating that the increase in the anionic alkyl chain length strongly affected the conformation of the lipase. The hydrophobicity and concentration of the [Ch]+ -ILs play an important role in the enzyme activity, and these parameters can be controlled by adjusting the anionic alkyl chain length. The inhibitory effects of [Ch][Pent] and [Ch][Hex] may be of great interest to the pharmaceutical industry to induce pharmacological inhibition of gastric and pancreatic lipases.

Made available in DSpace on 2019-10-06T15:18:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-08 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Lipopolysaccharide endotoxins (LPS) are the most common contaminant pyrogenic compounds found in intracellular recombinant biomolecules purified from Gram-negative bacteria, such as Escherichia coli. Thus, the purification downstream processing should guarantee the effective removal of LPS from the final bioproduct, particularly, therapeutic biopharmaceuticals. Aqueous two-phase micellar systems (ATPMS) appear to be an excellent strategy to purify recombinant biopharmaceuticals from the cell lysate of E. coli, reducing high LPS concentrations. In order to demonstrate the effectiveness of ATPMS as a biopharmaceutical purification platform, the influence of inorganic salt electrolytes (NaCl, Li2SO4, KI, or KNO3) on the partitioning of green fluorescent protein (GFP) and LPS removal using ATPMS composed of n-decyl tetraethylene oxide (C10E4) was evaluated. The impact of different LPS concentrations on GFP partitioning was also studied. The addition of electrolytes (i.e., NaCl or Li2SO4) to the C10E4-based ATPMS have reduced the phase forming temperatures to very mild conditions (ca. 17.00 and 13.00 °C, for NaCl and Li2SO4, respectively). The selective partitioning ability of the proposed ATPMS was further demonstrated, where a complete removal of the LPS from the micelle-poor phase (REMLPS > 98%) and a preferential GFP recovery (RECGFP = 97%, KGFP > 7) to the micelle-poor phase was obtained. The GFP partitioning was even enhanced by increasing LPS loading (104–106 EU/mL), probably due to the formation of mixed micelles between LPS and C10E4. It is here demonstrated that a C10E4/buffer + salt-based ATPMS can be a useful and straightforward platform for the removal of endotoxin contaminants and the purification of recombinant biopharmaceuticals from E. coli cell lysates. Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical Sciences University of São Paulo – FCF/USP Ronin Institute Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University – UNESP Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University – UNESP FAPESP: #2005/60159-7 FAPESP: #2007/51978-0 FAPESP: #2014/19793-3 CAPES: 0366/09-9

Green fluorescent protein (GFP) is a globular protein used as biosensor and biomarker in medical and industrial fields. However, due to the expensive production costs of expressing proteins using high-cost inducers like isopropyl-β-d-1-thiogalactopyranoside (IPTG), the number of GFP applications are still scarce. This work studied the production of enhanced GFP (EGFP) using Escherichia coli BL21 (DE3) [pLysS; pET28(a)], aiming to increase its yield and reduce costs. First, the influence of agitation rate, induction time, and concentration of IPTG in the production of EGFP was evaluated, but only the first two parameters were significant. Subsequently, aiming to reduce costs related to the use of inducer, the IPTG concentration (0.005, 0.010, and 0.025 mM) was decreased and, interestingly, the production levels were maintained or increased. These results show that a proper choice of production conditions, particularly through the decrease of inducer concentration, is effective to reduce the upstream production costs and guarantee high EGFP expression.

Made available in DSpace on 2019-10-06T15:28:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Lipases are enzymes with a large number of applications at the industrial level. New producers and the development of bioprocess with high productivity are the major challenges of these enzymes industrially. Regarding the production process, oils from agro-industrial waste can emerge as efficient enzymes inductor reducing the production cost. Moreover, this process can minimize environmental problems related to agro-industrial disposal. Thus, this work proposes to evaluate a new Aspergillus specie from Amazon forest in Brazil as lipase producer using oils from agro-industrial waste (grape seed oil (GSO) and cotton seed oil (CSO)) as an inductor. It was used olive and soybean oil as standard. The microorganism metabolized all oils studied; however, the ones with a higher percentage of fatty acid esters (>80%), namely, soybean, olive, and GSO, promoted the highest lipase production. By comparing the activity of the lipase produced, GSO allowed an enzyme activity 2 fold higher than CSO. The enzyme produced using GSO was characterized and pre-purified by precipitation, showing an optimum pH of 7.5 and stability for pH between 7 and 7.5 values; an optimum temperature of 45 °C and a thermal stability between 30 and 50 °C was achieved. In conclusion, a new isolated from Amazon, Aspergillus sp., can produce lipase for industrial purpose using GSO. Moreover, other oils from agro-industrial waste with a high percentage of fatty acid esters can be used as an inductor for lipase production. Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) Rodovia Araraquara-Jaú/ Km 01 Centro de Ciências Biológicas State University of Londrina Culture Collection DPUA/UFAM Federal University of Amazonas Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) Rodovia Araraquara-Jaú/ Km 01 FAPESP: 2014/1580-3

