Fungal endo and exochitinase production, characterization, and application for Candida biofilm removal.

Chitinases are promising enzymes for a multitude of applications, including chitooligosaccharide (COS) synthesis for food and pharmaceutical uses and marine waste management. Owing to fungal diversity, fungal chitinases may offer alternatives for chitin degradation and industrial applications. The rapid reproduction cycle, inexpensive growth media, and ease of handling of fungi may also contribute to reducing enzyme production costs. Thus, this study aimed to identify fungal species with chitinolytic potential and optimize chitinase production by submerged culture and enzyme characterization using shrimp chitin. Three fungal species, Coriolopsis byrsina, Trichoderma reesei, and Trichoderma harzianum, were selected for chitinase production. The highest endochitinase production was achieved in C. byrsina after 168 h cultivation (0.3 U mL- 1). The optimal temperature for enzyme activity was similar for the three fungal species (up to 45 and 55 ºC for endochitinases and exochitinases, respectively). The effect of pH on activity indicated maximum hydrolysis in acidic pH (4-7). In addition, the crude T. reesei extract showed promising properties for removing Candida albicans biofilms. This study showed the possibility of using shrimp chitin to induce chitinase production and enzymes that can be applied in different industrial sectors.

Production, Extraction, and Solubilization of Exopolysaccharides Using Submerged Cultures of Agaricomycetes.

Macrofungi, also known as mushrooms, can produce various bioactive compounds, including exopolysaccharides (EPS) with distinct biological properties and subsequent industrial applications in the preparation of cosmetics, pharmaceuticals, and food products. EPS are extracellular polymers with diverse chemical compositions and physical properties secreted by macrofungi in the form of capsules or biofilms into the cellular medium. Submerged cultivation is an industrially implemented biotechnological technique used to produce a wide variety of fungal metabolites, which are of economic and social importance due to their food, pharmaceutical, and agronomic applications. It is a favorable technique for cultivating fungi because it requires little space, minimal labor, and low production costs. Moreover, it allows for control over environmental variables and nutrient supply, essential for the growth of the fungus. Although this technique has been widely applied to yeasts, there is limited knowledge regarding optimal growth conditions for filamentous fungi. Filamentous fungi exhibit different behavior compared to yeast, primarily due to differences in cell morphology, reproductive forms, and the type of aggregates generated during submerged fermentation. Furthermore, various growing conditions can affect the production yield of metabolites, necessitating the development of new knowledge to scale up metabolite production from filamentous fungi. This protocol implements the following culture conditions: an inoculum of three agar discs with mycelium, agitation at 150 rpm, a temperature of 28 °C, an incubation time of 72 h, and a carbon source concentration of 40 g/L. These EPS are precipitated using polar solvents such as water, ethanol, and isopropanol and solubilized using water or alkaline solutions. This protocol details the production procedure of EPS using submerged culture; the conditions and culture medium used are described. A detailed description of the extraction is performed, from neutralization to lyophilization. The concentrations and conditions necessary for solubilization are also described. Key features • Production and extraction of EPS from submerged cultures of mycelial forms of macrofungi. • Modification of the method described by Fariña et al. (2001), extending its application to submerged cultures of mycelial forms of the macrofungi. • Determination of EPS production parameters in submerged cultures of mycelial forms of macrofungi. • EPS solubilization using NaOH (0.1 N). Graphical overview.

Iron-enriched mycelia of edible and medicinal basidiomycetes.

Iron bioaccumulation in basidiomycetes is an alternative to recover ferrous sulphate from titanium dioxide pigment production and to produce an iron-enriched mycelial biomass. This study aimed to evaluate iron bioaccumulation capacity in vegetative mycelium of edible and medicinal fungi grown in malt extract liquid medium with different ferrous sulphate contents. Five basidiomycetes were grown in malt extract liquid medium with different iron contents from 0.116 to 100 mg L-1 iron. The iron content of dried mycelial biomass bioaccumulated with iron was determined by flame atomic absorption spectrophotometry. All fungi grew on the iron culture media and the mycelial biomass growth ranged from 3.24 ± 0.65a mg mL-1 to 12.46 ± 0.29 mg mL-1. Iron addition to culture media increased the iron content in the mycelial biomass from 4000-13,000-fold compared with control. Pleurotus ostreatus (2181 ± 218 mg kg-1) presented the greatest iron content in the mycelial biomass, followed by Schizophyllum commune (1769 ± 131 mg kg-1), Agaricus subrufescens (1272 ± 8.84 mg kg-1), and Ganoderma lucidum (840 ± 75 mg kg-1). P. ostreatus, followed by S. commune, and G. lucidum at 90 and 100 mg L-1 iron in the culture medium are the best choices to produce iron-enriched mycelial biomass. This extensive study of several edible and medicinal basidiomycetes grown in different iron contents was effective in recovering ferrous sulphate byproduct and transferring it to mycelium to produce a new nutraceutical food of iron-enriched mycelial biomass.

Agro-industrial residues for the production of red biopigment by Monascus ruber: rice flour and sugarcane molasses.

Three culture media were studied for red pigment production by Monascus ruber in submerged cultivation: rice flour (20 g L-1), sugarcane molasses (30 g L-1), and, finally, molasses + rice flour (10 g L-1+10 g L-1); all culture media were added of 5 g L-1 glycine as nitrogen source. Rice flour showed pigment production of 7.05 UA510nm and molasses 5.08 UA510nm, and the mixture of rice flour and molasses showed the best result of 16.38 UA510nm. Molasses culture presented good results for cell biomass production of 11.09 g L-1. With these results, it was observed that one substrate presented good pigment production (rice flour) and another attained better results for cell biomass growth (molasses), and a third medium containing 10 g L-1 of rice flour + 10 g L-1 of molasses was formulated. The results for this mixture showed satisfactory results, with global pigment productivity of 0.097 UA510nm h-1 and maximum productivity rate of 0.17 UA510nm h-1. The high production and productivity obtained for the mixture of rice flour and molasses indicated that the production of red pigment by submerged fermentation, using the mixture of these low-cost culture media, may be promising in terms of commercial production.

Growth of Leucoagaricus gongylophorus Möller (Singer) and production of key enzymes in submerged and solid-state cultures with lignocellulosic substrates.

The aim of this study was to characterize the growth of the fungus Leucoagaricus gongylophorus LEU18496, isolated from the fungus garden of the nest of leaf cutter ants Atta mexicana. The fungus garden was cultivated in an artificial laboratory nest and the fungus further grown in submerged (SmC) and solid state (SSC) cultures with sugarcane bagasse, grass or model substrates containing CM-cellulose, xylan or lignin. The CO2 production rate with grass in SmC (Vmax 34.76 mg CO2 Lgas-1 day- 1) was almost four times than SSC (Vmax 9.49 mg CO2 Lgas-1 day- 1), while the production rate obtained in sugarcane bagasse in SmC (Vmax 16.02 mg CO2 Lgas-1 day- 1) was almost three times than that for SSC (Vmax 5.42 mg CO2 Lgas-1 day- 1). In addition, the fungus grew with defined carbon substrates mixtures in SmC, but at different rates, first xylan, followed by CM-cellulose and lignin. Endoglucanase and xylanase activities (U mgprotein-1) were detected in all cultures, the specific activity was higher in the fungus-garden, 5.2 and 1.8; followed by SSC-grass, 1.5 and 0.8, and SSC-bagasse, 0.9 and 0.8, respectively. Laccase activity in the fungus-garden was 44.8 U L- 1 and 10.9 U L- 1 in the SSC-grass. The gongylidia structures observed by environmental scanning electron microscopy were ca. 40 µm and the hyphae width ca. 5 µm. The results show that L. gongylophorus from A. mexicana have promising applications for the treatment of plant residues to release fermentable sugars and the production of high value lignocellulolytic enzymes such as endoglucanase, xylanase or laccases.

Water-soluble fluorescent red colorant production by Talaromyces amestolkiae.

The replacement of synthetic colors in food products by natural alternatives has been boosted by consumers willing to pay more for healthier products. However, the success of microbial colorants depends not only on its acceptability on the market but also its production costs. Talaromyces species can produce water-soluble red colorants induced by glucose and monosodium glutamate (MSG). In this study, the influence of several conditions was evaluated to produce natural red colorants by submerged culture of Talaromyces amestolkiae. Under optimal conditions (g/L: glucose 10, MSG 25, MgSO4 0.012, FeSO4 0.01, CaCl2 0.015; and initial pH of 5.0), a 30-fold increase in the production was achieved, reaching a red colorant production of 13.44 UA500nm. Depending on the initial pH, colorants with different hues and chroma values were obtained. Deep yellow colorants were derived from neutral and basic pH, while deep red colors were derived from acidic pH. The fluorescence spectrum of culture broth obtained before and after complexation with salts presented red colorants with yellow fluorescence spectra. The information generated in this study would be useful for the formulation of industrial media for large-scale cultivation of T. amestolkiae, which have the potential to produce Talaromyces fermented colorants for use in health foods and pharmaceutics.

Estudio comparativo de la producción de biomasa en fermentación superficial y en estado líquido de macromicetos con diferentes fuentes nutrimentales / Comparative study of the production of biomass in surface fermentation and in the liquid state of macromycetes with different nutrient sources

RESUMEN Los macromicetos han adquirido gran interés por su importancia alimenticia, terapéutica y económica, razón por la cual es necesario enfocar esfuerzos en la búsqueda de optimizar su producción de biomasa. El presente trabajo buscó determinar y comparar el efecto de la fermentación líquida (FEL) y superficial (FeSup) (medio solido) sobre la producción de biomasa de Flammulina velutipes, Hypsizigus tessulatus y Grifola frondosa empleando seis fuentes de nutrientes de bajo costo: harina de soja, trigo integral, maíz blanco, maíz amarillo precocido, salvado de trigo y semillas de linaza molida. Los resultados de la FeSup permitieron determinar que la especie de mayor crecimiento radial, indistintamente de la fuente de nutrientes, es F. velutipes seguida de H. tessulatus y por ultimo G. frondosa. Adicionalmente, la mayor producción de biomasa con FeSup se observa para F. velutipes y H. tessulatus (6,4480 g/L y 5,7320 g/L, respectivamente) Por el contrario, para la FEL, G. frondosa (11,4620 g/L) es la especie de mayor producción. La comparación en la producción de biomasa empleando FEL y FeSup, evidenció que los resultados son dependientes de la técnica de cultivo y que la FeSup no puede ser empleada para la selección preliminar de hongos macromicetos, enfocada en la producción de biomasa por FEL.

ABSTRACT Macromycetes have acquired great interest because of their nutritional, therapeutic and economic importance, which is why it is necessary to focus efforts in the search to optimize their biomass production. The present work sought to determine and compare the effect of liquid fermentation (FEL) and surface fermentation (FeSup) (solid medium) on the biomass production of Flammulina velutipes, Hypsizigus tessulatus and Grifola frondosa using six sources of low cost nutrients: soybeans, whole wheat, white corn, precooked yellow corn, wheat bran and ground flax seed. The results of the FeSup allowed to determine that the species with the highest radial growth, indistinctly from the nutrient source, is F. velutipes followed by H. tessulatus and finally G. frondosa. Additionally, the highest biomass production with FeSup is observed for F. velutipes and H. tessulatus (6.4480 g/L and 5.7320 g/L, respectively) On the contrary, for the FEL, G. frondosa (11.4620 g/L) is the species with the highest production. The comparison in the production of biomass using FEL and FeSup, showed that the results are dependent on the culture technique and that the FeSup cannot be used for the preliminary selection of fungi macromycetes, focused on the production of biomass by FEL.

Producción de unidades infectivas de Isaria fumosorosea (Hypocreales: Cordycipitaceae) a partir de aislados nativos del noreste de México mediante 3 estrategias de propagación / Production of infectious units of Isaria fumosorosea ( Hypocreales: Cordycipitaceae) from different indigenous isolates of northeastern Mexico using 3 propagation strategies

El objetivo del presente estudio fue evaluar la producción de blastosporas y conidios de diferentes aislados nativos de México del hongo entomopatógeno Isaria fumosorosea y de una cepa de colección mediante diferentes técnicas de propagación. En la producción de blastosporas se utilizaron 2 medios de cultivo líquidos (sumergidos), uno a base de casaminoácidos y el otro a base de peptona de colágeno como fuentes de nitrógeno, con glucosa como fuente de carbono en ambos. Para la producción de conidios, los hongos se cultivaron en agar papa dextrosa, a partir de esos cultivos se prepararon suspensiones de 1 x 10(6) conidios/ml para inocular matraces con caldo dextrosa Sabouraud, para iniciar así la fase líquida del cultivo bifásico, denominado también precultivo. Posteriormente con el precultivo y las suspensiones de conidios se inocularon bolsas con granos de arroz, que se incubaron durante 14 días para el cultivo bifásico y para la fermentación sólida, respectivamente. El aislado HIB-23 fue el que logró la más elevada concentración de blastosporas obtenida en el cultivo sumergido: 4,90 x 10(8) blastosporas/ml en el medio casaminoácidos; y en el medio con peptona de colágeno se obtuvieron 2,15 x 10(8) blastosporas/ml. La máxima producción de conidios en fermentación sólida la logró la cepa Pfr-612 (1,58 x 10(9) conidios/g), mientras que la máxima en cultivo bifásico correspondió al aislado HIB-30 (9,00 x 10(6) conidios/g). La fermentación sólida resultó ser el método más efectivo, con un promedio de 1,09 x 10(9) conidios/g, mientras que el cultivo bifásico fue el menos efectivo, con un promedio de 2,76 x 10(6) conidios/g. Para la producción de blastosporas en los medios sumergidos no se obtuvo diferencia significativa alguna.

The aim of this study was to evaluate the production of blastospores and conidia of different native isolates and a strain of Isaria fumosorosea using different propagation techniques. Two liquid culture media of casamino acids and peptone as nitrogen sources and glucose as carbon source for both media cultures were respectively used in the production of blastospores, while for the production of conidia, the fungi were grown in potato dextrose agar; from these cultures, solutions of conidia to a concentration of 1 x 10(6) per milliliter were prepared to inoculate flasks with Sabouraud dextrose broth for the liquid phase of the biphasic culture, also known as preculture. Subsequently, rice grain bags were inoculated with the preculture and the conidia solutions, which were incubated for 14 days for solid fermentation and biphasic culture, respectively. The HIB-23 isolate recorded a concentration of 4.90 x 10(8) blastospores/ml in the casamino acid medium, while a concentration of 2.15 x 10(8) blastospores/ml was obtained in the peptone collagen medium. For the Pfr-612 strain, the conidia production in solid-state fermentation was 1.58 x 10(9) conidia/g, and for HIB-30 in the biphasic culture of 9.00 x 10(6) conidia/g. Solid-state fermentation proved to be the most effective method with an average of 1.09 x 10(9) conidia/g, whereas the biphasic culture was the least effective method with 2.76 x 10(6) conidia/g; no significant difference was reported for the submerged production media.

[Production of infectious units of Isaria fumosorosea (Hypocreales: Cordycipitaceae) from different indigenous isolates of northeastern Mexico using 3 propagation strategies]. / Producción de unidades infectivas de Isaria fumosorosea (Hypocreales: Cordycipitaceae) a partir de aislados nativos del noreste de México mediante 3 estrategias de propagación.

The aim of this study was to evaluate the production of blastospores and conidia of different native isolates and a strain of Isaria fumosorosea using different propagation techniques. Two liquid culture media of casamino acids and peptone as nitrogen sources and glucose as carbon source for both media cultures were respectively used in the production of blastospores, while for the production of conidia, the fungi were grown in potato dextrose agar; from these cultures, solutions of conidia to a concentration of 1×106 per milliliter were prepared to inoculate flasks with Sabouraud dextrose broth for the liquid phase of the biphasic culture, also known as preculture. Subsequently, rice grain bags were inoculated with the preculture and the conidia solutions, which were incubated for 14 days for solid fermentation and biphasic culture, respectively. The HIB-23 isolate recorded a concentration of 4.90×108 blastospores/ml in the casamino acid medium, while a concentration of 2.15×108 blastospores/ml was obtained in the peptone collagen medium. For the Pfr-612 strain, the conidia production in solid-state fermentation was 1.58×109 conidia/g, and for HIB-30 in the biphasic culture of 9.00×106 conidia/g. Solid-state fermentation proved to be the most effective method with an average of 1.09×109 conidia/g, whereas the biphasic culture was the least effective method with 2.76×106 conidia/g; no significant difference was reported for the submerged production media.

Batch and Pulsed Fed-Batch Cultures of Aspergillus flavipes FP-500 Growing on Lemon Peel at Stirred Tank Reactor.

Aspergillus flavipes FP-500 grew up on submerged cultures using lemon peel as the only carbon source, developing several batch and pulsed fed-batch trials on a stirred tank reactor. The effect of carbon source concentration, reducing sugar presence and initial pH on exopectinase and endopectinase production, was analyzed on batch cultures. From this, we observed that the highest substrate concentration favored biomass (X max) but had not influence on the corresponding specific production (q p) of both pectinases; the most acid condition provoked higher endopectinase-specific productions but had not a significant effect on those corresponding to exopectinases; and reducing sugar concentrations higher than 1.5 g/L retarded pectinase production. On the other hand, by employing the pulsed fed-batch operation mode, we observed a prolonged growth phase, and an increase of about twofold on endopectinase production without a significant raise on biomass concentration. So, pulsed fed-batch seems to be a good alternative for obtaining higher endopectinase titers by using high lemon peel quantities without having mixing and repression problems to the system. The usefulness of unstructured kinetic models for explaining, under a theoretic level, the behavior of the fungus along the batch culture with regard to pectinase production was evident.

Screening of wild type Streptomyces isolates able to overproduce clavulanic acid.

The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a ß-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of ß-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of ß-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

Improvement of submerged culture conditions to produce colorants by Penicillium purpurogenum.

Safety issues related to the employment of synthetic colorants in different industrial segments have increased the interest in the production of colorants from natural sources, such as microorganisms. Improved cultivation technologies have allowed the use of microorganisms as an alternative source of natural colorants. The objective of this work was to evaluate the influence of some factors on natural colorants production by a recently isolated from Amazon Forest, Penicillium purpurogenum DPUA 1275 employing statistical tools. To this purpose the following variables: orbital stirring speed, pH, temperature, sucrose and yeast extract concentrations and incubation time were studied through two fractional factorial, one full factorial and a central composite factorial designs. The regression analysis pointed out that sucrose and yeast extract concentrations were the variables that influenced more in colorants production. Under the best conditions (yeast extract concentration around 10 g/L and sucrose concentration of 50 g/L) an increase of 10, 33 and 23% respectively to yellow, orange and red colorants absorbance was achieved. These results show that P. purpurogenum is an alternative colorants producer and the production of these biocompounds can be improved employing statistical tool.

Screening of wild type Streptomyces isolates able to overproduce clavulanic acid

The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

Screening of wild type Streptomyces isolates able to overproduce clavulanic acid

The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

Improvement of submerged culture conditions to produce colorants by Penicillium purpurogenum

Safety issues related to the employment of synthetic colorants in different industrial segments have increased the interest in the production of colorants from natural sources, such as microorganisms. Improved cultivation technologies have allowed the use of microorganisms as an alternative source of natural colorants. The objective of this work was to evaluate the influence of some factors on natural colorants production by a recently isolated from Amazon Forest, Penicillium purpurogenum DPUA 1275 employing statistical tools. To this purpose the following variables: orbital stirring speed, pH, temperature, sucrose and yeast extract concentrations and incubation time were studied through two fractional factorial, one full factorial and a central composite factorial designs. The regression analysis pointed out that sucrose and yeast extract concentrations were the variables that influenced more in colorants production. Under the best conditions (yeast extract concentration around 10 g/L and sucrose concentration of 50 g/L) an increase of 10, 33 and 23% respectively to yellow, orange and red colorants absorbance was achieved. These results show that P. purpurogenum is an alternative colorants producer and the production of these biocompounds can be improved employing statistical tool.

Improvement of submerged culture conditions to produce colorants by Penicillium purpurogenum

Safety issues related to the employment of synthetic colorants in different industrial segments have increased the interest in the production of colorants from natural sources, such as microorganisms. Improved cultivation technologies have allowed the use of microorganisms as an alternative source of natural colorants. The objective of this work was to evaluate the influence of some factors on natural colorants production by a recently isolated from Amazon Forest, Penicillium purpurogenum DPUA 1275 employing statistical tools. To this purpose the following variables: orbital stirring speed, pH, temperature, sucrose and yeast extract concentrations and incubation time were studied through two fractional factorial, one full factorial and a central composite factorial designs. The regression analysis pointed out that sucrose and yeast extract concentrations were the variables that influenced more in colorants production. Under the best conditions (yeast extract concentration around 10 g/L and sucrose concentration of 50 g/L) an increase of 10, 33 and 23% respectively to yellow, orange and red colorants absorbance was achieved. These results show that P. purpurogenum is an alternative colorants producer and the production of these biocompounds can be improved employing statistical tool.

