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Made available in DSpace on 2022-05-01T09:47:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Non-biodegradable petroleum-based plastics are still widely used by the food industry for packaging applications and, due to that bio-based raw materials such as starch, have been attracting growing interest from the packaging industries as a biocompatible alternative material. During the industrial processing of potato, a large amount of residual starch is produced from the washing of the raw material. Starch-like biopolymers have been proposed for the formulation of biodegradable materials. The application of starch in the packaging production depends on chemical, physical and functional properties to form gels and films. The aim of this study was to develop packaging films made from potato processing residual starch combined with polar organic solvents (glycerol and acetic acid) that can be fully obtained from renewable sources. The characterization of the material obtained was performed evaluating the microbiological stability, mechanical properties, formed bonds and barrier characteristics and, in order to demonstrate the real application in fruits preservation the material was direct applied into papaya fruits (Carica papaya) by the immersion method. The present study exhibits the potential of starch residual from potato industrialization to manufacture packaging films. The films achieved the expected results for microbiological analysis, water vapor permeability and Fourier Transform Infrared spectroscopy. The qualitative analysis of the shelf stability test showed the potential of the films in fruit preservation envisioning the application in food industry for packaging. Postgraduate Program in Biomaterials and Bioprocess Engineering School of Pharmaceutical Sciences São Paulo State University (UNESP) School of Pharmaceutical Sciences São Paulo State University (UNESP) Embrapa Agroindústria Tropical/Embrapa Instrumentação Brazilian Agricultural Research Corporation (Embrapa) Postgraduate Program in Biomaterials and Bioprocess Engineering School of Pharmaceutical Sciences São Paulo State University (UNESP) School of Pharmaceutical Sciences São Paulo State University (UNESP)

Made available in DSpace on 2022-04-28T19:44:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Aqueous two-phase systems (ATPS) have been demonstrated to be a simple and robust worldwide applied bioprocessing technology. From high value microscopic to macroscopic biological molecules obtained from a wide broad of matrices, ATPS have shown a high efficiency capacity for their concentration, enrichment, recovery, and purification. Particularly in Latin America, four relevant countries have paved the way and played important leading roles in this transformation history. This perspective article aims to present a critical analysis of the practical evolution of the main implementations of ATPS in Argentina, Brazil, Chile, and Mexico. Special attention is given to novel applications of this biphasic bioprocess that are being explored to accomplish visionary therapeutic tasks for the healthcare, biotechnology, and pharmaceutical sectors, as well as other environmental purposes. © 2021 Society of Chemical Industry (SCI). Tecnologico de Monterrey Escuela de Medicina y Ciencias de la Salud Institute of Biotechnological and Chemistry Processes Faculty of Biochemical and Pharmaceutical Sciences Nacional University of Rosario Tiradentes University (UNIT) Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) School of Pharmaceutical Sciences University of São Paulo Univ. Coimbra CIEPQPF Department of Chemical Engineering Rua Sílvio Lima Pólo II – Pinhal de Marrocos Department of Life Science & Chemistry Focus Area Health Jacobs University Centre for Biotechnology and Bioengineering University of Chile Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP)

The search for new, biotechnologically useful yeast strains has been carried out in many research centers in the world. Sporobolomyces and Sporidiobolus are examples of such useful yeasts, that can be used as a source of many valuable metabolites in industries. This article describes the modern taxonomy of these yeasts, which resulted from many years of research, including both classical microbiology and genetic analyses. Subsequently, the paper presents a review of scientific studies on the biosynthesis of various extracellular and intracellular metabolites produced by Sporobolomyces and Sporidiobolus yeasts, which are of great importance in the contemporary food, feed, and pharmaceutical industries. Such metabolites include exopolysaccharides, lipids, carotenoids, enzymes, and γ-decalactone. Aiming at developing a sustainable circular bioeconomy, this study considers two directions of use of these yeasts, i.e., as a feed additive and as an antagonist in the biocontrol of plant materials. This article is one of the first to organize the knowledge collected from published studies and present the contemporary scientific achievements and prospects for the biotechnological use of Sporobolomyces and Sporidiobolus yeasts.

