Evaluación in vivo de la pirogenicidad de bioproductos fúngicos con potencial prebiótico / In vivo pyrogenicity evaluation of fungal bioproducts with prebiotic potential / Avaliação in vivo da pirogenicidade de bioprodutos fúngicos com potencial prebiótico

RESUMEN Introducción: Los productos naturales con actividad farmacológica requieren de evaluaciones preclínicas que justifiquen su empleo sobre una base científica. El ensayo de pirógenos es una prueba dentro de la Farmacología de Seguridad que se realiza para determinar la presencia de endotoxinas y constituye un método valioso, para demostrar la seguridad de bioderivados con potencial prebiótico en el campo de la inmunonutrición. Objetivo: Evaluar la pirogenicidad de bioproductos fúngicos de Pleurotus ostreatus (extractos acuosos del micelio y cuerpos fructíferos) y un biopreparado de levadura Kluyveromyces marxianus, empleando el ensayo de pirógenos en conejos Nueva Zelanda. Método: Se ensayaron concentraciones de 1,0 y 10,0 mg/mL de cada muestra por vía endovenosa en dosis de 0,5 y 5,0 mg/kg de peso. El diseño experimental cumplió las buenas prácticas de laboratorio según lo establecido por el International Council for Laboratory Animals Science y se realizó de acuerdo a los procedimientos normalizados de trabajo del Centro de Toxicología y Biomedicina, Santiago de Cuba. Resultados: Los extractos de Pleurotus ostreatus y el biopreparado de levadura (0,5 mg/kg) no mostraron signos de pirogenicidad. En los resultados del biopreparado (5,0 mg/kg), los valores de temperatura caen en un rango de incertidumbre, según la Farmacopea de Estados Unidos (USP) y se sugirió repetir el estudio. Conclusiones: Los extractos de Pleurotus ostreatus y el biopreparado de Kluyveromyces marxianus (0,5 mg/kg) no indujeron un aumento de temperatura significativo en los animales, lo cual sugiere que en estos bioproductos no existen niveles de endotoxinas que puedan provocar pirogenicidad.

ABSTRACT Introduction: Natural products with pharmacological activity require preclinical evaluations to justify their uses scientifically. The pyrogen assay is a safety pharmacology test performed to determine the presence of endotoxins and it is a valuable method to demonstrate the bio-derivative products safety and their prebiotic potential in the field of immunonutrition. Objective: To evaluate the pyrogenicity of fungal bioproducts from Pleurotus ostreatus (aqueous extracts from mycelium and fruiting bodies) and a biopreparation from Kluyveromyces marxianus yeast, using a pyrogen assay in New Zealand rabbits. Method: Concentrations of 1.0 and 10.0 mg/mL of each sample were tested intravenously at doses of 0.5 and 5.0 mg/kg body weight. The experimental design complied with good laboratory practices as established by the International Council for Laboratory Animal Science and was carried out according to the standard work procedures of the Centro de Toxicología y Biomedicina, Santiago de Cuba. Results: Pleurotus ostreatus extracts and the yeast biopreparation (0.5 mg/kg) showed no signs of pyrogenicity. In the biopreparation results (5.0 mg/kg), temperature values fall in the uncertainty range according to the United States Pharmacopoeia (USP), and therefore it was suggested to repeat the study. Conclusions: Pleurotus ostreatus extracts and Kluyveromyces marxianus biopreparation (0.5 mg/kg) did not induce a significant temperature increase in the animals, thereby suggesting that there are no endotoxin levels in such bioproducts that could cause pyrogenicity.