Made available in DSpace on 2021-06-25T10:14:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-15 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Recent environmental and health concerns have been pushing the society for the consumption of natural and sustainable products, as for example, the use of natural colorants as additives to impart color. Talaromyces amestolkiae are natural producers of red colorants at both extracellular and cell-bond state, so are very important for the dye industry. T. amestolkiae cells are constituted by a complex and rigid cell wall structure, which contain a major fraction of the total colorants content. The use of environmentally friendly, biocompatible and high-efficiency cell disruption procedures is thus critical for the recovery of the intracellular colorants. An alternative approach using imidazolium-based ionic liquids (ILs) was applied to recover the intracellular colorants from T. amestolkiae fungi. First, the cell disruption ability of a series 1-alkyl-3-methylimidazolium chloride ILs ([Cnmim]Cl) aqueous solutions was determined, assessing both the effect of increasing cationic alkyl chain length (n = 1, 2, 4, 6, 8 or 10) and IL concentration (30% and 45% w/v). [C4mim]Cl showed the highest extraction ability to recover red, orange and yellow colorants. In the second part, the extraction ability of [C4mim]Cl (at 45% w/v in water) was improved, by combining the IL solution chemical treatment with a mechanical (ultrasound-temperature-assisted) cell disrupting method. At this point, the incubation time (at 60 °C and 150 rpm) after sonication, number sonication pulses (5 min ON) and re-extraction effects were screened as parameters for increasing the cell disruption. The mechanical-assisted method allowed an increase of almost 3-fold in the extractive performance of [C4mim]Cl solution, achieving a maximum recovery of 76.07 UA490nm/mL of red colorants. The results demonstrate the potential of [C4mim]Cl solution as chemical cell-disrupting aid in ultrasound-temperature-based methods to improve the recovery of intracellular colorants from complex filamentous fungi cells. Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) Culture Collection DPUA/UFAM Universidade Federal do Amazonas Univ Coimbra CIEPQPF Department of Chemical Engineering Rua Sílvio Lima, Polo II - Pinhal de Marrocos Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) FAPESP: 2014/16424-7 FAPESP: FAPESP 2014/01580-3

Made available in DSpace on 2021-06-25T10:14:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-15 Rhodotorula strains can produce industrial valuable bioproducts. In this work, the production of carotenoids, and lipids by Rhodotorula glutinis using different nitrogen sources was evaluated. Two statistical experimental designs were applied to improve carotenoid production: a first 25−1 fractional factorial design evaluating the influence of independent variables pH, nitrogen source, glucose, KH2PO4 and MgSO4 concentration; a second 22 central factorial design to optimize the effect of pH and nitrogen sources. After the optimization using two statistical designs, a culture mediium composed of (in g/L) glucose (10), asparagine (10), NH4NO3 (4), KH2PO4 (0.52), MgSO4 (0.52) was found as the best for the production of carotenoids at a pH 5 and 30 °C. The best bioprocess was scaled-up to a 5 L stirred-tank bioreactor. The change to a bioreactor allowed to improve aeration and agitation conditions, and consequently, increasing the production yields (m/v) in, approximately, 25.83 %, 11.88 %, 24.50 % and 10.32 % for β-carotene, torularhodin, torulene and lipids, respectively. The combined supplementation of the culture media with both organic (asparagine) and inorganic nitrogen (ammonium nitrate) sources was primordial for enhancing the carotenogenesis. R. glutinis are very efficient in the production of valuable carotenoids and lipids, presenting high potential of yeast for the industrial production of more sustainable cosmetic, pharmaceutical, and food products. Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences Sao Paulo State University (UNESP) CICECO-Aveiro Institute of Materials Department of Chemistry University of Aveiro Department of Chemical Engineering and Analytical Science The University of Manchester Univ Coimbra CIEPQPF Department of Chemical Engineering Rua Sílvio Lima Pólo II – Pinhal de Marrocos Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences Sao Paulo State University (UNESP)

