Heterologous expression of a rice metallothionein isoform (OsMTI-1b) in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance

Abstract Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b) on the tolerance of Saccharomyces cerevisiae to Cd2+, H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd2+ and accumulated more Cd2+ ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

Antioxidant potential of yerba mate (Ilex paraguariensis St. Hil.) extracts in Saccharomyces cerevisae deficient in oxidant defense genes / Potencial antioxidante de extratos de erva-mate (Ilex paraguariensis St. Hil.) em Saccharomyces cerevisiae deficientes para genes de defesa oxidante

Abstract Yerba-mate (Ilex paraguariensis St. Hil) is mainly consumed as “chimarrão”, a hot drink highly appreciated in Brazil, Argentina, Paraguay and Uruguay. This study evaluated the antioxidant potential of aqueous extracts of I. paraguariensis precipitated with ethanol. The leaves were processed as for tea product (TM) and oxidized (OX). The antioxidant potential was evaluated in cells of Saccharomyces cerevisiae deficient in antioxidant defense genes. Three strains evaluated were: a wild (EG) and two mutants (ctt1Δ e ctt1Δsod1Δ). These strains were pre-treated with the yerba-mate extracts (TM e OX) and submitted to oxidative stress induced by hydrogen peroxide. None of the extracts produced loss of cell viability. The extracts exerted antioxidant activity, protecting the strains (except sod1∆ctt1∆). The TM extract was more effective than OX. I. paraguariensis extracts showed a potential to be explored in the development of new products.

Resumo A erva-mate (Ilex paraguariensis St. Hil) é consumida principalmente como “chimarrão”, uma bebida quente muito apreciada no Brasil, Argentina, Paraguai e Uruguai. Este estudo avaliou o potencial antioxidante de extratos aquosos de I. paraguariensis precipitado com etanol. Folhas de erva-mate foram processados de maneira semelhante ao processamento do chá-preto (OX) e na forma de mate (TM). O potencial antioxidante foi avaliado sobre células de Saccharomyces cerevisiae deficientes para genes de defesa antioxidante. Três linhagens celulares foram estudadas: uma selvagem (EG) e duas mutantes (ctt1Δ e ctt1Δsod1Δ). As linhagens foram pré-tratadas com os extratos de erva-mate (TM e OX) e submetidos ao estresse oxidativo induzido por peróxido de hidrogênio. Nenhum dos extratos produziu perda de viabilidade celular. Os extratos exerceram atividade antioxidante, protegendo as linhagens (exceto a sod1Δctt1Δ). O extrato TM foi mais eficaz em relação ao OX. Extratos de I. paraguariensis apresentaram potencial para ser explorado no desenvolvimento de novas formulações.

Screening and characterization of β-glucosidase production by Saccharomyces cerevisiae.

Beta-Glucosidases (BGS) are the group of hydrolase enzymes, involved in the degradation processes and many biological processes. Due to demand, intensive screening of BGS is required to explore the natural microbial source of BGS. The current study deals with isolation and identification of BGS producing S. cerevisiae from Thai fruits & beverages and assessment of impact of pH, temperature, and salt concentration on BGS production. About 34 samples were collected. Yeast cells were isolated by plate method and characterized. About ten different strains were isolated and identified. The strain has been confirmed as S. cerevisiae through ribosomal sequencing. The optimization of BGS production was achieved by Box-Behnken design and Response Surface Methodology and confirmed that pH 4.0, temperature at 40 C, and 0.5% of NaCl are optimum conditions. The kinetic analysis suggested that 24 h of incubation achieve the maximum yield. The reported S. cerevisiae strain could be the safer source for BGS. Further studies on enzyme recovery and purification will unbolt the way to attain high-quality microbial enzyme.

Capacidade antioxidante celular da rutina frente ao dano oxidativo induzido em linhagens mutantes de Saccharomyces cerevisiae / Cellular antioxidant capacity of rutin against the oxidative damage induced in mutant strains of Saccharomyces cerevisiae

A rutina é um tipo de flavonoide encontrado nas plantas e de grande interesse farmacológico, já que muitas propriedades têm sido atribuídas a rutina, incluindo antialérgica, anti-inflamatória, antitumoral e principalmente antioxidante. O objetivo deste estudo foi proporcionar um maior conhecimento sobre a capacidade antioxidante celular da rutina utilizando linhagens de S. cerevisiae proficiente e deficiente em defesas antioxidantes. As linhagens de S. cerevisiae (Sodwt, Sod1Δ, Sod2Δ, Sod1ΔSod2Δ, Cat1Δ, Sod1ΔCat1Δ) foram expostas as várias concentrações da rutina em três diferentes condições de tratamento (prétratamento, co-tratamento e pós-tratamento) e assim verificar se a rutina inibe o efeito oxidativo do peróxido de hidrogênio, permitindo o aumento da sobrevivência das linhagens testadas. Os resultados obtidos mostram que a rutina diminui significativamente os danos oxidativos nas linhagens de S. cerevisiae nas condições de pré-tratamento, co-tratamento e pós-tratamento, demonstrando que a rutina apresenta uma elevada capacidade antioxidante celular, sendo importante na proteção ao estresse oxidativo induzido.