RESUMO Introdução: Produtos naturais com atividade farmacológica requerem avaliações pré-clínicas que justifiquem seu uso em bases científicas. O ensaio de pirogênio é um teste dentro da Farmacologia de Segurança que é realizado para determinar a presença de endotoxinas e é um método valioso para demonstrar a segurança de bioderivados com potencial prebiótico no campo da imunonutrição. Objetivo: Avaliar a pirogenicidade de bioprodutos fúngicos de Pleurotus ostreatus (extratos aquosos do micélio e corpos de frutificação) e de uma biopreparação da levedura Kluyveromyces marxianus, utilizando o ensaio pirogênico de coelho da Nova Zelândia. Método: Concentrações de 1,0 e 10,0 mg/mL de cada amostra foram testadas por via intravenosa nas doses de 0,5 e 5,0 mg/kg de peso. O desenho experimental obedeceu às boas práticas laboratoriais estabelecidas pelo Conselho Internacional para a Ciência dos Animais de Laboratório e foi realizado de acordo com os procedimentos de trabalho padrão do Centro de Toxicologia e Biomedicina, Santiago de Cuba. Resultados: Os extratos de Pleurotus ostreatus e a biopreparação de leveduras (0,5 mg/kg) não apresentaram sinais de pirogenicidade. Nos resultados da biopreparação (5,0 mg/kg), os valores de temperatura estão dentro de uma faixa de incerteza, segundo a Farmacopeia dos Estados Unidos (USP) e foi sugerido repetir o estudo. Conclusões: Os extratos de Pleurotus ostreatus e a biopreparação de Kluyveromyces marxianus (0,5 mg/kg) não induziram um aumento significativo da temperatura nos animais, o que sugere que não há níveis de endotoxinas nesses bioprodutos que possam causar pirogenicidade.

Determination of Bioethanol Production from Apricot (Prunus armeniaca) Pomace

Abstract Fruit juice industry generates massive amount of lignocellulosic by-products annually which are excellent raw materials for bioethanol production. In the current study, bioethanol production from apricot (Prunus armeniaca) pomace by Kluyveromyces marxianus was investigated for the first time. Some key parameters for fermentation such as pretreatment methods, biomass and cellulase loading and time, were optimized. Kluyveromyces marxianus produced 30.09 g/L ethanol in the 20% washed apricot pomace and 120 FPU/g cellulose enzyme loading. The highest theoretical yield and Y P/S values were also observed as 94.7% and 0.50 g/g, respectively, when 15 FPU/g cellulose enzyme was used. These results depict that apricot pomace is a promising feedstock for bioethanol production.

Native yeasts for alternative utilization of overripe mango pulp for ethanol production / Levaduras nativas para la utilización alternativa de pulpa de mango senescente para la producción de etanol

Mango fruits (Mangifera indica L.) are highly perishable, causing postharvest losses and producing agroindustrial waste. In the present work, native yeasts were used to evaluate ethanol production in overripe mango pulp. The two isolated strains showed similar sequences in the 18S rDNA region corresponding to Kluyveromyces marxianus, being different to the data reported in the NCBI database. Values of up to 5% ethanol (w/v) were obtained at the end of fermentation, showing a productivity of 4g/l/day, a yield of up to 49% of ethanol and a process efficiency of 80%. These results represent a viable option for using the surplus production and all the fruits that have suffered mechanical injury that are not marketable and are considered as agroindustrial waste, thus achieving greater income and less postharvest losses.

Las frutas de mango (Mangifera indica L.) son altamente perecederas, lo cual causa pérdidas poscosecha y produce desechos agroindustriales. En el presente trabajo, se utilizaron 2 levaduras nativas para evaluar la producción de etanol en pulpa de mango senescente. Las 2 cepas aisladas mostraron similitud en la región 18S ADNr, correspondiente a Kluyveromyces marxianus, la cual es diferente a lo reportado en la base de datos del NCBI. Se obtuvieron valores de hasta el 6% de etanol (v/v) al final de la fermentación, con una productividad de hasta 4g/l/día, un rendimiento de hasta 49% de etanol y una eficiencia en el proceso fermentativo del 80%. Esto representa una opción viable para utilizar excedentes de producción o frutos que han sufrido daño mecánico y no son comercializables, al lograr más ingresos y menos pérdida poscosecha.

Fermentations of xylose and arabinose by Kluyveromyces marxianus / 生物工程学报

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.