Microbial carotenoids have attracted rising interest from several industries as a sustainable alternative to substitute the synthetic ones. Traditionally, carotenoids available in the market are obtained by the chemical route using nonrenewable sources (petrochemicals), revealing the negative impact on the environment and consumers. The most promising developments in the upstream and downstream processes of microbial carotenoids are reviewed in this work. The use of agro-based raw materials for bioproduction, and alternative solvents such as biosolvents, deep eutectic solvents, and ionic liquids for the recovery/polishing of microbial carotenoids were also reviewed. The principal advances in the field, regarding the biorefinery and circular economy concepts, were also discussed for a better understanding of the current developments. This review provides comprehensive overview of the hot topics in the field besides an exhaustive analysis of the main advantages/drawbacks and opportunities regarding the implementation of microbial carotenoids in the market.

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

The demand for food, feed, cosmeceutical, and nutraceutical supplements/additives from natural sources has been rapidly increasing, with expectations for a faster expansion than the growth of the global markets in the coming years. In this framework, a particular interest is given to carotenoids due to their outstanding antioxidant activities, particularly the xanthophylls class. Torularhodin is one of these carotenoids that stands out for its multifunctional role as: antioxidant, anticancer and antimicrobial, yet its commercial potential is still unexplored. Although most xanthophylls can be naturally found in: microbial, plant and animal sources, torularhodin is only produced by microbial species, especially red oleaginous yeast. The microbial production of xanthophylls has many advantages as compared to other natural sources, such as: the need for low production area, easier extraction, high yields (at optimum operating conditions), and low (or no) seasonal, climatic, and geographic variation dependency. Due to the importance of natural products and their relevance to the market, this review provides a comprehensive overview of the: properties, characteristics and potential health benefits of torularhodin. Moreover, the most promising developments in both upstream and downstream processing to obtain this colorant from microbial sources are considered. For this purpose, the main microorganisms used for torularhodin production are firstly reviewed, including biosynthesis pathway and torularhodin properties. Following, an overall analysis of the processing aspects related with its: extraction, separation and purification is provided. Lastly, current status and future trends of torularhodin-based processes and products such as therapeutic agents or biomaterials are discussed, indicating promising directions toward biorefinery and circular economy.

Gold nanoparticles (AuNPs) have shown interesting properties and specific biofunctions, providing benefits and new opportunities for controlled release systems. In this research, we demonstrated the use of natural rubber latex (NRL) from Hevea brasiliensis as a carrier of AuNPs and the antibiotic metronidazole (MET). We prepared AuNP-MET-NRL and characterized by physicochemical, biological and in vitro release assays. The effect of AuNPs on MET release was evaluated using UV-Vis and Laser-Induced Breakdown Spectroscopy (LIBS) techniques. AuNPs synthesized by Turkevich and Frens method resulted in a spherical shape with diameters of 34.8 ± 5.5 nm. We verified that there was no emergence or disappearance of new vibrational bands. Qualitatively and quantitatively, we showed that the MET crystals dispersed throughout the NRL. The Young's modulus and elongation values at dressing rupture were in the range appropriate for human skin application. 64.70% of the AuNP-MET complex was released within 100 h, exhibiting a second-order exponential release profile. The LIBS technique allowed monitoring of the AuNP release, indicating the Au emission peak reduction at 267.57 nm over time. Moreover, the dressing displayed an excellent hemocompatibility and fibroblast cell viability. These results demonstrated that the AuNP-MET-NRL wound dressing is a promising approach for dermal applications.

