Alternative strategy for pigments production using solid-statefermentation and cheese whey

Natural pigments have several industrial applications, namely in the textile industry for dyeing cloths, or in the food industry, as coloring agents. Pigments can also be used in cosmetics, leather or in the pharmaceutical industry. More recently, other applications were found for pigments like in histological staining, in solar cells or as pH indicators [1]. Microbial production of natural pigments has been considered a promising alternative to synthetic pigments. Filamentous fungi are known to produce many different pigments. Recently, some Penicillium species, such as P. chrysogenum and P. purpurogenum, were described as effective pigment producers. Solid-state fermentation (SSF) has gained attention and is preferred for fungi cultivation due to betters yields and easier and less expensive product recovery [2]. Preliminary results indicated that pigment production was favored when lactose was used as carbon source under submerged fermentation (SmF). Since CW, a by-product from cheese industry, contains high lactose content, it was used as an inexpensive alternative fermentation medium to induce the pigment production. In this work, the production of pigments by Penicillium sp. was evaluated under SSF conditions using an inert support and the results obtained were compared with those obtained under SmF conditions. The studied culture media comprised a synthetic medium [3] and alternative media containing cheese whey (CW). A mixture of three pigments (yellow, orange and red with max=400, 470 and 500 nm, respectively) was obtained. To evaluate the best conditions for pigments production, the sum of absorbances was determined and the synthetic medium under SmF conditions was used as reference medium to calculate the relative absorbance. After 12 days of fermentation, the synthetic medium presented the highest pigment production (Figure 1) for both SmF and SSF conditions. The supplementation of CW with yeast extract and peptone considerably improved the production of pigments under SmF conditions. On the other hand, under SSF conditions an increase in supplementation was followed by a decrease in pigments production. These results suggest that SSF combined with a more concentrated CW medium can be a promising strategy to improve pigment production using a less expensive fermentation condition and alternative media totally composed of an industrial by-product.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Alternative strategy for pigments production using solid-state fermentation and cheese whey

Natural pigments have several industrial applications, namely in the textile industry for dyeing cloths, or in the food industry, as coloring agents. Pigments can also be used in cosmetics, leather or in the pharmaceutical industry. More recently, other applications were found for pigments like in histological staining, in solar cells or as pH indicators [1]. Microbial production of natural pigments has been considered a promising alternative to synthetic pigments. Filamentous fungi are known to produce many different pigments. Recently, some Penicillium species, such as P. chrysogenum and P. purpurogenum, were described as effective pigment producers. Solid-state fermentation (SSF) has gained attention and is preferred for fungi cultivation due to betters yields and easier and less expensive product recovery [2]. Preliminary results indicated that pigment production was favored when lactose was used as carbon source under submerged fermentation (SmF). Since CW, a by-product from cheese industry, contains high lactose content, it was used as an inexpensive alternative fermentation medium to induce the pigment production. In this work, the production of pigments by Penicillium sp. was evaluated under SSF conditions using an inert support andthe results obtained were compared with those obtained under SmF conditions. The studied culture media comprised a synthetic medium [3] and alternative media containing cheese whey (CW). A mixture of three pigments (yellow, orange and red with max=400, 470 and 500 nm, respectively) was obtained. To evaluate the best conditions for pigments production, the sum of absorbances was determined and the synthetic medium under SmF conditions was used as reference medium to calculate the relative absorbance. After 12 days of fermentation, the synthetic medium presented the highest pigment production (Figure 1) for both SmF and SSF conditions. The supplementation of CW with yeast extract and peptone considerably improved the production of pigments under SmF conditions. On the other hand, under SSF conditions an increase in supplementation was followed by a decrease in pigments production. These results suggest that SSF combined with a more concentrated CW medium can be a promising strategy to improve pigment production using a less expensive fermentation condition and alternative media totally composed of an industrial by-product.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Production of natural pigments by Penicillium brevicompactum using agro-industrial byproducts