Produção e extração de colorantes naturais de Penicillium purpurogenum DPUA 1275 / Production and extraction of natural colorants from Penicillium purpurogenum DPUA 1275

Há interesse mundial no desenvolvimento de pesquisas envolvendo produção e extração de colorantes naturais, devido a sérios problemas de segurança industrial associados ao uso de colorantes sintéticos. Este trabalho objetivou produzir colorantes naturais de Penicillium purpurogenum DPUA 1275 por cultivo submerso (em frascos agitados e em biorreator) e estudar a extração dos colorantes vermelhos. Para a produção, os estudos iniciais mostraram que 5 discos de micélio, sacarose e extrato de levedura como fontes de carbono e nitrogênio, respectivamente, e 336 horas de cultivo eram condições adequadas para a produção dos colorantes. Visando à otimização da produção, realizaram-se planejamentos fatoriais, com as variáveis independentes: tempo de cultivo; velocidade de agitação; pH; temperatura; concentração de sacarose e de extrato de levedura. As variáveis-respostas foram produção de colorantes amarelos, laranjas e vermelhos. Dos resultados obtidos, as variáveis mais significativas ao processo foram concentrações de extrato de levedura e de sacarose. A produção dos colorantes vermelhos foi otimizada, alcançando a produção de 2,97 UA490nm, nas condições 48,90 e 11,80 g/L de sacarose e extrato de levedura, respectivamente, 30°C, pH 4,5 150 rpm e 336 horas de cultivo. Nos experimentos em biorreator, o melhor resultado foi obtido na frequência de agitação de 500 rpm e na mudança do pH do meio para 8,0, após 96 horas de bioprocesso. Ademais, avaliou-se a estabilidade dos colorantes vermelhos presentes no meio fermentado em diferentes condições (pH, temperatura, sais, polímeros e tensoativos). Referente a pH e temperatura, os colorantes vermelhos mostraram-se mais estáveis nas condições alcalinas e a 70 °C. Tanto os sais (NaCl e Na2SO4) quanto os polímeros (PEG 1.000, 6.000 e 10.000 g/mol e NaPA 8.000 g/mol a 5 e 15%) e os tensoativos (Tween 20, CTAB e SDS) não causaram perda da cor nas condições avaliadas. Estudos de solubilidade e de coeficiente de partição octanol-água mostraram que os colorantes vermelhos apresentam solubilidade superior em solventes polares e característica mais hidrofílica. Nos estudos de extração, as técnicas avaliadas foram Sistemas Poliméricos de Duas Fases Aquosas (SPDFA) formados pelo sistema PEG/NaPA e Colloidal Gas Aphrons (CGA). Pela primeira técnica, os colorantes vermelhos migraram preferencialmente para a fase PEG. Os polímeros PEG 6.000 g/mol, na presença de NaCl 0,1 e 0,5 M, e PEG 10.000 g/mol, com Na2SO4 0,5M, se destacaram dentre as condições analisadas com coeficiente de partição (K) próximo a 13, em ambos os casos, e seletividade de proteínas (SeP) próximas a 3. Para a técnica de CGA, o CTAB proporcionou os melhores resultados, seguido do Tween 20. Porém, o valor de K foi inferior ao obtido com SPDFA, com um máximo de 5 (CTAB 2 mM/pH 9,0). Os resultados obtidos demonstram um novo produtor de colorantes naturais, as quais têm potencial de aplicação em diversos segmentos industriais. Ademais, os resultados obtidos mostraram a eficiência das técnicas utilizadas para extração dos colorantes vermelhos, com destaque para SPDFA, que apresentou maiores valores de K