In the last years, there is a trend towards the replacement of synthetic colorants by natural ones, mainly due to the increase of consumer demand for natural products. The natural colorants are used to enhance the appearance of pharmaceutical products, food, and different materials, making them preferable or attractive. This review intends to provide and describe a comprehensive overview of the history of colorants, from prehistory to modern time, of their market and their applications, as well as of the most important aspects of the fermentation process to obtain natural colorants. Focus is given to colorants produced by filamentous fungal species, aiming to demonstrate the importance of these microorganisms and biocompounds, highlighting the production performance to get high yields and the aspects of conclusion that should be taken into consideration in future studies about natural colorants.

Made available in DSpace on 2020-12-12T01:29:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fungi bioproducts have recently become commercially important to the food and pharmaceutical industry. However, different structural forms exhibited by filamentous fungi throughout their life cycles are often a bottleneck for productivity in many industrial processes. In this work, the relationship among mycelium accumulation, morphology, and colorant production was evaluated. Experiments were performed in an incubator shaker and in a stirred-tank bioreactor. First, a series of nitrogen sources and pure vitamins were investigated as additives to the monosodium glutamate-glucose medium in order to improve Talaromyces amestolkiae red colorant production in the incubator shaker. MSG and pH played an important role in generating stress, which affected cell growth and colorants production. The colorant synthesis seems to be is triggered under metabolic stress. Next, the effects of starter culture composition (MSG-glucose and nitrogen complex starter media) and airflow (2.0 and 8.0 Lmin−1) were investigated in the bioreactor. After 120 h of cultivation in the bioreactor, a maximum red colorant production of 9.33 UA500nm, 72.38 % glucose consumption, and 4.53 g L-1 biomass concentration were achieved under complex nitrogen as starter medium at 8 Lmin−1. Growth remained at moderate level, probably affected by the synergistic effect of MSG and pH as stress factors. Moreover, pellets formation favored suitable oxygenation of the culture broth leading to the best consumption of the carbon source. Department of Bioprocess Engineering and Biotechnology School of Pharmaceutical Sciences Universidade Estadual Paulista - UNESP Culture Collection DPUA/UFAM Universidade Federal do Amazonas Department of Bioprocess Engineering and Biotechnology School of Pharmaceutical Sciences Universidade Estadual Paulista - UNESP

Green fluorescent protein (GFP) has excellent properties as a biosensor and biomarker; however, its widespread use is limited by its purification costs. Alternative low-cost purification techniques can overcome this issue. The aim of this work was to evaluate aqueous biphasic systems (ABS) composed of cholinium chloride ([Ch]Cl) and different polymers as effective platforms to recover GFP from cell lysate of recombinant Escherichia coli BL21. All systems completely extracted GFP from cell lysate (>99%) into the polymeric- or [Ch]Cl-rich phases. In general, [Ch]Clbased ABS allowed a good purification capacity (GFP 80− 100% pure), with the best results (approximately 100% pure GFP) achieved with a polypropylene glycol (PPG)-400/[Ch]Cl ABS in a single-step extraction or in a two-step extraction (backextraction) by the integration of a polyethylene glycol (PEG)/sodium polyacrylate+[Ch]Cl ABS with a following stage using a PEG/[Ch]Cl-based ABS. Additionally, to demonstrate the potential of the PPG-400/[Ch]Cl ABS in downstream processing, solvent recycling and GFP polishing were carried out using ultrafiltration. Finally, the capacity of the PPG-400/[Ch]Cl ABS to extract other fluorescent proteins was also confirmed. The results clearly demonstrated that the PPG-400/[Ch]Cl ABS can be applied in downstream processing for the purification of proteins, not only enhancing purification yields but also providing simpler, quicker, cost-effective, and biocompatible processes. published

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two-phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG-rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (KC) was obtained in the presence of NaCl 0.1 M (KC = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na2SO4 0.5 M showed the highest KC (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%ηTOP) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0–3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1295–1304, 2015

Xanthan gum (XG) is a microbial exopolysaccharide that occupies a prominent place in the market because of its unique properties compared with other microbial gums. When lignocellulosic materials such as sugarcane bagasse (SCB) are used as a carbon source, the degradation of the SCB material can be partial, retaining microfibrils of cellulose in the fermentation broth. These microfibrils can be incorporated into the XG structure. The aim of this work was to select the best concentration of SCB to produce XG and to analyze the characteristics of the biopolymer obtained. The XG produced in the best condition was added to biodegradable films as a reinforcing agent. The SCB was evaluated at the following concentrations: 2, 4, 6, 8 and 10% w/w. The XG produced with 4% w/w SCB provided the best XG, not only for production but also for polymer characteristics. Visualization with transmission electron microscopy (TEM) established the presence of microfibrils in the polysaccharide structure, and the incorporation of XG with microfibrils provided a flexible film with enhanced mechanical properties.