The demand for natural pigments for industrial applications has significantly increased. Penicillium brevicompactum was recently reported as a promising pigments producer using submerged fermentation and a synthetic culture medium containing lactose. In this work, pigment production by P. brevicompactum was studied under different fermentation conditions, namely, submerged fermentation with free (SmF) and immobilized mycelium (SmFi), and solid-state fermentation (SSF). The potential of culture media composed of agro-industrial byproducts (cheese-whey (CW) and corn steep liquor (CSL)) was investigated for the first time as low-cost alternatives to pigment production by P. brevicompactum. The fungus showed great adaptability to the different culture media and types of fermentation, being able to synthesize pigments under all the tested conditions. A culture medium composed of 34.6 g/L of CW and 8 g/L of CSL proved to be the most suitable alternative to the synthetic medium, especially under SmF and SmFi. Our data also show that different mixtures of pigments (yellow, orange, and red) can be produced depending on the medium composition and the type of fermentation. Additionally, the immobilization and reuse of biomass to produce pigments by P. brevicompactum were demonstrated for the first time, suggesting the possibility of operating under repeated batch mode at an industrial scale.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems

Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEGamine) or conjugation with positively charged amino acids (PEGlysine, PEGarginine, and PEGhistidine) were studied to increase the selectivity of PEGdextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEGdextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination.Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the NMR Portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho) and strategic funding for CQUM (UID/QUI/00686/2020).info:eu-repo/semantics/publishedVersio

More than coloring agents: natural pigments with antimicrobial activity

Natural pigments interest has rapidly increased due to a risen awareness concerning sustainability and green economy policies, and as an attempt to comply with changes in consumer demands. Health and safety concerns due to synthetic pigments also prompt the crescent global market trend for natural pigments, valued at 5862 million USD in 2022 and estimated to reach 9824.5 million USD by 2032. Natural pigments are non-toxic, biocompatible coloring agents that can also provide additional biological properties (antioxidant, antimicrobial, anti-inflammatory, anticancer activity, etc.). For this reason, they have been attracting several industrial sectors and have gradually replaced synthetic pigments and dyes. Among natural sources, microbes, particularly Fungi, have gained special attention over the last years as valuable producers of natural pigments. Recently, we have described the ability of a Penicillium strain to produce pigments under different growth conditions (fermentation type and medium). Also, its ability to produce pigments in low-cost, alternative media composed only of agroindustrial byproducts like cheese whey and corn steep liquor was demonstrated. Interestingly, according to the growth condition, different pigment mixtures were produced, presenting distinct antioxidant potential and antimicrobial activity. Outstanding results were achieved with the mixture of pigments ethanolic extracted from the Penicillium mycelium grown in agar plates. This crude extract had the highest concentration of flavonoids and phenolic compounds, despite its low antioxidant potential. Nevertheless, it was effective in inhibiting (0.31-0.62 mg/mL) and/or killing (> 0.62 mg/mL) gram-negative and grampositive bacteria, yeast, and filamentous fungi, according to the agar diffusion method and the minimum inhibitory concentration (MIC) assays. Our research showed alternative and sustainable approaches to produce natural pigments with proven antimicrobial activity and antioxidant potential. Those can be attractive for the food, beverages, pharmaceutical, and cosmetics industries not only as coloring agents but also as alternatives to the common antimicrobial and disinfectant agents.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020info:eu-repo/semantics/publishedVersio

Partitioning of fibrinolytic protease from Bacillus sp. UFPEDA 485 by aqueous two-phase systems using PEG/sodium sulfate