Volatile metabolites produced from agro-industrial wastes by Na-alginate entrapped Kluyveromyces marxianus

Abstract The aim of this study was to evaluate the effects of alginate entrapment on fermentation metabolites of Kluyveromyces marxianus grown in agrowastes that served as the liquid culture media. K. marxianus cells entrapped in Na-alginate were prepared using the traditional liquid-droplet-forming method. Whey and pomaces from processed tomatoes, peppers, and grapes were used as the culture media. The changes in the concentrations of sugar, alcohol, organic acids, and flavor compounds were analyzed using gas chromatography-mass spectrometry (GC-MS) and high pressure liquid chromatography (HPLC). Both free and entrapped, K. marxianus were used individually to metabolize sugars, organic acids, alcohols, and flavor compounds in the tomato, pepper, grape, and acid whey based media. Marked changes in the fermentation behaviors of entrapped and free K. marxianus were observed in each culture. A 1.45-log increase was observed in the cell numbers of free K. marxianus during fermentation. On the contrary, the cell numbers of entrapped K. marxianus remained the same. Both free and entrapped K. marxianus brought about the fermentation of sugars such as glucose, fructose, and lactose in the agrowaste cultures. The highest volume of ethanol was produced by K. marxianus in the whey based media. The concentrations of flavor compounds such as ethyl acetate, isoamyl alcohol, isoamyl acetate, 2-phenylethyl isobutyrate, phenylethyl acetate, and phenylethyl alcohol were higher in fermented agrowaste based media compared to the control.

Hypocholesterolemic effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rat

To investigate the effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rats, female Wistar rats were fed a high-cholesterol diet (HCD) for 28 d to generate hyperlipidemic models. Hyperlipidemic rats were assigned to four groups, which were individually treated with three different dosages of K. marxianus M3+HCD or physiological saline+HCD via oral gavage for 28 d. The total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels in the serum and liver of the rats were measured using commercially available enzyme kits. In addition, the liver morphology was also examined using hematoxylin and eosin staining and optical microscopy. According to our results, the serum and liver TC, TG, LDL-C levels and atherogenic index (AI) were significantly decreased in rats orally administered K. marxianus M3 (p <0.01), and the HDL-C levels and anti atherogenic index (AAI) were significantly increased (p <0.01) compared to the control group. Moreover, K. marxianus M3 treatment also reduced the build-up of lipid droplets in the liver and exhibited normal hepatocytes, suggesting a protective effect of K. marxianus M3 in hyperlipidemic rats.

Evaluación de dos métodos de conservación para Kluyveromyces marxianus CCEBI 2011 en la Colección de Cultivos del CEBI / Evaluation of two conservation methods for Kluyveromyces marxianus CCEBI 2011 at the CEBI Culture Collection

La elección de un método de conservación debe permitir mantener, entre otros parámetros, la viabilidad, pureza y estabilidad de las propiedades de una cepa. Kluyveromyces marxianus CCEBI 2011 es una levadura con atractivas potencialidades biotecnológicas, conservada en la Colección de Cultivos del CEBI mediante dos métodos: transferencia periódica y agua destilada estéril (método de Castellani). En este trabajo, para determinar la eficacia y calidad de los dos métodos utilizados para la conservación de K. marxianus CCEBI 2011, se evaluaron parámetros como viabilidad, pureza e identidad y propiedades biotecnológicas. Por ambos métodos se obtuvo una elevada pureza y una viabilidad de 5,31x1010 células/mL y 6,8x1010 células/mL por el método de Castellani y transferencia periódica, respectivamente. En este último se alcanzaron los mayores valores de tolerancia a etanol (11%), producción de etanol (6,08 ± 0,11 g/L) y actividad pectinolítica (15,29 ± 1,71 U/mL). En los dos métodos se corroboró la clasificación taxonómica de esta cepa como K. marxianus, según el análisis de las regiones ITS1-gen 5,8S ARNr-ITS2. Se demostró que los métodos de conservación empleados son efectivos, y que han mantenido con calidad las potencialidades biotecnológicas de este microorganismo por más de 10 años.