There is worldwide interest in developing research projects involving the production and extraction of natural colorants due to serious safety problems associated with industrial use of synthetic ones. The aim of this work was to investigate the production of natural colorants from Penicillium purpurogenum DPUA 1275 by submerged culture (rotatory shaker and bioreactor) besides studying the red colorants extraction. To the production step, initial studies showed that 5 agar mycelial discs, sucrose and yeast extract as carbon and nitrogen sources, respectively, and 336 hours of bioprocess promoted the best results. To optimize the colorants production a serie of factorial designs were performed. The independent variables studied were: fermentation time, agitation speed, pH, temperature, sucrose and yeast extract concentration under the responses production of yellow, orange and red colorants. From these results, the most significant variables for the process were sucrose and yeast extract concentration. The red colorants production was optimized achieving 2.97 UA490nm, in the following conditions: 48.90 and 11.80 g/L of sucrose and yeast extract, respectively, 30 °C, 4.5 pH, 150 rev min-1 and 336 hours of culture. In the experiments performed in bioreactor, the condition that promoted the best results was 500 rpm and pH adjusted for 8.0 after 96 hours of bioprocess. Furthermore, we evaluated the red colorants stability at different conditions (pH, temperature, salts, polymers and surfactants). Concerning to pH and temperature, the red colorants were more stable under basic conditions and 70 °C; not only the salts (NaCl and Na2SO4) but also the polymers (PEG 1000, 6000 and 10000 g/mol and NaPA 8000 g/mol) and the surfactants (Tween 20, CTAB and SDS) not promoted loss of color upon the conditions evaluated. Studies of red colorants solubility and octanol water coefficient showed that these compounds exhibit a higher solubility in polar solvents and present hydrophilic characteristics. Subsequently, the extraction of red colorant was evaluated through two extraction methods: Polymeric Systems Aqueous Two Phase (ATPS) composed by PEG and NaPA and Colloidal Gas Aphrons (CGA). For the first technique, the red colorant preferentially migrated to the PEG phase. The best results were obtained with PEG 6000 g/mol in the presence of 0.1 to 0.5 M NaCl and with PEG 10000 g/mol with 0.5 M Na2SO4. To both cases the partition coefficient (K) was close to 13 and the Selectivity in terms of proteins (SeP) was close to 3. For the CGA technique, CTAB gave the best results followed by Tween 20. However, the K values were lower than the ones obtained with ATPS with a maximum of 5 in the following condition: CTAB 2 mM/pH 9.0. For the SeP, the values obtained for both techniques were close. The results above show a new producer of natural colorants which have potential application in various industries. Moreover, the results show the efficiency of the techniques used to extract the red colorants, especially to ATPS that presented higher K values

Optimization of exopolysaccharides production from a novel strain of Ganoderma lucidum CAU5501 in submerged culture

This study aimed at optimizing the medium of a new Ganoderma lucidum strain CAU5501 to enhance the yield of exopolysaccharides (EPS) and mycelial growth. Firstly, the suitable level of glucose, magnesium, phosphate and C/N ratio was determined by single factor experiment. Subsequently, the optimum concentrations of these medium components were investigated using the orthogonal matrix method. The results indicated that the higher levels of EPS were correlated with the level of cell growth when glucose concentration was studied (data no show). The optimum medium for EPS yield was found to be 70 g/l glucose, 5 C/N ratio, 2.5 g/l KH2PO4, 0.75 g/l MgSO4·7H2O, and for mycelial growth was 50 g/l glucose, 5 C/N ratio, 1.5 g/l KH2PO4, 0.5 g/l MgSO4·7H2O. When cultivated in the obtained optimal media in 3 L shake flask, compared to the basal medium, the EPS yield increased markedly from 1.003 to 1.723 g/l, and the mycelium formation was also markedly improved from 2.028 to 7.235 g/l. Results obtained in this study are beneficial to further study for enhancing the production of Ganoderma lucidum polysaccharides in large scale commercialized production.

Exopolysaccharides, proteins and lipids in Pleurotus pulmonarius submerged culture using different carbon sources.

For many years mushrooms have been consumed and appreciated by their nutritional value, and medicinal properties. The traditional mushroom cultivation takes too long and the macrofungi biotechnology has not been explored in its full potential yet. The goal of this work was to observe if different carbon sources could improve the yield and diversify fungi nutrient composition in submerged culture. Pleurotus pulmonarius mycelia and exopolysacharide productions were evaluated using glucose, galactose, xylose and arabinose. The mycelia yield varied depending on the culture medium, and galactose showed to be the best carbon source to produce EPS. Samples that showed the highest protein contents were grown with xylose (19.44%) and arabinose (26.05%). Furthermore, the biomass cultivated with these carbohydrates and with galactose showed five essential amino acids. All cultured biomass showed low lipid contents (∼1%), being composed mainly of unsaturated fatty acids. All EPS fractions showed as main structures glucans and mannogalactans.