Occurrence and diversity of yeast species isolated from fish feed and tambatinga gut

The present study aimed to identify yeasts species isolated from fish feed and the intestinal tracts of tambatinga fish (Colossoma macropomum × Piaractus brachypomum) cultivated in a Brazilian fish farm. Twenty tambatingas and 30 fish feed samples from different brands were acquired from two commercial establishments in the state of Piauí, Brazil. The sampled guts were divided into three equal parts, namely the anterior, medium and posterior portions, totaling 60 samples. Molecular identification was performed by PCR amplification and sequencing of the D1/D2 regions of the large rRNA subunit gene. Sixteen Candida nivariensis strains in the gut were identified. The fish feed samples showed a higher variability of yeast species, with the following species isolated: Hyphopichia burtonii (23.08%), Lodderomyces elongisporus (15.39%) and Trichosporon asahii (15.39%), Candida nivariensis (7.69%), C. orthopsilosis (7.69%), C. parapsilosis (7.69%), Rhodotorula minuta (7.69%), Sterigmatomyces elviae (7.69%), Cryptococcus liquefaciens (7.69%). Yeast found in feed, however, was not always isolated from tambatinga gut. Molecular identification allowed for the isolation of yeast species not previously reported in fish feed and gut samples.Fil: Pinheiro, Raizza Eveline E.. Universidade Federal Do Piaui;Fil: Dourado Rodrigues, Aline Maria. Universidade Federal Do Piaui; BrasilFil: Santos, Julliet Teixeira de O.. Universidade Federal Do Piaui; BrasilFil: Costa, Juliana de A.. Universidade Federal Do Piaui; BrasilFil: Pereyra, Carina Maricel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Torres, Adriana Mabel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Rosa, Carlos A.. Universidade Federal de Minas Gerais; BrasilFil: Santos, Ana Raquel de O.. Universidade Federal de Minas Gerais; BrasilFil: Muratori, Maria Christina S.. Universidade Federal Do Piaui; Brasi

Recent strategies and applications for l-asparaginase confinement

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

Use of tetramethylbenzidine for the spectrophotometric sequential injection determination of free chlorine in waters

A sequential injection (SI) method was developed for the spectrophotometric determination of chlorine based on the reaction between tetramethylbenzidine (TMB) and free chlorine. The advantages resulting from the use of TMB are considerable: TMB is highly selective for chlorine, it enables a fairly low quantification limit and represents a less toxic alternative to reagents such as tolidine. The use of this reaction in SI adds other advantages as it enhances the degree of automation, minimisation of reagent consumption (6.8 g TMB/assay) and low effluent production (2.5 mL/determination). The developed method allowed a quantification limit of 90 g/L with a working range of 0.09–1.30 mg OCl−/L and a determination rate of 60 det./h. Based on these features, the system was applied to tap-water and surface water samples with no previous treatment required. The results obtained with the developed system were compared to the reference method, diethyl-p-phenylelediamine (DPD) colorimetric method, and proved not to be statistically different

L-asparaginase-based biosensors

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

Unveiling the Influence of Carbon Nanotube Diameter and Surface Modification on the Anchorage of L-Asparaginase

L-asparaginase (ASNase, EC 3.5.1.1) is an amidohydrolase enzyme known for its anti-cancer properties, with an ever-increasing commercial value. Immobilization has been studied to improve the enzyme’s efficiency, enabling its recovery and reuse, enhancing its stability and half-life time. In this work, the effect of pH, contact time and enzyme concentration during the ASNase physical adsorption onto pristine and functionalized multi-walled carbon nanotubes (MWCNTs and f-MWCNTs, respectively) with different size diameters was investigated by maximizing ASNase relative recovered activity (RRA) and immobilization yield (IY). Immobilized ASNase reusability and kinetic parameters were also evaluated. The ASNase immobilization onto f-MWCNTs offered higher loading capacities, enhanced reusability, and improved enzyme affinity to the substrate, attaining RRA and IY of 100 and 99%, respectively, at the best immobilization conditions (0.4 mg/mL of ASNase, pH 8, 30 min of contact time). In addition, MWCNTs diameter proved to play a critical role in determining the enzyme binding affinity, as evidenced by the best results attained with f-MWCNTs with diameters of 10–20 nm and 20–40 nm. This study provided essential information on the impact of MWCNTs diameter and their surface functionalization on ASNase efficiency, which may be helpful for the development of innovative biomedical devices or food pre-treatment solutionspublishe