The election of a conservation method should allow maintaining, among other parameters, the viability, purity and stability of the properties of a strain. Kluyveromyces marxianus CCEBI 2011 is a yeast with attractive biotechnological potentialities, conserved at the CEBI Culture Collection by two methods: periodic transfer and sterile distilled water (Castellani method). In this study for determining the efficacy and quality of the two methods being used for the conservation of K. marxianus, we evaluated parameters such as viability, purity, identity and biotechnological properties. For both methods we obtained a high purity and a viability of 5.31x1010 cells/mL and 6.8x1010 cells/ mL, for the Castellani method and the periodic transfer method respectively. In this last method we obtained the highest values for ethanol tolerance (11%), ethanol production (6.08 ± 0.11 g/L) and pectonolitic activity (15.29 ± 1.71 U/mL). For the two methods the taxonomic classification of this strain as K. marxianus was corroborated according to the analysis of the ITSI-gen 5.8S ARNr-ITS2 regions. It was shown that the conservation methods being used are effective and that they have maintained the quality of the biotechnological potentialities of this microorganism during more than 10 years.

Whole cells in enantioselective reduction of benzyl acetoacetate

The β-ketoester benzyl acetoacetate was enantioselectively reduced to benzyl (S)-3-hydroxybutanoate by seven microorganism species. The best result using free cells was obtained with the yeast Hansenula sp., which furnished 97% ee and 85% of conversion within 24 h. After immobilization in calcium alginate spheres, K.marxianus showed to be more stable after 2 cycles of reaction.

Optimization of the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 by response surface methodology

The aim of this work was to study the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 using optimized nutritional and cultural conditions in a complex yeast medium under aerobic batch fermentation. By applying the conventional "one-variable-at-a-time" approach and the response surface methodology, the effect of four fermentation parameters (type of carbon source, initial culture pH, temperature, and incubation time) on the growth and α-amylase production was evaluated. The production of α-amylase during 60 h of fermentation increased 13-fold under optimized conditions (1% starch, pH 6.0, 30ºC) in comparison to the conventional optimization method. The initial pH value of 6.13 and temperature of 30.3ºC were optimal conditions by the response surface methodology, leading to further improvement (up to 13-fold) in the production of extracellular α-amylase. These results constituted first evidence that K. marxianus could be potentially used as an effective source of extracellular α-amylase.

Study on Conversion of Conjugated Anthraquinone in Radix et Rhizoma Rhei by Yeast Strain / 世界科学技术-中医药现代化

A yeast strain KM12 , which can convert conjugated anthraquinone of rhub arb to free anthraquinone , was screened. And it is identified as Kluyveromyces marxianus by 26S rDNA. The percentage of free an-thraquinone in total anthraquinone of rhub arb as an indicator was used to investigate the effect of culture medium component and fermentation processing conditions, such as seed age, fermentation time and liquid volume on con-version of conjugated anthraquinone by KM12. The optimal fermentation medium was determined by orthogonal test L9(43) and the composition (%) is yeast extract 1, glucose 2, and rhubarb 2. The 5% (V/V) of the seed culture inoculated 36-48 h was inoculumed to fermentation medium in shake flask to ferment at 30℃, 200 r?min-1 for 4 days. TLC analysis showed most of conjugated anthraquinone decomposed or converted into free anthraquinone in fermented rhub arb . It was concluded that the side effect of severe diarrhea caused by Chinese medicine rhub arb can be alleviated through fermentation processing by KM12 strain .

Efecto de la concentración de lactosa sobre la producción de β-D-galactosidasa a partir de Kluyveromyces marxianus ATCC 8554 en cultivo por lote alimentado / Effect of lactose concentration over β-D-galactosidase production by Kluyveromyces marxianus ATCC 8554 in fed batch cultures

Se estudió el efecto de la concentración de lactosa sobre la producción de β-D-galactosidasa, a partir de Kluyveromyces marxianus var. marxianus ATCC 8554 en cultivo por lote alimentado, utilizando suero de leche como sustrato. Los bioprocesos se llevaron a cabo por triplicado a dos concentraciones de lactosa: 2,3% y 4,7%, pH 5 y temperatura de 35 ºC en un biorreactor Bioflo 4000. La extracción de la enzima de las células se realizó empleando tolueno al 2%. La actividad enzimática se determinó evaluando la hidrólisis del o-nitrofenil-β-D-galactopiranósido (ONPG). En el cultivo por lote alimentado y suero de leche sin diluir (4,7% de lactosa) se logró obtener una alta densidad celular. Sin embargo, con el suero de leche diluido (2,3% de lactosa) se obtuvo un rendimiento superior en biomasa (YX/S). El cultivo por lote alimentado permitió prolongar la fase de producción de la enzima y mantener la producción (509 µmol/L/10 min) por un período de tiempo superior al referenciado en el cultivo por carga.