Astaxanthin (AXT) is a ketocarotenoid with several properties, including antioxidant, antidiabetic and anticancer with a wide range of applications in cosmeceutical, feed, food and pharmaceuticals sectors. The large fraction of AXT available in the market is obtained by chemical route, but the consumers preference for natural products are changing the global market of AXT, and due to that several companies are looking for potential alternative sources such as Gram-negative bacteria Paracoccus carotinifaciens (P. carotinifaciens) to obtain natural AXT. The aim of this critical review is to provide a comprehensive overview of the latest AXT research findings and characteristics of the hyperproducer-AXT P. carotinifaciens. Moreover, a brief description of the potential application of P. carotinifaciens for the production of natural AXT at industrial scale for commercial purposes and the latest advancements in the upstream and downstream procedures following the biorefinery and circular economy percepts are considered.

Microorganisms such as bacteria, microalgae and fungi, are natural and rich sources of several valuable bioactive antioxidant's compounds, including carotenoids. Among the carotenoids with antioxidant properties, astaxanthin can be highlighted due to its pharmaceutical, feed, food, cosmetic and biotechnological applications. The best-known producers of astaxanthin are yeast and microalgae cells that biosynthesize this pigment intracellularly, requiring efficient and sustainable downstream procedures for its recovery. Conventional multi-step procedures usually involve the consumption of large amounts of volatile organic compounds (VOCs), which are regarded as toxic and hazardous chemicals. Considering these environmental issues, this review is focused on revealing the potential of unconventional extraction procedures [

Made available in DSpace on 2022-04-29T08:41:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 The number of mammalian animals killed for research purposes has increased significantly in recent years. For this reason, several alternative animal models have been widely used. In this study, two phases of calcium phosphates synthesized by the sol-gel method were added by precipitation to the surface of the natural rubber latex membranes for bone applications. The toxicity eluates from the membrane were evaluated by in vitro assays through cell viability and hemolysis; and by in vivo models using Caenorhabditis elegans worms and zebrafish embryos through survival analysis. Hemolysis levels were less than 3%, which indicates that there was no significant hemolytic activity. It was demonstrated that latex presents enzymes that can dissociate osteoblasts MC3T3, so it was necessary to eliminate these enzymes to obtain a good adhesion and cellular proliferation without morphological alterations. The eluates of membranes did not reduce the survival of Caenorhabditis elegans larvae for 48 h of analysis, except for a hydroxyapatite sample incubated for 5 days. In the zebrafish embryotoxicity assay for 96 hpf of analysis, 60% of embryos survived and there was no evidence of embryos with malformation or developmental delay. In summary, alternative animal models, which are not commonly used to assess biocompatibility, have provided reliable in vivo results that allow suggesting the use of these membranes in the bone biomedical. Department of Biochemistry and Chemical Technology Chemistry Institute UNESP - Univ. Estadual Paulista Department of Chemistry & CESAM Universidade de Aveiro, Campus Universitário de Santiago Department of Biotechnology and Bioprocess Engineering School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista Department of Clinical Analysis School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista Area of Exact Sciences and Engineering UCS - University of Caxias do Sul, Rio Grande do Sul Department of Biochemistry and Chemical Technology Chemistry Institute UNESP - Univ. Estadual Paulista Department of Biotechnology and Bioprocess Engineering School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista Department of Clinical Analysis School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista

Made available in DSpace on 2022-04-28T19:49:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-07 In order to replace conventional organic solvents with more benign equivalents such as ionic liquids or eutectic solvents, in this work we attempt to develop a simple and ecofriendly process using these alternative solvents for the extraction of astaxanthin and β-carotene from Phaffia rhodozyma biomass, which could simultaneously be used as a colorant and plasticizer agent for the preparation of bioactive starch-based biodegradable films without further purification. The use of cholinium-based eutectic solvents appears to be a promising solution envisioning the development of functionalized carotenoid-rich biofilms. Department of Pharmaceutical-Biochemical Technology University of São Paulo School of Pharmaceutical Sciences Department of Engineering of Bioprocesses and Biotechnology São Paulo State University (UNESP) School of Pharmaceutical Sciences, Araraquara Terasaki Institute for Biomedical Innovation (TIBI) CICECO-Aveiro Institute of Materials University of Aveiro Department of Chemistry Univ. Coimbra Department of Chemical Engineering CIEPQPF Department of Engineering of Bioprocesses and Biotechnology São Paulo State University (UNESP) School of Pharmaceutical Sciences, Araraquara

Made available in DSpace on 2022-04-29T08:36:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) In this work we synthesize and characterize AuNPs using two chemical routes: via sodium citrate and via sodium citrate/borohydride. The cytotoxicity of AuNPs was tested by traditional in vitro assays and the in vivo toxicity was evaluated using alternative animals (Galleria mellonella and Caenorhabditis elegans). Avoidance tests with Folsomia candida were carried out to evaluate possible toxic effects in the soil ecosystem. Through Scanning Electron Microscopy and Transmission Electron Microscopy we verified the spherical shape of the AuNPs, with diameters of 34.8 ± 5.5 nm (via sodium citrate) and 7.9 ± 2.2 nm (via sodium citrate/borohydride). The hydrodynamic diameters values were compatible with those obtained microscopically, 30.1 ± 10.1 nm (method A) and 8.3 ± 2.8 nm (method B). Stability of up to 6 months was verified for AuNPs synthesized via sodium citrate, while synthesis via citrate/sodium borohydride was stable for only 1 month. The AuNPs from method A showed better efficacy in the NPs formation with higher absorbance in the plasmonic band at 523 nm, higher concentration and size, spherical shape, homogeneity and stability. The cytotoxicity of the two AuNPs exhibited a slight dose- and size-dependent behavior, with cell viability greater than 80%. The in vivo toxicity in the G. mellonella model showed a survival rate of 100% for 7 days, however, with C. elegans, the percent of survival increased as the concentration of AuNPs decreased. All concentrations of AuNPs, except for the AuNPs synthesized by the citrate pathway (1 mg/kg), showed non-avoidance responses when exposed to contaminated soil. School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry & Chemical Technology São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Bioprocesses and Biotechnology Engineering São Paulo State University (UNESP) School of Chemistry and Molecular Biosciences The University of Queensland Postgraduate Program in Agricultural and Natural Ecosystems (PPGEAN) Federal University of Santa Catarina (UFSC) Area of Exact Sciences and Engineering University of Caxias do Sul (UCS) Institute of Exact Sciences Federal University of Southern and Southeastern Pará (UNIFESSPA) School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry & Chemical Technology São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Bioprocesses and Biotechnology Engineering São Paulo State University (UNESP) FAPESP: 2016/11836-0 FAPESP: 2017/19603-8 CNPq: 470261/2012-9

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 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

The number of animals killed used for research purposes has increased significantly in recent years. For this reason, several alternatives of animal models are widely used. In this study, two phases of calcium phosphates synthesized by the sol-gel method were added by precipitation to the surface of the natural rubber latex membranes for bone applications. Membranes cytotoxicity and toxicity was evaluated by in vitro and in vivo methods. Hemolysis levels were less than 3%, which indicates that there was no significant hemolytic activity. It was demonstrated that latex presents enzymes that can dissociate osteoblasts MC3T3, so it was necessary to eliminate these enzymes to obtain a good adhesion and cellular proliferation without morphological alterations. The in vivo toxicity test with Caenorhabditis elegans worms did not show evidence of toxicity with a sample of hydroxyapatite incubated for 5 days, exhibiting the lowest survival rate of the worms. In the Zebrafish embryotoxicity assay, 60% of embryos survived and there was no evidence of embryos with malformation or developmental delay. In summary, alternative animal models, which are not commonly used to assess biocompatibility, have provided reliable in vivo results that allow suggesting the use of these membranes in the bone biomedical.

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)

Submitted by Cassamo Ussemane Mussagy (cassamo.mussagy@unesp.br) on 2021-01-26T23:12:01Z No. of bitstreams: 1 Cassamo_Mussagy_2021_PhD_Thesis.pdf: 8991428 bytes, checksum: c3cc6e25bae0eeb15c21f4893cbbbe64 (MD5) Approved for entry into archive by Maria Irani Coito (irani@fcfar.unesp.br) on 2021-01-27T13:14:44Z (GMT) No. of bitstreams: 1 mussagy_cu_dr_arafcf_int.pdf: 8448341 bytes, checksum: 3226fe2f8822dd25b3424db1daf48749 (MD5) Made available in DSpace on 2021-01-27T13:14:44Z (GMT). No. of bitstreams: 1 mussagy_cu_dr_arafcf_int.pdf: 8448341 bytes, checksum: 3226fe2f8822dd25b3424db1daf48749 (MD5) Previous issue date: 2021-01-21 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) A crescente busca por produtos naturais e os desafios de sua produção industrial, tem motivado pesquisadores a investigar estratégias para o incremento da produção a partir de fontes microbianas e o desenvolvimento de procedimentos sustentáveis e eficientes para a sua extração. Os carotenoides naturais como o β-caroteno, toruleno e torularodina além de serem poderosos antioxidantes naturais, são corantes que vem merecendo destaque devido às suas propriedades biológicas com aplicações relevantes nas indústrias alimentar, cosmética e farmacêutica. No presente trabalho estudou-se um processo integrado que engloba o cultivo da levedura Rhodotorula glutinis CCT- 2186 (R. glutinis) para o incremento da produção de carotenoides e a extração destes produtos empregando líquidos iónicos (LIs) e bio-solventes como solventes alternativos. Inicialmente, diferentes planejamentos fatoriais foram aplicados para incrementar a produção de carotenoides e para a melhor condição foi realizado um aumento de escala em biorreator de 5 L. Posteriormente, estudos usando soluções aquosas de LIs em processos de extração sólido-líquido foram realizados, seguidos pelo processo de purificação empregando um sistema de partição trifásico composto por soluções aquosas de LIs e sais inorgânicos. Para reunir informações adicionais sobre os mecanismos de separação das fases, sistemas aquosos bifásicos (SAB) compostos por LIs e sais inorgânicos foram determinados e caracterizados. O potencial dos bio-solventes para a recuperação de carotenoides de células de R. glutinis foram também avaliados, a fim de se obter um processo de extração/purificação sustentável, mais eficiente e ecologicamente correto. Este último estudo permitiu a compreensão e otimização de uma abordagem downstream sustentável para a extração usando misturas ternárias (acetato de etilo/etanol/água) com posterior isolamento e polimento de carotenoides, e reciclagem dos solventes da mistura. A sustentabilidade geral da tecnologia proposta neste trabalho foi avaliada pelo impacto ambiental do processo alternativo em termos de avaliação do ciclo de vida (pegada de carbono). Esta tese revela que o uso combinado de fontes de nitrogênio orgânico e inorgânico é recomendado para a suplementação do meio de cultura para o cultivo de R. glutinis e que o uso de LIs e bio-solventes como agentes para o rompimento da parede celular pode ser um procedimento simples, eficiente, sustentável e viável para a recuperação de carotenoides intracelulares da biomassa microbiana. This thesis consists of the investigation and optimization of sustainable strategies to enhance carotenoids production from yeast Rhodotorula glutinis CCT- 2186 (R. glutinis) and their extraction by means of ionic liquids (ILs) and bio-based solvents. The interest on carotenoids such as β-carotene, torulene and torularhodin relies on the plethora of relevant properties of these products and their commercial value for food, feed, cosmetic and pharmaceutical industries. However, to make carotenoids more accessible in a sustainable way, their production from microbial sources is of paramount importance. Concerning the production of carotenoids from R. glutinis yeast there are still processual challenges, such as the improvement of production yields and development of efficient and sustainable extraction platforms. Initially, different statistical experimental designs were applied to improve carotenoids production and the best bioprocess was scaled-up to a 5 L stirred-tank bioreactor. Since the carotenoids are produced intracellularly, requiring appropriate cell-disrupting and extraction methodologies for their recovery, subsequently, the development of more benign and effective extraction/purification platforms was evaluated. A comprehensive study using aqueous solutions of ionic liquids (ILs) for solid-liquid extraction (SLE) processes was carried out, following the carotenoids purification using a three-phase partitioning system composed of aqueous solutions of ILs and inorganic salts. To gather additional information on the phase separation mechanisms, aqueous biphasic systems (ABS) composed of ILs and inorganic salts were determined and characterized. Afterward, the potential of bio-based solvents was evaluated, with the purpose of designing a more efficient and ecofriendly extraction process for recovery of the intracellular carotenoids from R. glutinis. In this study it was designed and optimized an integrated downstream platform using a ternary mixture of bio-based solvents (ethyl acetate/ethanol/water) with isolation and polishing of carotenoids as well as the recycling of the solvents. The overall sustainability of the proposed technology was assessed in terms of solvents recyclability and carotenoids polishing, and the environmental impact of the platform through a life cycle assessment (carbon footprint). This thesis demonstrates the importance of combined organic and inorganic nitrogen sources to supplement the nutritional media for cultivation of R. glutinis and production of carotenoids, and that ILs and mixed bio-based solvents can be used to design simple, efficient and sustainable platforms for the recovery of intracellular carotenoids from microbial biomass. 88887.367977/2019-00 88882.330111/2019-01

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 2021-06-25T10:11:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-15 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) Aqueous two-phase micellar systems (ATPMS) are more than simple amphiphilic copolymer or surfactant aqueous solutions since due to the formation of biphasic regimes, at certain temperatures, these can be successfully used for the separation, encapsulation, and formulation of several bio-based compounds. The recent use of ionic liquids (ILs) as adjuvants in the formation of ATPMS have enlarged its “working window”, but the measurement of respective binodal curves is still very empiric and dependent of operator accuracy through the visual determination of cloud-point temperature. In this work, the range of ATPMS using ILs as adjuvants was extended, providing more insights about their phase formation mechanisms, as well as into determination of cloud-point values of the lower critical solution temperature (LCST)-type demixing transition by using the new temperature modulated optical refractometry approach. The binodal curves of ATPMS composed of Pluronic® L35/PBS buffer at pH 7.4 + cholinium ILs were determined, by using two different methods, i.e.: cloud-point temperature (TCP) visual determination versus temperature modulated optical refractometry (TMOR). TMOR method was more sensitive to the determination of biphasic transition, particularly, in highly diluted solutions (< 3.0 wt% of Pluronic L35). The results also established that the addition of cholinium ILs decreased significantly the phase separation temperatures of Pluronic L35/PBS, with an increase of biphasic region (viz. lower of TCP values) as a function of the relative hydrophobicity of the IL anion, i.e.: [Hex]− > [But]− > [Pro]− > [Ac]− > Cl−. The use of cholinium ILs as adjuvants is particularly important to provide mild conditions for ATPMS-based separation and encapsulation strategies, while TMOR-based methods allow an improvement of phase separation determination accuracy. Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) University of Coimbra CIEPQPF Department of Chemical Engineering, Rua Sílvio Lima, Pólo II – Pinhal de Marrocos Faculty of Pharmaceutical Sciences University of Campinas Department of Engineering of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) CAPES: 001 FAPESP: 2014/16424-7 FAPESP: 2017/10789-1 FAPESP: 2018/05111-9 FAPESP: 2018/10799-0 FAPESP: 2019/05624-9 FAPESP: 2019/08549-8

Made available in DSpace on 2020-12-12T01:24:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Ionic liquids (ILs)-based aqueous biphasic systems (ABS) are efficient platforms for the extraction and purification of biomolecules. To find more biocompatible ABS, propylammonium-based ILs, phosphate salts and water were mixed at three temperatures, evaluating the influence of IL and temperature on the liquid-liquid equilibria (LLE). The biphasic region increases with the increase of IL’ anionic/cationic chain and decreases with the increase of temperature. There is good agreement between the IL’ ability to form ABS and the hydrogen bonding interaction predicted by COSMO-RS. The subtle balance of hydrogen bonding between salt anion-water and IL anion-water governs the phase separation. Department of Engineering Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP) CICECO-Aveiro Institute of Materials Department of Chemistry University of Aveiro Department of Chemical Engineering Federal University of Paraná (UFPR), Polytechnic Center Department of Marine Faculty of Fisheries and Marine Universitas Airlangga Jalan Mulyorejo Kampus C 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 São Paulo State University (UNESP) FAPESP: 2014/16424-7 FAPESP: 2014/19793-3 FAPESP: 2015/50058-0 CNPq: 310182/2018