Made available in DSpace on 2015-02-02T12:39:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-01Bitstream added on 2015-02-02T13:08:42Z : No. of bitstreams: 1 S0104-66322014000400023.pdf: 626628 bytes, checksum: e6750a1bef390c705465a35d12ff00aa (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Aqueous two-phase micellar systems (ATPMS) can be exploited in separation science for the extraction/purification of desired biomolecules. Prior to phase separation the surfactant solution reaches a cloud point temperature, which is influenced by the presence of electrolytes. In this work, we provide an investigation on the cloud point behavior of the nonionic surfactant C10E4 in the presence of NaCl, Li2SO4 and KI. We also investigated the salts' influence on a model protein partitioning. NaCl and Li2SO4 promoted a depression of the cloud point. The order of salts and the concentration that decreased the cloud point was: Li2SO4 0.5 M > NaCl 0.5 M ≈ Li2SO4 0.2 M. On the other hand, 0.5 M KI dislocated the curve to higher cloud point values. For our model protein, glucose-6-phosphate dehydrogenase (G6PD), partitioning experiments with 0.5 M NaCl or 0.2 M Li2SO4 at 13.85 °C showed similar results, with KG6PD ~ 0.46. The lowest partition coefficient was obtained in the presence of 0.5 M KI (KG6PD = 0.12), with major recovery of the enzyme in the micelle-dilute phase (%Recovery = 90%). Our results show that choosing the correct salt to add to ATPMS may be useful to attain the desired partitioning conditions at more extreme temperatures. Furthermore, this system can be effective to separate a target biomolecule from fermented broth contaminants. University of São Paulo School of Pharmaceutical Sciences Department of Biochemical and Pharmaceutical Technology Universidade Estadual Paulista School of Pharmaceutical Sciences Department of Bioprocess and Biotechnology Universidade Estadual Paulista School of Pharmaceutical Sciences Department of Bioprocess and Biotechnology

Liquid–liquid extraction for the purification of molecules has been central to many advances in the pharmaceutical industry. These processes were developed based on the property that some polymer and/or micellar solutions present to separate into a concentrated phase and a diluted phase. Based on the differences in the physical and chemical environments of the two coexisting phases, and since both phases contain approximately 60–90% of water, liquid–liquid extraction provides a powerful alternative to both extract and solubilize a molecule. This paper examines the partition behavior of the synthetic drug, 2-[(3,4-dichlorine-benzylidene)-amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (5CN05), in an aqueous two-phase polymer system (ATPPS) and also in an aqueous two-phase micellar system (ATPMS). The results showed that both systems are favorable for extraction the 5CN05 drug high partition coefficient values (K5CN05 > 200) and yield (Y5CN05 > 99.48%) in the concentrated phase were achieved with the systems. However, the ATPPS generated a partition coefficient (K5CN05) higher than the one obtained with ATPMS. The results suggest that both processes may be used for the extraction and concentration of molecules with hydrophobic characteristics, such as 5CN05. They also provide an optimal environment for the solubilization of such molecules, allowing for greater efficiency when purifying many classes of drugs.

Made available in DSpace on 2015-02-02T12:39:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-09-01Bitstream added on 2015-02-02T13:08:32Z : No. of bitstreams: 1 S1517-83822014000300022.pdf: 1163361 bytes, checksum: f6578eb61c12ca4b15b58b2bb5a98706 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture. Universidade Federal Rural de Pernambuco Departamento de Morfologia e Fisiologia Animal Universidade Estadual Paulista Júlio de Mesquita Filho Escola de Ciências Farmacêuticas Departamento de Bioprocessos e Biotecnologia Universidade Federal de Pernambuco Department of Antibiotics Universidade Federal de Pernambuco Laboratorio de Biotecnologia University of Genoa Department of Civil, Chemical and Environmental Engineering Universidade de São Paulo Departamento de Bioquimica e Tecnologia Farmacêutica Universidade Estadual Paulista Júlio de Mesquita Filho Escola de Ciências Farmacêuticas Departamento de Bioprocessos e Biotecnologia