The effect of lactose concentration over β-D-galactosidase production by Kluyveromyces marxianus ATCC 8554 in fed batch cultures was studied using milk serum as substrate. The bioprocesses were carried out in triplicate at two lactose concentrations: 2.3% and 4.7%, pH 5 and a 35oC temperature in a Bioflo 4000 bioreactor. The enzyme extraction from the cells was done using 2% toluene. Enzymatic activity was determined evaluating the hydrolysis of o-nitrophenil-β-D-galactopiranoside (ONPG). With fed batch cultures and undiluted milk serum (4.7% lactose) we were able to obtain a higher biomass yield (Yx/s). The fed batch cultures allowed prolonging the production phase of the enzyme (509 µmol/L/10min) and maintain production for a longer period of time, higher than that mentioned for load cultures.

Production of inulinase from Kluyveromyces marxianus using Dahlia tuber extract

Various carbon sources were evaluated for production of inulinase by yeast, Kluyveromyces marxianus MTCC 3995. Highest inulinase activity was observed with Dahlia extract (25.3 nkat mL-1) as carbon source. The enzyme activity was 1.4 folds higher than that observed in media containing pure chicory inulin (17.8 nkat mL-1). The yeast showed good growth on a simple medium containing dahlia extract (20% w/v) and yeast extract (2%w/v) as carbon and nitrogen source respectively, in 96 h. at 28°C and 120 rpm. Lowest inulinase yield (4.8 nkat mL-1) was seen in the medium containing glucose as C-source. Although varied inulinase levels were noticed on different C- sources, Inulinase: Sucrase (I/S) ratios were noticed to be similar. Among various protein sources tested, yeast extract was found to be the best source followed by beef extract (17.9 nkat mL-1) and peptone (13.8 nkat mL-1). The enzyme was optimally active at pH (4.0) and 50°C. TLC analysis of end product revealed that inulinase hydrolyzed inulin exclusively into fructose. Results suggest that the dahlia extract induced exoinulinase synthesis in Kluyveromyces marxianus and can be utilized as a potential substrate for inulinase production.

Enhanced exoinulinase production from Kluyveromyces marxianus YS-1 using response surface methodology

Optimization of critical medium components for exoinulinase production by Kluyveromyces marxianus YS-1 at shake-flask was investigated using response surface methodology (RSM) based on a central composite rotatable design (CCRD). A five-level with five factors CCRD was used to evaluate the influence of related factors including concentration of inulin, meat extract, calcium chloride, sodium dodecyl sulphate and medium pH. Optimum values obtained by RSM were 2 percent inulin, 2.17 percent meat extract, 0.65 mM calcium chloride, 0.10 mM sodium dodecyl sulphate and pH 5.5. Optimized medium projected a theoretical exoinulinase production of 63.61 IU/mL and biomass yield of 0.965 (OD600/10). Multiple correlation coefficient R was 0.9976 and 0.9605 for exoinulinase production and biomass yield, respectively, which being close to one, justified an excellent correlation between the predicted and experimental values. Maximum productivity of exoinulinase (64.05 IU/mL) obtained experimentally by RSM was more than double in comparison to earlier findings using classical one-variable-at-a-time technique.

Foi investigada a optimização de componentes criticos do meio de cultivo para a produção de exoinulinase por Kluyveromyces marxianus YS-1 em frascos agitados utilizando a metodologia de superficie de resposta (RSM), com base em um delineamento composto central rotativo. As variáveis independentes, com cinco niveis, foram as concentrações de inulina, de extrato de carne, de cloreto de cálcio e de dodecil sulfato de sódio, bem como o pH do meio de cultivo. Os valores ótimos, obtidos pela RSM, foram com 2 por cento de inulina, 2.17 por cento de extrato de carne, 0.65 mM de cloreto de cálcio, 0.10 mM de dodecil sulfato de sódio e pH 5.5. As estimativas de produção de exoinulinase e de rendimento em biomassa no meio otimizado foram de 63.61 UI/ml e de 9.65 (em termos de OD600/10), respectivamente. Os coeficientes de correlação múltipla R foram de 0.9976 e 0.9605 para produção de exoinulinase e biomassa, respectivamente, e, sendo próximos de um, indicam uma excelente correlação entre os valores estimados e experimentais. A máxima productividade de exoinulinase (64.05 UI/ml) obtida experimentalmente por RSM foi mais que o dobro em comparação com nossos resultados anteriores utilizando uma técnica de otimização clássica de uma variável por vez.