Rutin is a type of flavonoid found in plants and of great pharmacological interest since many properties have been attributed rutin, including antiallergic, antiinflammatory, antitumor and antioxidant primarily. The aim of this study was to provide greater insight into the cellular antioxidant capacity of rutin using strains of S. cerevisiae proficient and deficient in antioxidant defenses. The strains of S. cerevisiae (Sodwt, Sod1Δ, Sod2Δ, Sod1ΔSod2Δ, Cat1Δ, Sod1ΔCat1Δ) were exposed to various concentrations of rutin in three different conditions of treatment (pre-treatment, co-treatment and post-treatment) and thus determine whether the rutin inhibits the oxidative effect of hydrogen peroxide, allowing increased survival of the strains tested. The results show that rutin significantly reduces oxidative damage in strains of S. cerevisiae under the conditions of pre-treatment, co-treatment and post-treatment, demonstrating that rutin has a high cellular antioxidant capacity, being important in protection to induced oxidative stress.

In vitro ability of beer fermentation residue and yeast-based products to bind aflatoxin B1

This study aimed to verify the in vitro ability of beer fermentation residue (BFR) containing Saccharomyces cerevisiae cells and five commercial products that differed in the viability and integrity of S. cerevisiae cells to remove aflatoxin B1 (AFB1) from a citrate-phosphate buffer solution (CPBS). BFR was collected at a microbrewery and prepared by drying and milling. The commercial yeast-based products were as follows: inactive intact yeast cells from beer alcoholic fermentation, inactive intact yeast cells from sugarcane alcoholic fermentation, hydrolyzed yeast cells, yeast cell walls and active yeast cells. Adsorption assays were performed in CPBS spiked with 1.0 μg AFB1/mL at pH 3.0 and 6.0 for a contact time of 60 min at room temperature. Analysis of AFB1 in the samples was performed by high performance liquid chromatography. AFB1 adsorption by the products ranged from 45.5% to 69.4% at pH 3.0 and from 24.0% to 63.8% at pH 6.0. The higher percentages (p < 0.05) of AFB1 binding at both pH values were achieved with products containing hydrolyzed yeast cells or yeast cell walls rather than intact cells. The AFB1 binding percentages of BFR were 55.0 ± 5.0% at pH 3.0 and 49.2 ± 4.5% at pH 6.0, which was not significantly different (p > 0.05) from commercial products containing inactive intact yeast cells. The results of this trial indicate that the yeast-based products tested, especially the BFR, have potential applications in animal feeds as a suitable biological method for reducing the adverse effects of aflatoxins.

Avaliação da capacidade antioxidante in vitro e in vivo do extrato etanólico da Copernicia prunifera (Mill.) H. E. Moore / Assessment of antioxidant capacity in vitro and in vivo of the ethanol extract of Copernicia prunifera (Mill.) H. E. Moore

The aim of this study was to assess the in vitro andin vivo antioxidant capacity of the ethanol extract ofCopernicia prunifera (Mill.) H. E. Moore leaves. Theleaves were collected in the city of Teresina, Piauístate (Brazil), dried for 4 days, crushed and extractedby maceration in 95% ethanol for 16 days, with achange of solvent every four days. Afterwards, theethanol extract was filtered, concentrated in a rotaryevaporator, lyophilized and weighed. Its in vitroantioxidant activity was determined as the capacity toscavenge the radicals 2,2-diphenyl-1-picrylhydrazyl(DPPH?) and 2,2?-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS?+). To determine the antioxidantcapacity in vivo, Saccharomyces cerevisiae strains withnormal and defective antioxidant defenses were usedas a study model. The quantitative phytochemicalanalysis revealed the following components: phenoliccompounds (1454.54 ± 12.56 mgGAE/g of extract and167.27 ± 1.44 mgGAE/g of dry material), flavonoids(645.73 ± 2.42 MgR/g of extract and 74.26 ± 0.27 mgR/gdry material), hydrolysable tannins (64 mgGAE/gof extract) and alkaloids (28.86% dry material). Theantioxidant analysis showed that the ethanol extract ofC. prunifera has a high capacity to reduce the radicalsDPPH? and ABTS?+, demonstrating its antioxidantcapacity. The in vivo study results confirmed the in vitroantioxidant action of the ethanol extract of C. prunifera,which protected S. cerevisiae strains against oxidativedamage induced by hydrogen peroxide. Therefore, itwas concluded that the ethanol extract of C. pruniferamay be a potential source of antioxidant compounds.

O objetivo deste trabalho foi avaliar a capacidade antioxidante in vitro e in vivo do extrato etanólico das folhas da Copernicia prunifera (Mill.) H. E. Moore. As folhas foram coletadas na cidade de Teresina-PI, secas por quatro dias, trituradas, moídas e extraídas por maceração por 16 dias com troca do solvente (etanol 95%) a cada quatro dias. Depois o extrato etanólico foi filtrado, concentrado em evaporador rotatório, liofilizado e a sua massa determinada. Foi avaliada a capacidade antioxidante in vitro utilizando os métodos do 1,1difenil-2-picrilhidrazil (DPPH?) e ácido 2,2?-azinobis-3 etilbenzotiazolina-6-sulfónico (ABTS?+). Para determinar a capacidade antioxidante in vivo, foram utilizadas linhagens de Saccharomyces cerevisiae proficientes e deficientes em defesas antioxidantes como modelo de estudo. A análise fitoquímica quantitativa resultou nos seguintes valores de compostos fenólicos (1454,54±12,56 mgEAG/g de extrato e 167,27±1,44 mgEAG/g de material seco), flavonoides (645,73±2,42 mgR/g de extrato e 74,26±0,27 mgR/g material seco), taninos hidrolisáveis (64 mgEAG/g de extrato) e alcaloides (28,86% presentes no material vegetal seco). A análise antioxidante demonstrou que o extrato etanólico da C. prunifera tem uma capacidade elevada de reduzir os radicais DPPH? e ABTS?+, demonstrando ação antioxidante. Os estudos in vivo vêm afirmar a ação antioxidante in vitro do extrato etanólico da C. prunifera ao proteger cepas de S. cerevisiae contra o dano oxidativo induzido pelo peróxido de hidrogênio. Concluiu-se que o extrato etanólico da C. prunifera pode ser uma potencial fonte de compostos antioxidantes.

Balance de Flujos Metabólicos en Saccharomyces cerevisiae basado en Compartimentalización Intracelular / Metabolic Flows Balance in Saccharomyces cerevisiae based on Intracellular Compartmentalization

Una de las técnicas más utilizadas para la predicción de producción de bioproductos y distribución intracelular de flujos de microorganismos es el Análisis de Balance de Flujos - FBA por sus siglas en inglés. El FBA requiere de una función objetivo que represente el objetivo biológico del microorganismo estudiado. En este trabajo se propone un nuevo tipo de funciones objetivo basada en la combinación de objetivos de compartimentos físicos presentes en el microorganismo estudiado. Este tipo de funciones objetivo son examinadas junto con un modelo estequiométrico extraído de la reconstrucción iMM904 del microorganismo S. cerevisiae. Su desempeño se compara con la función objetivo más usada en la literatura, la maximización de biomasa, en condiciones experimentales anaeróbicas en cultivos continuos y aeróbicas en cultivos tipo lote. La función objetivo propuesta en este trabajo mejora las predicciones de crecimiento en un 10% y las predicciones de producción de etanol en un 75% respecto a las obtenidas por la función objetivo de maximización de biomasa, en condiciones anaeróbicas. En condiciones aeróbicas tipo lote la función objetivo propuesta mejora en un 98% las predicciones de crecimiento y en un 70% las predicciones de etanol con respecto a la función objetivo de biomasa.

Flux Balance Analysis - FBA - is one of the most used techniques in prediction of microorganism bioproducts. It requires an objective function that represents biological objective of the studied microorganism. This paper presents a new kind of objective functions based on individual physical compartment objetives in the studied microorganism. These kind of functions was tested with a stoichiometric model extracted from iMM904 reconstruction of S. cerevisiae and its performance is compared with the most used objective function in literature, growth maximization, in anaerobic and aerobic batch conditions. The presented objective function outperform growth predictions in 10% and ethanol predictions in 75% compared with obtained by maximization of growth objective function, in anaerobic conditions. In aerobic batch conditions the presented objective function outperforms in 98% growth preditions and 70% ethanol predictions compared with growth maximization.

Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation

Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains ("52" -rough and "PE-02" smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone.

Effect of saccharomyces cerevisiae on survival, growth, biochemical constituents and energy utilization in the prawn macrobrachium rosenbergii.