This work aims at investigating the production of yellow, orange, and red natural colorants in a submerged culture of Penicillium purpurogenum DPUA 1275. For this purpose, different experimental conditions evaluating the effect of incubation time, type and size of inoculum, and different carbon and nitrogen sources were performed. Furthermore, the growth kinetics were obtained in the conditions of 10(8) spores/ml and 5 mycelia agar discs during 360 h. These experiments showed that 5 mycelia agar discs and 336 h promoted the highest yellow (3.08 UA400nm), orange (1.44 UA470nm), and red (2.27 UA490nm) colorants production. Moreover, sucrose and yeast extract were the most suitable carbon and nitrogen sources for natural colorants production. Thus, the present study shows a new source of natural colorants, which can be used as an alternative to others available in the market after toxicological studies.

Made available in DSpace on 2018-12-11T16:41:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-05-11 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) There is a worldwide interest in the development of processes for producing colorants from natural sources. Microorganisms provide an alternative source of natural colorants produced by cultivation technology and extracted from the fermented broth. The aim of the present work was to study the recovery of red colorants from the fermented broth of Talaromyces amestolkiae using the technique of colloidal gas aphrons (CGA) comprising surfactant-stabilized microbubbles. Preliminary experiments were performed to evaluate the red colorants' solubility in different organic solvents, octanol/water partitioning, and their stability in surfactant solutions, namely hexadecyl trimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and polyoxyethylenesorbitan monolaurate (Tween 20), which are cationic, anionic and nonionic surfactants, respectively. The first recovery experiments were carried out using CGA generated by these surfactants at different volumetric ratios (VR, 3-18). Subsequently, two different approaches to generate CGA were investigated at VR values of 6 and 12: the first involved the use of CTAB at pH 6.9-10.0, and the second involved the use of Tween 20 using red colorants partially dissolved in ethanol and Tween 20. The characterization results showed that red colorants have a hydrophilic nature. The highest recoveries were obtained with Tween 20 (78%) and CTAB (70%). These results demonstrated that the recovery of the colorants was driven by both electrostatic and hydrophobic interactions. The VR was found to be an important operating parameter and at VR 12 with CTAB (at pH 9) maximum recovery, partitioning coefficient (K = 5.39) and selectivity in relation to protein and sugar (SP = 3.75 and SS = 7.20 respectively) were achieved. Furthermore, with Tween 20, the separation was driven mainly by hydrophobic interactions. Overall CGA show promise for the recovery of red colorants from a fermented broth. Although better results were obtained with CTAB than with Tween 20 the latter may be more suitable for some application due to its lower toxicity. Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences UNESP Univ. Estadual Paulista, Rodovia Araraquara-Jaú/km 01 Department of Biochemical and Pharmaceutical Technology University of São Paulo, Avenida Prof. Lineu Prestes 580, Bl. 16 Culture Collection DPUA UFAM Universidade Federal Do Amazonas, Av. Gal. Rodrigo Octávio Jordão Ramos, 3000 Department of Food and Nutritional Sciences University of Reading, Whiteknights Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences UNESP Univ. Estadual Paulista, Rodovia Araraquara-Jaú/km 01 FAPESP: 0321/11-7 FAPESP: 08/58280-0

Production of bacterial nanocellulose (BNC) is becoming increasingly popular owing to its environmentally friendly properties. Based on this benefit of BNC production, researchers have also begun to examine the capacity for cellulose production through microbial hosts. Indeed, several research groups have developed processes for BNC production, and many studies have been published to date, with the goal of developing methods for large-scale production. During BNC bioproduction, the culture medium represents approximately 30 % of the total cost. Therefore, one important and challenging aspect of the fermentation process is identification of a new cost-effective culture medium that can facilitate the production of high yields within short periods of time, thereby improving BNC production and permitting application of BNC in the biotechnological, medical, pharmaceutical, and food industries. In this review, we addressed different aspects of BNC production, including types of fermentation processes and culture media, with the aim of demonstrating the importance of these parameters.

Bromelain is a cysteine protease found in pineapple tissue. Because of its anti-inflammatory and anti-cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid-liquid extractions by aqueous two-phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up-to-date compilation of the data reported on this enzyme.

