Biotechnological potential of yeasts from Ustilaginaceae family for production of glicolipid biosurfactants

Orientador: Glaucia Maria PastoreTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O presente trabalho de doutorado teve por objetivo principal explorar o potencial biotecnológico de leveduras da família Ustilaginaceae para a produção de biossurfactantes glicolipídicos. Inicialmente, o trabalho prático foi direcionado para um estudo de screening com 34 linhagens de leveduras dos gêneros Pseudozyma sp. e Moesziomyces sp. cultivadas em meio mineral com glicose como fonte de carbono e caldo YEPD. Dentre o total de linhagens testadas, quatro linhagens de Pseudozyma aphidis (UFMG-Y1387, UFMG-Y3468, UFMG-Y3533 e UFMG-Y5768) foram capazes de reduzir a tensão superficial do meio de cultivo para valores menores que 32 mN/m, com eficiência de redução de tensão superficial superior a 53% para o meio mineral com glicose e 37% para o meio YEPD, indicando o potencial dessas leveduras para a produção de compostos tensoativos. Na sequência, foram avaliados o perfil de crescimento e o perfil de redução da tensão superficial das quatro linhagens de P. aphidis selecionadas na etapa de screening em meio mineral contendo diferentes fontes de carbono, incluindo manipueira, glicose, óleo de soja e ácido oleico. De forma geral, foi observado que utilizando fontes de carbono hidrofóbicas foram alcançados os melhores perfis de crescimento, com produção de biomassa seca em maior que 15 g/L, e de redução de tensão superficial (valores menores que 31mN/m) após 192 horas de cultivo. Com base nos resultados dessa etapa do trabalho, a linhagem de P. aphidis UFMG-Y3468 foi considerada a mais promissora uma vez que apresentou os melhores perfis de redução de tensão superficial e de concentração micelar crítica (CMC) nos diferentes meios de cultivo avaliados. A partir das análises de espectrometria de massas e de ressonância magnética nuclear foi possível identificar a produção de manosileritritol lipídeos (MELs) do tipo A, B e C pela linhagem de P. aphidis UFMG-Y3468 quando cultivada em meio mineral com glicose, óleo de soja e ácido oleico como fontes de carbono. Além disso, pela primeira vez foi observada a produção de manosilmanitol lipídeos (MML) por uma linhagem de P. aphidis em meio de cultivo contendo ácido oleico. Os biossurfactantes purificados exibiram baixos valores de concentração micelar crítica (CMC) variando de 0,96 mg/L a 3,65 mg/L, indicando sua eficiência superior em relação aos valores reportados para os tensoativos sintéticos. O balanço hidrofílico-hidrofóbico (HLB) dos compostos tensoativos purificados determinado pelo método de Griffin variou entre 8,25 a 9,12, sendo esses valores condizentes aos reportados na literatura para MELs e comparáveis aos de tensoativos sintéticos utilizados em formulações cosméticas. Na última etapa do trabalho foram realizados três planejamentos experimentais para o estudo da influência e otimização das fontes de carbono e nitrogênio do meio de cultivo para a linhagem de P. aphidis UFMG-Y3468 seguida da análise quimiométrica dos dados utilizando a Análise Componentes Principais (PCA) para identificação das correlações entre as fontes de carbono e nitrogênio e as isoformas de MEL produzidas. Como resultado dessa etapa foi possível a obtenção de cinco modelos matemáticos preditivos válidos estatisticamente com coeficientes de determinação (R2) entre 80% a 96% e altos valores de F calculado no teste de Fisher, além da comprovação do papel determinante da fonte carbono hidrofóbica na discriminação de ensaios com melhor perfil de produção de biossurfactantes. Assim, o trabalho apresentado nessa tese abre caminho para novos estudos para a produção biotecnológica de biossurfactantes glicolipídicos, especificamente manosileritritol