A probiotic yeast, Saccharomyces cerevisiae was incorporated in basal diet prepared with fish meal, soybean meal, groundnut oil cake, corn flour, tapioca flour, egg albumin, cod liver oil and vitamin Bcomplex, at four different concentrations (10g, 20g, 30g and 40g kg-1) and fed to Macrobrachium rosenbergii post larvae (PL) for 90 days. The effect of this probiotic incorporation on the growth and survival performances, concentration of protein, amino acid, carbohydrate and lipid, and energy utilization was found to be significantly (P<0.05) higher at 40g kg-1 followed by 30g, 20g and 10g kg-1. 40g kg-1 S. cerevisiae incorporation was found to be established the highest rate of colony formation, 234x10-4 cfu (colony formation units). Actually, presence of Bacillus spp., Bacillus cereus, Pseudomonas spp., Escherichia coli, Streptococcus spp., and Klebsiella pneumoniae were deducted in water medium and the PL gut of control group. There is a general belief that Pseudomonas spp. and K. pneumonia are pathogenic to prawns. The establishment of S. cerevisiae colony in the gut of experimental PL has eradicated these pathogenic bacteria. Therefore, it is suggested that establishment of S. cerevisiae colony has led to better growth, survival and biochemical constituents in M. rosenbergii PL. Thus, S. cerevisiae could be taken as a useful probiotic in M. rosenbergii culture.

Influence of static magnetic fields on S. cerevisae biomass growth

Biomass growth of Saccharomyces cerevisiae DAUFPE-1012 was studied in eight batch fermentations exposed to steady magnetic fields (SMF) running at 23°C (± 1°C), for 24 h in a double cylindrical tube reactor with synchronic agitation. For every batch, one tube was exposed to 220mT flow intensity SMF, produced by NdFeB rod magnets attached diametrically opposed (N to S) magnets on one tube. In the other tube, without magnets, the fermentation occurred in the same conditions. The biomass growth in culture (yeast extract + glucose 2 percent) was monitored by spectrometry to obtain the absorbance and later, the corresponding cell dry weight. The culture glucose concentration was monitored every two hours so as the pH, which was maintained between 4 and 5. As a result, the biomass (g/L) increment was 2.5 times greater in magnetized cultures (n=8) as compared with SMF non-exposed cultures (n=8). The differential (SMF-control) biomass growth rate (135 percent) was slightly higher than the glucose consumption rate (130 percent) leading to increased biomass production of the magnetized cells.

O crescimento da biomassa da Saccharomyces cerevisiae DAUFPE-1012 foi estudado em oito bateladas de fermentação, cada uma exposta aos campos magnéticos contínuos (CMC), à 23°C (± 1°C), durante um período de 24 horas em um reator duplo com agitação sincrônica. Em cada batelada,um tubo foi exposto ao CMC, com 220mT de intensidade de fluxo, produzidos por imãs de NdFeB fixados diametralmente opostos (N para S) em um tubo do reator de fermentação. Em outro tubo, sem imãs, a fermentação ocorreu nas mesmas condições. O crescimento da biomassa nas culturas (extrato de fermento + glicose 2 por cento) foi monitorado através de espectrometria e correlacionado ao peso seco de levedura. A concentração de glicose nas culturas foi monitorada a cada duas horas e o pH foi mantido entre 4 e 5. Como resultado, a biomassa (g/L) aumentou 2,5 vezes nas culturas magnetizadas (n=8) quando comparadas com as culturas não expostas (n=8). A taxa de crescimento diferencial (CMC-controle) da biomassa (135 por cento) foi levemente maior que a taxa de consumo de glicose (130 por cento) sugerindo um ganho no processo de produção de biomassa nas células magnetizadas.

A Case of Saccharomyces cerevisiae Isolated from Stool of a Patient Administered Yeast Biotherapeutic Agent / 대한임상미생물학회지

A particular strain of Saccharomyces cerevisiae, Hansen CBS 5926 (syn. S. boulardii), is commonly used as biotherapeutic agent for the prevention and treatment of antibiotic associated diarrhea. Recently, cases of fungemia with S. cerevisiae have increased especially in immunosuppressed or critically ill patients treated with S. boulardii. And S. boulardii administered can simply pass into stool and be isolated as S. cerevisiae. In cases of S. cerevisiae isolated from clinical specimens, it should be required to confirm the relatedness with administration of biotherapeutic agent composed of S. boulardii. A 61-year-old woman who had persistent urinary tract infection by C. tropicalis, developed absolute neutopenia, fever and watery diarrhea and treated with Bioflor composed of S. boulardii, amikacin, ceftriaxone, metronidazole and fluconazole during 3rd chemotherapy against bladder cancer with liver metastasis. On KOH smear of stool specimen, yeasts were observed and the yeasts were identified as S. cerevisiae. She was diagnosed as fungal enterocolitis and sepsis and treated with amphotericin B, but considering her clinical course, S. cerevisiae was presumed not to be a pathogen, but to be a simple passage of S. boulardii administered from intestine. We report a case of S. cerevisiae isolated from stool specimen of a patient administered Bioflor with literatures review.