Effects of culture conditions on the production of inulinase by Kluyveromyces marxianus

The present study was conducted to investigate the influence of initial sucrose concentration, pH and aeration rate on biomass and inulinase production by Kluyveromyces marxianus var. bulgaricus in a stirred batch reactor. Maximum inulinase activity (15.29 UmL-1) was obtained at a sucrose concentration of 10 g L-1, pH 5.0 and aeration rate of 1 vvm. The 20 g L-1 sucrose concentration was suitable for cell growth; however, enzymatic activity at this concentration was inhibited due to catabolic repression. The increase in aeration rate caused a reduction in enzyme activity with no relevant biomass increase.

O estudo foi conduzido para investigar a influência da concentração inicial da sacarose, a taxa da aeração e do pH na biomassa e na produção da inulinase pela Kluyveromyces marxianus var. bulgaricus em um reator em batelada. A máxima atividade de inulinase, 15.29 UmL-1, foi obtida na concentração de 10 g L-1 de sacarose, no pH 5.0 e na taxa da aeração de 1 vvm. A concentração de sacarose de 20g L-1 foi apropriada para o crescimento celular, porém nesta concentração a atividade enzimática foi inibida, devido a repressão catabólica. O aumento na taxa da aeração propiciou redução da atividade enzimática, ao mesmo tempo em que não houve aumento considerável do biomassa.

Fermentación alcohólica del lactosuero por Kluyveromyces marxianus y solventes orgánicos como extractantes / Lactoserum alcoholic fermentation by Kluyveromyces marxianus and organic solvents as extractants

Las fermentaciones alcohólicas son realizadas a bajas concentraciones de azúcares, para evitar la inhibición por productos o substratos. La fermentación alcohólica extractiva es utilizada en procesos limitados por la concentración de etanol final. Se estudió la fermentación alcohólica del lactosuero evitando la inhibición del Kluyveromyces marxianus, con la adición de solventes de extracción (ácido oleico, hexadecano, butil laurato y aceite de soja) como extractantes del etanol. Se evaluó la biocompatibilidad, cinética de extracción de los solventes, consumo de sustrato, producción de etanol, crecimiento celular (sin solventes y con solventes seleccionados como mejores extractantes y biocompatibles) y rendimientos globales. El ácido oleico y el aceite de soja fueron los mejores solventes de extracción. La velocidad específica de crecimiento se incrementó con el uso de solventes, siendo mayor con el uso de aceite de soja. La lactosa consumida fue de 97 g/L para la fermentación extractiva con ácido oleico, 81,2 g/L con aceite de soja, y 64 g/L para la fermentación convencional. Se obtuvieron 53 y 44 g/L de etanol con ácido oleico y aceite de soja respectivamente, y 35 g/L de etanol con la fermentación convencional. Queda demostrado que la fermentación alcohólica usando acido oleico puede ser usada para reducir la concentración de etanol en el medio y evitar la inhibición de la levadura en el proceso, lo que conlleva a la posibilidad de usar un substrato con altas concentraciones de azúcares, produciendo un incremento en la productividad del sistema.

Alcoholic fermentations are done under low sugar concentrations to avoid product or substrate inhibition. Extractive alcoholic fermentation is used in processes limited by the final ethanol concentration. Lactoserum alcoholic fermentation avoiding the inhibition of Kluyveromyces marxianus by the addition of extraction solvents (oleic acid, hexadecane, butyl laureate and soy oil) as ethanol extractants was studied. Biocompatibility, extraction kinetic of the solvents, substrate use, ethanol production, cell growth (without solvents and with solvents selected as the best extractants and biocompatible), and global yield were evaluated. Oleic acid and soy oil were the best extraction solvents. Specific growth speed was increased by the use of solvents; the greatest increase was obtained with soy oil. Lactose consumption was 97 g/L for oleic acid extractive fermentation, 81.2 g/L for soy oil, and 64 g/L for conventional fermentation. The ethanol yield was 53 and 44 g/L with oleic acid and soy oil fermentation respectively, and 35 g/L with conventional fermentation. This study demonstrates that oleic acid alcoholic fermentation can be used to reduce the ethanol concentration in the medium and to avoid the inhibition of the yeasts during the process, which entails the possibility of using substrates with high sugar concentrations, obtaining an increase in the productivity of the system.

Obtención y Caracterización de dos Concentrados Proteicos a partir de Biomasa de Kluyveromyces marxianus var. marxianus cultivada en suero lácteo Desproteinizado / Attainment and Characterization of Two Protein Concentrates from Biomass of Kluyveromyces marxianus var. marxianus Grown in Deproteinized Whey

El presente trabajo se realizó con el propósito de obtener un concentrado proteico con un bajo contenido de ácidos nucleicos a partir de biomasa microbiana. Para ello se cultivó la levadura Kluyveromyces marxianus var. marxianus en lactosuero desproteinizado y suplementado con fuentes de nitrógeno y vitaminas a pH 4,5; 32 a 35°C y una tasa de aireación de 1,22 volúmenes de aire por volumen de medio por minuto (VVM). Se obtuvo un rendimiento promedio de: biomasa de 0,30 g/g lactosa, un consumo de lactosa del 93, 21% y concentración celular 4,15 g/L al final de la fermentación. A partir de la biomasa obtenida se elaboraron dos concentrados proteicos: uno por extracción alcalina y precipitación isoeléctrica (C1) y otro por extracción alcalina, fosforilación y precipitación isoeléctrica (C2). El análisis de los concentrados mostró diferencias significativas (P < 0,05) en los contenidos de cenizas, grasa, proteína cruda, nitrógeno no proteico, ARN y carbohidratos. Se evidenció una disminución significativa (P < 0,05) del contenido de ARN de 4,59% (C1) a 2,71% (C2) hecho importante para el uso de este concentrado proteico para el consumo humano. La fosforilación de la proteína (C2) incrementó la capacidad de absorción de agua (4,21 a 4,64 g agua/g proteína), la solubilidad (32,43 a 41,74 g proteína solubilizada/g proteína total), y la capacidad emulsionante (903,38 a 971,91 g aceite/g proteína), y disminuyó la capacidad de absorción de grasa (1,79 a 1,65 g grasa/g proteína), características éstas que deben ser consideradas para la incorporación de los concentrados proteicos como ingredientes de productos cárnicos y de panadería.

This work was carried out with the purpose of attain a protein concentrate from microbial biomass with low content of nucleic acids. The yeast Kluyveromyces marxianus var. marxianus was grown on deproteinized and supplemented whey with nitrogen and vitamins sources at pH 4.5; 32 to 35°C and an aeration rate of 1.22 volumes of air fed for volume of broth per minute (VVM). The average biomass yield obtained was 0.30 g/g lactose, a lactose consumption of 93.21% and a cellular concentration of 4.15 g/L at the end of the fermentation. Two protein concentrates were obtained: the first one by alkaline extraction and isoelectric precipitation (C1) and the second one by alkaline extraction, phosphorylation and isoelectric precipitation (C2). Chemical analysis of both concentrates shown significant differences (P < 0.05) in the content of ash, fat, crude protein, non protein nitrogen, RNA and carbohydrates. There was significant (P < 0.05) the decrease in the RNA content from 4.59% (C1) to 2.71% (C2), important fact for the use of this protein concentrates for human consumption. Phosphorylation of the protein (C2) produced an increase in the water holding capacity (4.21 to 4.64 g water/g protein), solubility (32.43 to 41.74 g solubilized protein/g total protein) and emulsifying capacity (903.38 to 971.91 g oil/g protein), as well as a decrease in the oil absorption capacity (1.79 to 1.65 g oil/g protein). These characteristics are important when considering the use of such protein concentrates as ingredients in bakery or meat products.