Immobilization of L-asparaginase towards surface-modified carbon nanotubes

L-asparaginase (LA) is an enzyme that catalyzes L-asparagine hydrolysis into L-aspartic acid and ammonia and is mainly applied in pharmaceutical and food industries. The LA currently commercialized for pharmaceutical purposes is produced from two main bacterial sources: recombinant Escherichia coli and Erwinia chrysanthemi. However, some disadvantages are associated with its free form, such as the shorter half-life. Immobilization of LA has been proposed as an efficient approach to overcome this limitation. In this work, a straightforward method, including the functionalization of multi-walled carbon nanotubes (MWCNTs) through a hydrothermal oxidation treatment and the immobilization of LA by adsorption over pristine and modified MWCNTs was investigated. Different operation conditions, including pH, contact time, ASNase/MWCNT mass ratio, and the operational stability of the immobilized LA, were evaluated. The characterization of the LA-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial LA above 95%, under the optimized adsorption conditions (pH 8, 60 min of contact, and 1.510–3 g.mL-1 of LA). The LA-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), proving its suitability for application in industrial processes.publishe

Immobilization of L-asparaginase towards surface-modified carbon nanotubes

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes L-asparagine hydrolysis into L-aspartic acid and ammonia and is mainly applied in pharmaceutical and food industries [1]. The ASNase currently commercialized for pharmaceutical purposes is produced from two main bacterial sources: recombinant Escherichia coli and Erwinia chrysanthemi. However, some disadvantages are associated with its free form, such as the shorter half-life [2]. Immobilization of ASNase has been proposed as an efficient approach to overcome this limitation [3]. In this work, a straightforward method, including the functionalization of multi-walled carbon nanotubes (MWCNTs) through a hydrothermal oxidation treatment with nitric acid, and the immobilization of ASNase by adsorption over pristine and modified MWCNTs was investigated. Different operation conditions, including pH, contact time, ASNase/MWCNT mass ratio, and the operational stability of the immobilized ASNase were evaluated. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95%, under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5´10–3 g.mL-1of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), proving its suitability for application in industrial processes.publishe

Superior operational stability of immobilized L-asparaginase over surface-modified carbon nanotubes

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes the L-asparagine hydrolysis into L-aspartic acid and ammonia, being mainly applied in pharmaceutical and food industries. However, some disadvantages are associated with its free form, such as the ASNase short half-life, which may be overcome by enzyme immobilization. In this work, the immobilization of ASNase by adsorption over pristine and modified multi-walled carbon nanotubes (MWCNTs) was investigated, the latter corresponding to functionalized MWCNTs through a hydrothermal oxidation treatment. Different operating conditions, including pH, contact time and ASNase/MWCNT mass ratio, as well as the operational stability of the immobilized ASNase, were evaluated. For comparison purposes, data regarding the ASNase immobilization with pristine MWCNT was detailed. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric Analysis (TGA) and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95% under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5 × 10-3 g mL-1 of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), paving the way for its use in industrial processes.publishe

Microbiological quality in the flour and starch cassava processing in traditional and model unit

O objetivo deste trabalho foi avaliar a qualidade microbiológica de farinha e fécula durante as diferentes etapas do processamento de mandioca, em unidades tradicionais e em uma unidade modelo. Foram determinados índices de coliformes totais e termotolerantes, Bacillus cereus, Salmonela spp., bactérias e fungos nas farinhas e féculas. Não foram observadas presenças de B. cereus e Salmonella spp. na farinha e fécula de mandioca produzidas nas unidades estudadas. A incidência microbiana diminui com o avanço da etapa do processamento para obtenção de farinha e foi menor na unidade modelo. Após o processo de torra, a carga microbiana estava de acordo com os valores preconizados pela legislação brasileira, concluindo-se que essa etapa pode ser considerada como crítica na obtenção de farinha. Na obtenção de fécula, a carga microbiana nas unidades tradicionais são maiores que na modelo, e o aumento do número de extrações promove o aumento da incidência de microrganismos, sendo recomendadas apenas quatro extrações. ______________________________________________________________________________________ ABSTRACT: The objective of this research was to evaluate microbiological contamination in the flour and starch during cassava processing in traditional and model units. The total and fecal coliforms indexes, Bacillus cereus, Salmonella, bacteria, yeast and fungi were determined. Bacillus cereus and Salmonella were not detected in any sample. The incidence of microorganisms decreased along the processing to obtain cassava flour, and is lower in model unit. After the roasting process, the microbial load was below the values established by the Brazilian legislation, and can be regarded as a critical step in obtaining cassava flour. Concerning starch production, the microbial load in the traditional units was higher than in the model units, and the increase of the extraction steps has promoted the growth of microorganisms. It’s recommended the used of only 4 extractions