XI Reunião Regional Nordeste da SBBq | 4th International Symposium in Biochemistry of Macromolecules and BiotechnologyThe fibrinolytic protease produced by bacteria of the genus Bacillus has attracted large interest in the pharmaceutical industry as a promising alternative in thrombolytic therapy due to their effectiveness in degrading fibrin, its production requiring the development of an efficient recovery process. Aqueous two-phase system (ATPS) have been recognized as an efficient and economical process for recovering enzymes due to their relative ease and low cost. The purpose of this work was to study the partition of fibrinolytic protease produced by Bacillus sp. UFPEDA 485 in a ATPS composed by Polyethylene glycol (PEG) and sodium sulfate using factorial design. The fibrinolytic protease production occurred in liquid culture medium containing 2% soy flour, pH 7.2, 150 rpm at 37 °C for 48 hours. To study the partitioning, was used a 23 full factorial design with four replicates at the central with the purpose of evaluating the effects and interactions of the independent variables: PEG molar mass (MMPEG), PEG concentration (CPEG) and sodium sulfate concentration (CNa2SO4) on the response variable: partition coefficient (K) of the fibrinolytic enzyme. In all the runs the enzyme partitioned to the top phase, indicating a significant interaction between the protein and the PEG. The best result was obtained at the central point, using MMPEG 6000 g/mol, CPEG 24 % and CNa2SO4 11,6 %. The partitions coefficients ranged between K = 327.63 and 2879.38. According to the results, the ATPS composed of PEG/sodium sulfate proved to be a promising method to extraction fibrinolytic protease.CAPES e CNPqinfo:eu-repo/semantics/publishedVersio

Análise de interações soluto-água por partição em múltiplos sistemas de duas fases aquosas

Programa doutoral em Engenharia Química e BiológicaO objetivo principal do presente trabalho é contribuir para a melhor compreensão dos mecanismos envolvidos na partição de solutos em sistemas de duas fases aquosas (SDFAs). Para este trabalho, foram selecionados sistemas PEG–Na2SO4 e PEG–DEX e foi estudado o efeito da adição de diferentes sais aditivos (NaCl, NaClO4, NaSCN e NaH2PO4) e/ou osmólitos (Sorbitol, Sacarose, Trealose e TMAO) nas propriedades do solvente no meio aquoso nas fases e na partição de solutos. Os parâmetros solvatocrómicos, polaridade (π*), capacidade do solvente em participar como dador (α) e como aceitador (β) de pontes de hidrogénio foram determinados e usados para caracterizar as fases em equilíbrio dos SDFAs. Os sistemas foram também caracterizados em termos das diferenças de hidrofobicidade relativa e das propriedades eletrostáticas das fases. Os coeficientes de partição (K) de várias biomoléculas (compostos orgânicos de baixo peso molecular iónicos e não iónicos e proteínas) foram determinados em todos os SDFAs. Verificou-se que os coeficientes de partição destes compostos podiam ser correlacionados de acordo com a equação de Collander. A partição dos solutos e as interações soluto-solvente foram descritas como uma combinação linear dos descritores específicos dos solutos, por uma relação linear de energia livre (LFER) baseada no modelo de Abraham, e previamente modificada. Os descritores específicos dos solutos obtidos em SDFAs com e sem aditivos foram comparados para verificar a existência de interações diretas aditivo-solutos ou se a presença dos aditivos aumentava a estabilidade estrutural dos solutos apenas através do seu efeito nas propriedades da água como solvente. O uso da equação de Collander e/ou da LFER para determinar os descritores específicos de moléculas biológicas e, consequentemente, para prever o comportamento da partição de solutos em SDFAs previamente caracterizados, mostrou ser um contributo importante para um conhecimento mais profundo dos SDFAs. Os resultados apresentados contribuem para uma melhor interpretação dos efeitos de diferentes aditivos na partição de solutos. Espera-se que estes contribuam para o design de SDFAs mais eficientes que possam ser usados numa vasta gama de aplicações.The main purpose of the present work is to contribute for a better understanding of the mechanisms governing solute partitioning in aqueous two‐phase systems (ATPSs). For this work, PEG–Na2SO4 and PEG–DEX systems were selected and the effect of addition of different salts additives (NaCl, NaClO4, NaSCN and NaH2PO4) and/or osmolytes (Sorbitol, Sucrose, Trehalose and TMAO) on phases solvent properties and in solute partitioning was assessed. The solvatochromic parameters characterizing the solvent's dipolarity/polarizability (π*), solvent hydrogen‐bond donor acidity (α), and solvent hydrogen‐bond acceptor basicity (β) of aqueous media were measured in the coexisting phases of each of the systems used. Also, all the systems were characterized in terms of the difference between the relative hydrophobicity and the electrostatic properties of the phases. Partition coefficients (K-values) of several biomolecules (ionic and nonionic small organic compounds and proteins) were obtained in all the ATPSs. It was found that the partition coefficients of the compounds were correlated according to the so-called Collander equation. Solutes partitioning and solutes-solvent interactions were described by a Linear Free Energy Relationship (LFER) based on the Abraham model, which was previously modified, as a linear combination of the so-called solute specific descriptors. The solute specific descriptors values obtained in ATPSs containing different additives were compared to those obtained in ATPSs without additives to verify if there were direct additive-solutes interactions or whether the additive presence enhances the solute structure stability due to its effect on the water solvent properties only. The use of the Collander equation and/or the LFER to determine solute specific descriptors for biological molecules and, consequently, to predict solute partitioning behavior in previously characterized ATPSs, was shown to be a valuable contribution to the understanding of ATPSs technology. The results presented not only contribute for better interpreting the effects of different additives on the solute partition in ATPSs but will also result in the design of more efficient ATPSs to be used in a wide range of applications.À Fundação para a Ciência e Tecnologia (FCT) agradeço pela atribuição da bolsa de doutoramento (ref. SFRH/BD/111825/2015) e por todo o apoio financeiro disponibilizado