lipídeos e manosilmanitol lipídeo utilizando a linhagem de P. aphidis UFMG-Y3468 bem como a potencial aplicação desses compostos nas áreas de cosméticos e de alimentosAbstract: The main objective of this work was to explore the biotechnological potential of yeasts from Ustilaginaceae family for production of glycolipid biosurfactants. Initially, the practical work was directed to a screening study with 34 strains of yeasts belonging the genus Pseudozyma sp. and Moesziomyces sp. cultivated in mineral medium with glucose as carbon source and YEPD broth. Among all tested microorganisms, four strains of Pseudozyma aphidis (UFMG-Y1387, UFMG-Y3468, UFMG-Y3533 and UFMG-Y5768) were able to reduce the surface tension of the culture medium to values lower than 32 mN/m, with efficiency for surface tension reduction more than 53% for the mineral medium supplemented with glucose and 37% for the YEPD broth, indicating the potential of these yeasts for the production biosurfactants. In sequence, the growth and the surface tension reduction profiles for selected P. aphidis strains selected were determineted in mineral medium containing different carbon sources, including cassava wastewater, glucose, soybean oil and oleic acid. In general, it was observed that the best growth profiles were obtained using hydrophobic carbon sources, with dry biomass values more than 15 g/L and of surface tension reduction less than 31 mN/m after 192 hours of cultivation. Based on the results of this step, the P. aphidis UFMG-Y3468 strain was considered the most promising because of their best ability for surface tension reduction and lower values of critical micelle concentration (CMC) on the different media tested. Considering the results mass spectrometry and nuclear magnetic resonance analysis, it was possible to identify a production of mannosylerythritol lipids (A, B and C) by P. aphidis UFMG-Y3468 strain when grown in mineral medium with glucose, soybean oil and oleic acid as carbon sources. In addition, for the first time was described the production of mannosylmannitol lipids (MML) by a of P. aphidis strain in culture medium containing oleic acid. The purified biosurfactants exhibited low values of critical micellar concentration (CMC) ranging from 0.96 mg/L to 3.65 mg/L, indicating their superior efficiency in relation to the reported values for synthetic surfactants. The hydrophilic and lipophilic balance (HLB) of purified biosurfactants determined by the Griffin¿s method rangend from 8,25 to 9,12, consistent with those reported in the literature for MELs and comparable to synthetic surfactants used in cosmetic formulations. In the last step of this work, three experimental designs were carried to study of influence and optimization of the carbon and nitrogen sources present in the culture medium for biosurfactant production by P. aphidis strain UFMG-Y3468, followed by the chemometric analysis of the data using the Principal Component Analysis (PCA) to identify the correlations between carbon and nitrogen sources and the produced MEL isoforms. In the final step of this thesis was possible to obtain five statistically valid mathematical predictive models with coefficients of determination (R2) between 80% and 96% and high values of F calculated in the Fisher test, besides proving the important role of the hydrophobic carbon source in the discrimination of assays with better biosurfactant production profile. Therefore, this work opens the way to further studies for biotechnological production of glycolipid biosurfactants, specifically lipid mannosylmitritol and mannosyl mannitol lipid using the P. aphidis UFMG-Y3468 strain as well as the potential applications of these compounds in cosmetics and food fieldsDoutoradoCiência de AlimentosDoutor em Ciência de Alimentos140513/2016-7CNP

Optimization of biotechnological processes for the production of aroma compounds from monoterpenic substrates

Orientador: Gláucia Maria PastoreDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: A crescente demanda por processos que tenham impacto ambiental reduzido, rendimentos consideráveis e ainda custos minimizados recorrem à biotecnologia como uma ciência que pode tornar essa necessidade uma realidade. Processos biotecnológicos para a produção de compostos de interesse industrial como biocorantes, açúcares funcionais, biosurfactantes e biodiesel são uma tendência no meio científico e ganham destaque pela inovação, resultados e aplicações obtidos. O apelo comercial dos produtos biotecnológicos é outro fator que influencia as pesquisas haja vista que esses produtos são considerados naturais e tem-se comprovado que muitos deles apresentam atividades biológicas relevantes. O aroma é um importante atributo dos alimentos e pode ser produzido via biotecnológica a partir de substratos de baixo custo. A utilização de resíduos agro-industriais com alto teor nesses substratos, como por exemplo, resíduos ricos em monoterpenos são de grande interesse na biotecnologia de produção de aromas. Além disso, a utilização de micro-organismos isolados de diferentes fontes como biocatalisadores na produção desses compostos é outra perspectiva, muito explorada e ainda desafiadora. A biotransformação de monoterpenos para a produção de compostos de aroma é vantajosa quando comparada com a síntese química clássica. Contudo, alguns problemas ainda devem ser minimizados, como sensibilidade dos biocatalisadores à toxicidade dos substratos ou condições operacionais e baixos rendimentos. Assim, a seleção de novos biocatalisadores, como micro-organismos endofíticos, o uso de ferramentas de engenharia genética e a utilização de métodos estatísticos de otimização de processos são promissores e caracterizam-se como a nova vertente na área de bioaromas, contornando as desvantagens até então limitantes dos processos de biotransformação. Eles permitem em alguns casos, o aumento de escala e dessa forma a real aplicação da bioconversão microbiana na indústria. Assim, trabalhos que visem selecionar novos biocatalisadores é ainda uma necessidade e quando aliados a otimização de processos podem fornecer caminhos para que os rendimentos da bioconversão de monoterpenos sejam aumentados. Nesse contexto, o presente trabalho visou selecionar micro-organismos com potencial para a bioconversão de monoterpenos de baixo custo e presentes em grandes quantidades em resíduos agro-industriais, como 'alfa'-pineno e R-(+)-limoneno, para a produção de compostos de aroma valorizados comercialmente, S-(-)-verbenona e 'alfa'-terpineol. O capítulo 1 traz uma revisão sobre os compostos monoterpênicos, abordando suas características químico-estruturais, biossíntese e recentes aplicações terapêuticas, além do seu uso como substratos na biotecnologia de produção de compostos de aroma. O capítulo 2 é dividido em duas partes: a parte I mostra os resultados de um screening realizado com 81 linhagens de fungos isolados de diferentes fontes, entre eles, fungos endofíticos, utilizados no estudo do potencial de biotransformação de R-(+)-limoneno e 'alfa'-pineno. A parte II descreve a otimização do processo de produção de S-(-)-verbenona biotecnológica a partir de ?-pineno por uma linhagem de fungo endofítico, ainda não identificado, isolado da romã (Punica granatum). O uso de um Delineamento Composto Central Rotacional (DCCR) de 4 variáveis proporcionou o estabelecimento de condições mais favoráveis ao processo, elevando a concentração de verbenona que era de 66 mg.L-1 para aproximadamente 108 mg.L-1 em um dos ensaios do DCCR e em torno de 98 mg.L-1 no ensaio de validação. O capítulo 3 aborda a otimização do processo biotecnológico de produção de ?-terpineol a partir de R-(+)-limoneno pela linhagem Bacillus tequilensis isolada de resíduos de processamento de frutas cítricas. Após a otimização e validação do processo nas condições consideradas ótimas através da análise de superfícies de resposta, a concentração de ?-terpineol passou de 25 mg.L-1 para 85 mg.L-1. Portanto, os resultados obtidos no trabalho mostraram um ganho de rendimento nos processos de bioconversão de substratos monoterpênicos e que a otimização de processos é uma importante ferramenta para a melhoria das condições experimentais possibilitando trabalhos futuros que envolvam aumento de escalaAbstract: The growing demand for processes that reduce environmental impacts, have high yields and costs minimized uses biotechnology as a science that can make this need into a reality. Biotechnological processes for the production of industrial interesting compounds, such as biocolorant, functional sugars, biosurfactants and biodiesel, are a trend in the scientific field and are highlighted by innovation, application and results obtained. The commercial appeal of biotechnological products is another factor that stimulates researches since these products are considered natural and has been proven that many of them presents significant biological activities. The flavor is an important attribute of food and can be produced through biotechnological processes from low-cost-substrates. The use of agro-industrial wastes with high content of these substrates, such as residues rich in monoterpenes, is of great interest in biotechnology. Furthermore, the use of micro-organisms isolated from several sources as biocatalysts in the production of flavor compounds is another well-explored and challenging perspective. The biotransformation of terpenes for aroma compounds production is advantageous when compared with chemical synthesis. However, there are still some problems that must be minimized like biocatalysts sensitivity, toxicity of substrates and low yields. Thus, the selection of new biocatalysts, such as endophytic micro-organisms, use of genetic engineering and statistical methods for optimization of processes are promising and characterize a new perspective in bioaromas field. Thereby, works that search for new biocatalysts are necessary and associated with optimization processes can improve the monoterpenes bioconvertion. In this context, the objective of this work was the screening and selection of micro-organisms that are able to realize the biotransformation of low cost monoterpenes, 'alfa'-pinene and limonene, into high-value aroma compounds, such as verbenone and 'alfa-terpineol. The chapter 1 presents a current review on monoterpenes, showing their chemical characteristics, biosynthesis and recent therapeutic applications, besides their potential use as substrates for bioflavor production. The chapter 2 is divides in two parts: the first one shows the results of the screening of 81 strains of fungi isolated from several sources, including endophytic strains, for biotransformation of limonene and ?-pinene. The second part describes the optimization process for biotechnological verbenone production from "alfa'-pinene by a non-indentified fungal strain, isolated from pomegranate (Punica granatum). The use of a central composite rotatable design (CCRD) of 4 variables provided the optimal conditions for this process, increasing the verbenone concentration from 66 mg.L-1 to 108 mg.L-1 in one of the CCRD assays and 98 mg.L-1 after validation. The chapter 3 also shows an optimization study, in this case using limonene as substrate and the product obtained was ?-terpineol. The biocatalyst used was a bacterial strain, identified as Bacillus tequilensis, isolated from citrus fruit processing waste. After the optimization and validation in the best conditions obtained through the surface response analysis, the concentration of ?-terpineol reached was 85 mg.L-1. Therefore, the results obtained in this work show an improve of the yields in the biotransformation of both monoterpenic substrates and that optimization of processes is an important tool for the progress of future works involving scale-up studiesMestradoCiência de AlimentosMestra em Ciência de Alimento

An overview on ultrafiltration in food processing

Generally speaking, purification represents the most costly step within food production and biotechnological processes. Membrane-based processes, such as ultrafiltration (pore size from 10 to 1000 Å) are widely used on an industrial scale. Membrane-based processes are aligned to green chemistry concepts, that is, they are environmentally-friendly, do not generate harmful residues, show a low consumption of energy and an easy scale-up, among others. The food industry applies ultrafiltration to a wide range of fields. For instance (i) dairy - milk treatment, production of ice cream, etc. As an alternative to pasteurization of milk, ultrafiltration can be used also as pretreatment of milk for cheese production, in which large molecular weight compounds such as caseins, whey proteins, etc., are in the retentate, whereas low molecular weight compounds such as lactose and peptides are in the permeate. Similarly, low lactose yogurts can be produced (ii) beverage - during the juice clarification using membranes, pulp, pectin and essential oils are retained, whereas the juice itself is permeate. Ultrafiltration is also used in the production of clear beer and wine (concentration) (iii) degumming edible oils - (e.g., crude soybean oil, sunflower seed oil), in which phospholipids are removed (retentate) by ultrafiltration as an equivalent first step of the oil refining process (traditionally, carried out by water or dilute acid that leads to precipitation phospholipids) (iv) fish, poultry and gelatin - ultrafiltration is largely used for wastewater treatment processes, in particular for high protein content residues. Nevertheless, over the past few decades, the recovery of bioactive peptides and proteins from these wastewaters has drawn significant attention, that is, doubly advantageous (waste treatment and recovery of high added-value compounds) (v) drinking water treatment - high quality potable water implies the absence of microorganisms (e.g., Giardia), organic matter (e.g., humic substances), inorganic particles, and others hazardous substances. This water quality can be achieved by ultrafiltration, in which the main limitation is related to long-term flux decline (membrane fouling). Thus, membrane materials and membrane filtration operating systems, etc., should be better investigated (vi) recovery of specific molecules - plant proteins, enzymes (e.g., lysosyme) and phenolic compounds can be recovery and purified by ultrafiltration well-defined methodologies. Membrane-based separations, in particular ultrafiltration, are extensively used by food industry. However, improvements are still needed in virtually all applications

High anti-SARS-CoV-2 antibody seroconversion rates before the second wave in Manaus, Brazil, and the protective effect of social behaviour measures: results from the prospective DETECTCoV-19 cohort

Background: The city of Manaus, Brazil, has seen two collapses of the health system due to the COVID-19 pandemic. We report anti-SARS-CoV-2 nucleocapsid IgG antibody seroconversion rates and associated risk factors in Manaus residents before the second wave of the epidemic in Brazil. Methods: A convenience sample of adult (aged ≥18 years) residents of Manaus was recruited through online and university website advertising into the DETECTCoV-19 study cohort. The current analysis of seroconversion included a subgroup of DETECTCoV-19 participants who had at least two serum sample collections separated by at least 4 weeks between Aug 19 and Oct 2, 2020 (visit 1), and Oct 19 and Nov 27, 2020 (visit 2). Those who reported (or had no data on) having a COVID-19 diagnosis before visit 1, and who were positive for anti-SARS-CoV-2 nucleocapsid IgG antibodies at visit 1 were excluded. Using an in-house ELISA, the reactivity index (RI; calculated as the optical density ratio of the sample to the negative control) for serum anti-SARS-CoV-2 nucleocapsid IgG antibodies was measured at both visits. We calculated the incidence of seroconversion (defined as RI values ≤1·5 at visit 1 and ≥1·5 at visit 2, and a ratio >2 between the visit 2 and visit 1 RI values) during the study period, as well as incidence rate ratios (IRRs) through cluster-corrected and adjusted Poisson regression models to analyse associations between seroconversion and variables related to sociodemographic characteristics, health access, comorbidities, COVID-19 exposure, protective behaviours, and symptoms. Findings: 2496 DETECTCoV-19 cohort participants returned for a follow-up visit between Oct 19 and Nov 27, 2020, of whom 204 reported having COVID-19 before the first visit and 24 had no data regarding previous disease status. 559 participants were seropositive for anti-SARS-CoV-2 nucleocapsid IgG antibodies at baseline. Of the remaining 1709 participants who were seronegative at baseline, 71 did not meet the criteria for seroconversion and were excluded from the analyses. Among the remaining 1638 participants who were seronegative at baseline, 214 showed seroconversion at visit 2. The seroconversion incidence was 13·06% (95% CI 11·52–14·79) overall and 6·78% (5·61–8·10) for symptomatic seroconversion, over a median follow-up period of 57 days (IQR 54–61). 48·1% of seroconversion events were estimated to be asymptomatic. The sample had higher proportions of affluent and higher-educated people than those reported for the Manaus city population. In the fully adjusted and corrected model, risk factors for seroconversion before visit 2 were having a COVID-19 case in the household (IRR 1·49 [95% CI 1·21–1·83]), not wearing a mask during contact with a person with COVID-19 (1·25 [1·09–1·45]), relaxation of physical distancing (1·31 [1·05–1·64]), and having flu-like symptoms (1·79 [1·23–2·59]) or a COVID-19 diagnosis (3·57 [2·27–5·63]) between the first and second visits, whereas working remotely was associated with lower incidence (0·74 [0·56–0·97]). Interpretation: An intense infection transmission period preceded the second wave of COVID-19 in Manaus. Several modifiable behaviours increased the risk of seroconversion, including non-compliance with non-pharmaceutical interventions measures such as not wearing a mask during contact, relaxation of protective measures, and non-remote working. Increased testing in high-transmission areas is needed to provide timely information about ongoing transmission and aid appropriate implementation of transmission mitigation measures. Funding: Ministry of Education, Brazil; Fundação de Amparo à Pesquisa do Estado do Amazonas; Pan American Health Organization (PAHO)/WHO.World Health OrganizationRevisión por pare