In this work, the effects of agitation and aeration rates on aqueous two-phase system (ATPS)-based extractive fermentation of clavulanic acid (CA) by Streptomyces variabilis DAUFPE 3060 were investigated through a 22 full factorial design, where oxygen transfer rate (OTR) and oxygen uptake rate (OUR) were selected as the responses. Aeration rates significantly influenced cell growth, OUR, and CA yield, while OTR was practically the same in all the runs. Under the intermediate agitation (950 rpm) and aeration conditions (3.5 vvm) of the central point runs, it was achieved OTR of 1.617 ± 0.049 mmol L-1 h-1 , OUR of 0.132 ± 0.030 mmol L-1 h-1 , maximum CA production of 434 ± 4 mg L-1 , oxygen mass transfer coefficient of 33.40 ± 2.01 s-1 , partition coefficient of 66.5 ± 1.5, CA yield in the top and bottom phases of 75% ± 2% and 19% ± 1%, respectively, mass balance of 95% ± 4% and purification factor of 3.8 ± 0.1. These results not only confirmed the paramount role of O2 supply, broth composition and operational conditions in CA ATPS-extractive fermentation, but also demonstrated the possibility of effectively using this technology as a cheap tool to simultaneously produce and recover CA. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1444-1452, 2016.

Há interesse mundial no desenvolvimento de pesquisas envolvendo produção e extração de colorantes naturais, devido a sérios problemas de segurança industrial associados ao uso de colorantes sintéticos. Este trabalho objetivou produzir colorantes naturais de Penicillium purpurogenum DPUA 1275 por cultivo submerso (em frascos agitados e em biorreator) e estudar a extração dos colorantes vermelhos. Para a produção, os estudos iniciais mostraram que 5 discos de micélio, sacarose e extrato de levedura como fontes de carbono e nitrogênio, respectivamente, e 336 horas de cultivo eram condições adequadas para a produção dos colorantes. Visando à otimização da produção, realizaram-se planejamentos fatoriais, com as variáveis independentes: tempo de cultivo; velocidade de agitação; pH; temperatura; concentração de sacarose e de extrato de levedura. As variáveis-respostas foram produção de colorantes amarelos, laranjas e vermelhos. Dos resultados obtidos, as variáveis mais significativas ao processo foram concentrações de extrato de levedura e de sacarose. A produção dos colorantes vermelhos foi otimizada, alcançando a produção de 2,97 UA490nm, nas condições 48,90 e 11,80 g/L de sacarose e extrato de levedura, respectivamente, 30°C, pH 4,5 150 rpm e 336 horas de cultivo. Nos experimentos em biorreator, o melhor resultado foi obtido na frequência de agitação de 500 rpm e na mudança do pH do meio para 8,0, após 96 horas de bioprocesso. Ademais, avaliou-se a estabilidade dos colorantes vermelhos presentes no meio fermentado em diferentes condições (pH, temperatura, sais, polímeros e tensoativos). Referente a pH e temperatura, os colorantes vermelhos mostraram-se mais estáveis nas condições alcalinas e a 70 °C. Tanto os sais (NaCl e Na2SO4) quanto os polímeros (PEG 1.000, 6.000 e 10.000 g/mol e NaPA 8.000 g/mol a 5 e 15%) e os tensoativos (Tween 20, CTAB e SDS) não causaram perda da cor nas condições avaliadas. Estudos de solubilidade e de coeficiente de partição octanol-água mostraram que os colorantes vermelhos apresentam solubilidade superior em solventes polares e característica mais hidrofílica. Nos estudos de extração, as técnicas avaliadas foram Sistemas Poliméricos de Duas Fases Aquosas (SPDFA) formados pelo sistema PEG/NaPA e Colloidal Gas Aphrons (CGA). Pela primeira técnica, os colorantes vermelhos migraram preferencialmente para a fase PEG. Os polímeros PEG 6.000 g/mol, na presença de NaCl 0,1 e 0,5 M, e PEG 10.000 g/mol, com Na2SO4 0,5M, se destacaram dentre as condições analisadas com coeficiente de partição (K) próximo a 13, em ambos os casos, e seletividade de proteínas (SeP) próximas a 3. Para a técnica de CGA, o CTAB proporcionou os melhores resultados, seguido do Tween 20. Porém, o valor de K foi inferior ao obtido com SPDFA, com um máximo de 5 (CTAB 2 mM/pH 9,0). Os resultados obtidos demonstram um novo produtor de colorantes naturais, as quais têm potencial de aplicação em diversos segmentos industriais. Ademais, os resultados obtidos mostraram a eficiência das técnicas utilizadas para extração dos colorantes vermelhos, com destaque para SPDFA, que apresentou maiores valores de K. There is worldwide interest in developing research projects involving the production and extraction of natural colorants due to serious safety problems associated with industrial use of synthetic ones. The aim of this work was to investigate the production of natural colorants from Penicillium purpurogenum DPUA 1275 by submerged culture (rotatory shaker and bioreactor) besides studying the red colorants extraction. To the production step, initial studies showed that 5 agar mycelial discs, sucrose and yeast extract as carbon and nitrogen sources, respectively, and 336 hours of bioprocess promoted the best results. To optimize the colorants production a serie of factorial designs were performed. The independent variables studied were: fermentation time, agitation speed, pH, temperature, sucrose and yeast extract concentration under the responses production of yellow, orange and red colorants. From these results, the most significant variables for the process were sucrose and yeast extract concentration. The red colorants production was optimized achieving 2.97 UA490nm, in the following conditions: 48.90 and 11.80 g/L of sucrose and yeast extract, respectively, 30 °C, 4.5 pH, 150 rev min-1 and 336 hours of culture. In the experiments performed in bioreactor, the condition that promoted the best results was 500 rpm and pH adjusted for 8.0 after 96 hours of bioprocess. Furthermore, we evaluated the red colorants stability at different conditions (pH, temperature, salts, polymers and surfactants). Concerning to pH and temperature, the red colorants were more stable under basic conditions and 70 °C; not only the salts (NaCl and Na2SO4) but also the polymers (PEG 1000, 6000 and 10000 g/mol and NaPA 8000 g/mol) and the surfactants (Tween 20, CTAB and SDS) not promoted loss of color upon the conditions evaluated. Studies of red colorants solubility and octanol water coefficient showed that these compounds exhibit a higher solubility in polar solvents and present hydrophilic characteristics. Subsequently, the extraction of red colorant was evaluated through two extraction methods: Polymeric Systems Aqueous Two Phase (ATPS) composed by PEG and NaPA and Colloidal Gas Aphrons (CGA). For the first technique, the red colorant preferentially migrated to the PEG phase. The best results were obtained with PEG 6000 g/mol in the presence of 0.1 to 0.5 M NaCl and with PEG 10000 g/mol with 0.5 M Na2SO4. To both cases the partition coefficient (K) was close to 13 and the Selectivity in terms of proteins (SeP) was close to 3. For the CGA technique, CTAB gave the best results followed by Tween 20. However, the K values were lower than the ones obtained with ATPS with a maximum of 5 in the following condition: CTAB 2 mM/pH 9.0. For the SeP, the values obtained for both techniques were close. The results above show a new producer of natural colorants which have potential application in various industries. Moreover, the results show the efficiency of the techniques used to extract the red colorants, especially to ATPS that presented higher K values.