Made available in DSpace on 2022-04-29T08:36:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-03-30 Soybean and its derivatives are rich sources of nutrients and bioactive compounds with antioxidant properties, however, the wastes with high nutritional value are discarded by the industry. This study aimed to evaluate centesimal composition, microbial safety and antioxidant activity of soybean processing wastes (okara and okara flour) and soymilk. High fiber, carbohydrate, energy and lipids contents were found. Antioxidant activity by spectrophotometric and Electron Paramagnetic Resonance assays showed values for soybean (72.4% and 83.5%), okara (9.6% and 7.7%), okara flour (30.7% and 11.5%) and soymilk (28.4% and 36.5%). The total phenolic content was an average of 3.33 mg of gallic acid equivalent.g−1. Infrared spectra revealed no significant changes in the absorption bands, guaranteeing non-alteration in the compounds composition after processing. Microbiological assays indicated that soybean derivatives are safe for consumption. These results reinforce that these wastes contain bioactive compounds of interest with great potential to generate high value added products. São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Biotechnology and Bioprocesses Engineering São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry and Chemical Technology Terasaki Institute for Biomedical Innovation (TIBI), 11570 West Olympic Boulevard University of Caxias do Sul (UCS) Area of Exact Sciences and Engineering Caxias do Sul São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Biotechnology and Bioprocesses Engineering São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry and Chemical Technology

The search for biotechnological alternatives for the use of residuals generated in the agro-industrial processing of coffee is a current problem. This study evaluated the biotransformation of the liquid residual of the humid coffee benefit using the yeast Kluyveromyces marxianus CCEBI 2011. It was demonstrated that this strain is able to effectively use the reducing and neutral sugars in 24 h, for a yield of 40% in the production of alcohol. Pectin oligomers were obtained with an average size between 5.4 and 5.8 units of anhydrogalacturonic acid, with potential biological activity. In the fermented residuals, a decrease of more than 30% in the concentration of caffeine was observed. The pectinolytic yeast K. marxianus CCEBI 2011 significantly reduces the content of soluble proteins in the liquid residual. The results indicate that the residuals of the coffee benefit are viable substrate to be used by means of alcoholic fermentation in the presence of the pectinolitic yeast K. marxianus CCEBI 2011, reducing its environmental impact and propitiating alternatives for their better use in the agricultural sector. Key words: Kluyveromyces marxianus, pectinolitic yeast, coffee residuals.

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)

The search of the biotechnological ways to the integral use of the wastes generated by the coffee and citric agroindustries, is a current problem. The biotransformation of coffee pulp and orange mesocarp during aerobic and anoxic fermentation with the pectinolític yeast Kluyveromyces marxianus CCEBI 2011 was studied in this research. It was found that coffee pulp is a better substrate to yeast growth, which was evidenced throughout a better conversion of the carbon and energy source. The yeast used in 24 h the reducing and neutral sugars from substrates efficiently, either under aerobic or oxygen-limited cultures. The excretion of the polygalacturonase enzyme by the yeast lead to the increasing in the pectic substances extraction, which reached the 225 % and 33 % for coffee pulp and orange mesocarp, respectively, compared with the amounts which were extracted during substrates sterilization. The enzyme production by the microorganism presented not significantly differences in relation to the substrate used, but the contrary occurred with respect to glucose concentration in the medium. These results point to the promissory use of the K. marxianus CCEBI 2011 yeast strain in the valorization of pectin-rich agricultural wastes by means of their conversion in potential plant elicitors and prebiotics oligogalacturonides-based.