Whey permeate as the raw material in galacto-oligosaccharide synthesis using commercial enzymes

Galacto-oligosaccharides (GOS), molecules with prebiotic properties are considered promising in the food industry. Its synthesis can be performed by enzymatic pathway, using commercial microbial enzymes. The reaction, known as transgalactosylation, is mediated by the enzyme beta-galactosidase and its catalysis is influenced during the process by substrate concentration present (in this case lactose), pH, and temperature, among others. The use of whey permeate, a by-product of the dairy industry, demonstrates the interest in making such processes viable from an economic and technological point of view. The main of this work was to use whey permeate as raw material in an enzymatic GOS synthesis, comparing three commercial enzymes of different microbial sources. For better performance, the results on lactose conversion, yield, and specific productivity were evaluated. The commercial enzyme of kluyveromyces laths (Lactozyme (TM) 2600 L) showed the best results for lactose conversion (89.27%), yield (25 g GOS/100 g lactose) and specific productivity (51 g GOS/g enzyme*h). Thus, it can be considered suitable for further technological development. Asperaus oryzae commercial enzyme also showed good results and could be used for other studies either. However, the Escherichia coli commercial enzyme did not present good results in GOS synthesis, being more appropriate to lactose hydrolysis reactions. All the three enzymes showed a decrease in the production and even depletion of GOS molecules, and therefore, smaller reaction times should be established. New stages of optimization and processes development should be considered in future works, in order to obtain best yields and productivities1247885CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140512/2016-0não temnão tem12th Latin American symposium on food science (SLACA) : food science and its impact on a changing worl

Current Status In Biotechnological Production And Applications Of Glycolipid Biosurfactants

Biosurfactants are natural compounds with surface activity and emulsifying properties produced by several types of microorganisms and have been considered an interesting alternative to synthetic surfactants. Glycolipids are promising biosurfactants, due to low toxicity, biodegradability, and chemical stability in different conditions and also because they have many biological activities, allowing wide applications in different fields. In this review, we addressed general information about families of glycolipids, rhamnolipids, sophorolipids, mannosylerythritol lipids, and trehalose lipids, describing their chemical and surface characteristics, recent studies using alternative substrates, and new strategies to improve of production, beyond their specificities. We focus in providing recent developments and trends in biotechnological process and medical and industrial applications.10024102651029

Reduced blood pressure and abdominal circumference and Improves Functional Capacity of older hypertensive submitted Functional Physical Therapy Program

The objective was to investigate the effect of functional physiotherapy program (FFP) on hemodynamic, anthropometric variables and functional capacity (FC) in hypertensive elderly women. Study subjects were seven elderly hypertensive women (68.1 ± 7.7 years). Anthropometry and blood pressure measurement were performed. To measure the FC were applied the six-minute walk test (6 MWT), the Timed Up &amp; Go (TUG) and the step test (ST). After the FFP, reduction in diastolic blood pressure (DBP) of 77.7 ± 5.6 mmHg to 70.1 ± 8.2 mmHg and abdominal circumference (AC) of 95.6 ± 7.8 cm to 93.6 ± 7, 3 cm and increased 6 MWT of 438 ± 58 m to 506 ± 61 m were observed. It was concluded that the FFP contributed in DBP reduction and AC and FC, evaluated by the 6 MWT test in hypertensive elderly. <br /