The population interest in health products is increasing day-by-day. Thus, the demand for natural products to be added in food and pharmaceutical commodity is also rising. Among these additives, colorants, which provides color to products, can be produced by microorganism through bioprocess. Looking for new source of natural colorants, fungi have been employed to this purpose producing novel and safer natural colorants. So, the main goal of this study was to describe a Talaromyces species able to produce natural colorants and investigate nutritional parameters of colorants production using statistical tool. The taxonomy classified the microorganism as Talaromyces amestolkiae. The statistical design evaluated pH and glucose, meat extract and meat peptone concentration as independent variables, and red colorants production as main response. Under the best condition (g/L: glucose 30, meat extract 1, meat peptone 10, and initial pH of 7.0) an increase of 229% in the red colorant production was achieved as compared with the initial media used. The dried fermented broth containing red colorants showed low cytotoxicity against fibroblasts cells (IC50 > 187.5 g/L) and effective antimicrobial activity against S. aureus (MIC of 2.5 g/L). Thus, T. amestolkiae colorants can be attractive to food and pharmaceutical applications as it does not produce toxic compounds and can promote protection against microorganism contaminants. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2684, 2019.

The development of efficient and cost-effective downstream processes is essential for any pharmaceutical products. Clavulanic acid (CA) is a β-lactamic drug used combined with antibiotics to inhibit bacterial resistance mediated by β-lactamase enzymes. CA industrial purification involves techniques such as liquid–liquid extraction with organic solvents and chromatographic techniques, resulting in low recovery yields. In this way, techniques more suitable for biomolecules such as liquid–liquid extraction based on aqueous two-phase systems, which contains 60–90% of water, are of great interest. In this work, aqueous two-phase micellar systems (ATPMS) formed by the nonionic surfactants Triton X-114 and Triton X-100 in the presence or absence of dextran sulfate (Dx-S) were investigated as a first step of CA purification. The drug was found to be stable in the presence of polymers and surfactants with mass balances higher than 80% for all the conditions evaluated. CA partitioning in ATPMS was investigated in the presence of concentrations of Dx-S lower than 4%, CA partitioned more to micelle-poor phase. However, increasing the polymer concentration (8 and 14%), we observed a phase inversion with both surfactants and the drug partitioned more pronounced to the micelle rich-phase, with partition coefficients up to 1.83 with TX-100. The results show that Dx-S/Triton X systems can be used as a first step of CA extraction and the conditions can be adjusted depending on the subsequent extraction methods.

Bacterial cellulose (BC) is used in different fields as a biological material due to its unique properties. Despite there being many BC applications, there still remain many problems associated with bioprocess technology, such as increasing productivity and decreasing production cost. New technologies that use waste from the food industry as raw materials for culture media promote economic advantages because they reduce environmental pollution and stimulate new research for science sustainability. For this reason, BC production requires optimized conditions to increase its application. The main objective of this study was to evaluate BC production by Gluconacetobacter xylinus using industry waste, namely, rotten fruits and milk whey, as culture media. Furthermore, the structure of BC produced at different conditions was also determined. The culture media employed in this study were composed of rotten fruit collected from the disposal of free markets, milk whey from a local industrial disposal, and their combination, and Hestrin and Schramm media was used as standard culture media. Although all culture media studied produced BC, the highest BC yield60 mg/mLwas achieved with the rotten fruit culture. Thus, the results showed that rotten fruit can be used for BC production. This culture media can be considered as a profitable alternative to generate high-value products. In addition, it combines environmental concern with sustainable processes that can promote also the reduction of production cost., The authors would like to acknowledge the Brazil National Council of Technological and Scientific Development (CNPq, FAPESP, and CAPES), the financial support from FAPESP 2009/14897-7, and Fundacao para a Ciencia e a Tecnologia (FCT)/Portugal through the project PTDC/EBB-EBI/112170/2009 for the financial support and scholarship. Special thanks to Talita Almeida Vicentin for technical support.

Bromelain is a mixture of proteolytic enzymes present in all tissues of the pineapple (Ananas comosus Merr.), and it is known for its clinical therapeutic applications, food processing, and as a dietary supplement. The use of pineapple waste for bromelain extraction is interesting from both an environmental and a commercial point of view, because the protease has relevant clinical potential. We aimed to study the optimization of bromelain extraction from pineapple waste, using the aqueous two-phase system formed by polyethylene glycol (PEG) and poly(acrylic acid). In this work, bromelain partitioned preferentially to the top/PEG-rich phase and, in the best condition, achieved a yield of 335.27% with a purification factor of 25.78. The statistical analysis showed that all variables analyzed were significant to the process.

Clavulanic acid is a drug obtained by Streptomyces sp. Fermentation and therapeutically employed in association with β-lactamic antibiotics to avoid bacterial resistance. Its industrial purification involves several steps including liquid–liquid extraction with organic solvents. Therefore, new economical and ecological alternatives for this drug purification, such as aqueous two-phase micellar system (ATPMS), are of great interest. In this work we investigated clavulanic acid partitioning in ATPMS composed only by the nonionic surfactant, C 10 E 4 , as well as mixed systems with the addition of the cationic CTAB or anionic AOT surfactants. According to the results, a lowering in the partition coefficient ( K ) value was observed with increasing temperature to the C 10 E 4 micellar system, with K going from 0.87 ± 0.03 ( T = 21 °C) to 0.67 ± 0.18 ( T = 28 °C), probably due to the more hydrophobic environment in the top micelle-rich phase as compared to the bottom micelle-poor phase. For the mixed micellar systems, electrostatic attraction or repulsion influenced drug partitioning. A K = 1.44 ± 0.02 was observed with CTAB/C 10 E 4 (the drug was attracted to the micelle-rich phase) and K = 0.78 ± 0.30 was observed with AOT/C 10 E 4 (the drug was slightly repelled to the micelle-poor phase). These results indicate the potential of mixed micellar systems for clavulanic acid purification.