Conventional and alternative microbial sources of food-grade pigments and their production processes

Food colorant regulation has been updated and improved over the years according to the latest knowledge in the field. Aiming to avoid carcinogenicity and toxicological effects on human health, synthetic pigments have been de-authorized. In recent decades, their gradual substitution with alternative pigments obtained from natural sources has increased. Natural pigments are frequently associated with greener, more sustainable production. Furthermore, these pigments can present additional biological properties such as antioxidant, anticancer, anti-inflammatory, and antimicrobial activity. Overall, these features are highly appreciated by consumers, contributing to the increasing demand by the food industry for novel natural pigments. Microorganisms are considered the most promising source of natural pigments, over plants and animals, due to their non-seasonality, high availability and easy scalability. In this chapter, an overview of conventional and alternative sources of food-grade pigments will be presented, and details of their production processes and extraction will be discussed. Additionally, the regulatory status and the main examples of their incorporation into food products will be highlighted.(undefined)info:eu-repo/semantics/publishedVersio

Penicillium brevicompactum as a novel source of natural pigments with potential for food applications

Synthetic colorants have gradually been replaced by natural pigments, mainly in the food industry. Among the several natural sources of pigments, filamentous fungi have gained special attention. However, the eventual biosynthesis of mycotoxins is a limitation for the practical use of this kind of microorganisms as pigment producers. In this work, Penicillium brevicompactum was studied for the first time as a potential pigment producer using submerged fermentation. The effect of several experimental parameters (culture medium composition, agitation speed, type of carbon source, temperature, concentration of supplements and carbon source, natural light and initial pH) in the production of pigments was evaluated. Under the optimal conditions (culture medium I containing lactose (20g/L) and peptone/yeast extract (8/8g/L), 23°C, initial pH 7.0, 150rpm, and natural light), a mixture of pigments (yellow, orange, and red) was obtained. This mixture is mycotoxin-free (ochratoxin A and mycophenolic acid) and presented promising antioxidant activity (FRAP: 58.58±4.58µmol Fe(II)/g; DPPH IC50: 18.48±0.26µmol TE/g and ABTS IC50: 28.38±3.79µmol TE/g). Furthermore, these pigments were proved to be very stable for a wide range of pH and temperatures. Overall, it was demonstrated that P. brevicompactum can be a safe source of natural pigments with interesting properties for the food industry.This work was financially supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. Luís Abrunhosa acknowledges FCT for the assistant research contract CEECIND/00728/2017, obtained under CEEC Individual 2017.info:eu-repo/semantics/publishedVersio

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit