Matrix-assisted laser desorption ionization mass spectrometry based quantitative analysis of cordycepin from Cordyceps militaris / 药物分析学报

Cordycepin,which has great immunomodulatory activities such as anticancer,antifungal,antivirus,antileukemia and lipid-lowering ones,is the secondary metabolite of Cordyceps militaris (C.militaris).Liquid submerged fermentation is the common cultivation process to produce cordycepin.To optimize the fermentation process and improve production,monitoring the cordycepin secretion in the fermen-tation is essential.The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation,so more rapid and convenient methods are required.Matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS) is more attractive for faster and direct detection.Therefore,MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium.This method made accurate quantification of cordycepin in the range of 5-400 μg/mL with a relative standard deviation of 5.6％.The recovery rates of fermentation samples after the 1,13,and 25 days were 90.15％,94.27％,and 95.06％,respectively.The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20th culture day,respectively.The cordycepin secretion curve of the liquid fermentation of C.militaris was real-time traced over 25 days.

Advances in biosynthesis of cordycepin from Cordyceps militaris / 生物工程学报

Cordycepin as the main active ingredient of Cordyceps militaris, a traditional medicinal fungus in China, has many physiological functions such as anti-cancer, anti-tumor and anti-virus activity. The most potential route for effective cordycepin production has been considered as liquid fermentation of C. militaris though with low productivity at present. Thus, it is urgent to apply both process engineering strategy and metabolic engineering strategy to enhance the productivity of cordycepin. In this review, the effects of medium components (i.e. the carbon/nitrogen source, precursor substances and metal ions) and operation factors (i.e. pH, dissolved oxygen and light) on cordycepin biosynthesis in liquid fermentation system are summarized. Besides, separation of cordycepin, the gene cluster involved and predicted biosynthesis pathways of cordycepin are also discussed, providing possible solutions of finally realizing efficient production of cordycepin.

Selection, isolation, and identification of fungi for bioherbicide production

Abstract Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture.

Changes of chemical components and anti-tumor activity of total ginsenosides from ginseng stems and leaves tranformed by submerged fermentation of ganoderma lucidum / 吉林大学学报(医学版)

Objective:To study the biotransformation rule of the chemical components of total ginsenosides from ginseng stems and leaves tranformed by submerged fermentation of ganoderma lucidum,and to lay the foundation for further development and research of the medicinal fungus of ginseng stems and leaves.Methods:The total ginsenosides from ginseng stems and leaves were fermented by ganoderma lucidum,the changes of chemical components before and after fermentation were measured by thin-layer chromatography;the levels of total ginsenosides from ginseng stems and leaves before and after fermentation were detected by ultraviolet spectrophotometry;the levels of ginsenoside Rb1,Rg3,Rd,CK,Re,Rg,and Rh1 before and after fermentation were measured by high performance liquid chromatography.The human hepatocellular carcinoma SMMC-7721 cells were divided into blank control group,low,medium and high doses of total ginsenosides from ginseng stems and leaves groups and medicinal fungus of ginseng stems and leaves groups.The dosages of total ginsenosides from ginseng stems and leaves and medicinal fungus of ginseng stems and leaves were 5,10 and 20 mg·L-1,respectively.The inhibitory rate(IR) of the cells was determined by MTT assay.Results:The results of thin-layer chromatography showed that the saponins before and after fermentation occured mutual transformation;the levels of total ginsenosides from ginseng stems and leaves before and after fermentation were 749.98 and 602.26 mg·g-1,respectively;the levels of panaxadiol saponins Rb1,Rg3,Rd and CK before fermentation were 24.54,2.21,87.22 and 0 mg·g-1,and the levels of panaxatriol saponins Re,Rg and Rh1 were 151.34,77.02,and 3.06 mg·g-1.After fermentation,the level of Rb1 was decreased by 61.45%,the levels of Rg3,Rd and CK were increased by 238.91%,34.43% and 268.00%.The levels of Re and Rg1 were decreased by 63.75% and 64.41%,and the level of Rh1 was increased by 408.88%;the IR of the SMMC-7721 cells in medium dose of medicinal fungus of ginseng stems and leaves group was higher than that in medium dose of total ginsenosides from ginseng stems and leaves group(P<0.05);the IR of the SMMC-7721 cells in high dose of medicinal fungus of ginseng stems and leaves group was higher than that in high dose of total ginsenosides from ginseng stems and leaves group(P<0.01).Conclusion:The total ginsenosides from ginseng stems and leaves transformed by the submerged fermentation of ganoderma lucidum can significantly change its chemical components,which have stronger anti-proliferation activity in the SMMC-7721 cells.

PRODUCCIÓN DE XILITOL POR Candida guilliermondii A PARTIR DE FERMENTACIÓN DE RESIDUOS DE PALMA DE ACEITE

La hidrólisis ácida diluida del residuo lignocelulósico de raquis de palma de aceite produce azúcares fermentables, como la xilosa, principal fuente de carbono para la producción de xilitol, por Candida guilliermondii En este estudio, se evaluó el efecto de diferentes medios de cultivo y de condiciones de fermentación sobre la producción de xilitol, a partir de raquis de palma de aceite, utilizando C guilliermondii. El hidrolizado ácido de raquis de palma suplementado con 4g/L extracto de levadura, 3g(NH4)2SO4/L, 0,5g/ MgSO4.7H2O/L y 0,1gCaCl2.2H2O/L mostró ser el mejor medio para el crecimiento de la levadura en cultivo sumergido, debido a que presentó los valores mayores, estadísticamente significativos, de la velocidad específica máxima de crecimiento, de 0,12h-1 y producción de biomasa, de 5,5g/L (p<0,05). Las condiciones de fermentación más apropiadas se obtuvieron con hidrolizado de raquis suplementado a pH de 5,5, concentración inicial de xilosa de 17g/L y un inóculo de 3g/L. La microaerobiosis mostró ser un factor importante en el proceso de fermentación, con un volumen de 40mL de medio, en un matraz de 100mL, se produjo la mayor concentración de xilitol de 6,7 g/L (p<0,05).

The dilute-acid hydrolysis of oil palm empty fruit bunch produces fermentable sugars such as xylose, main carbon source for xylitol production by Candida guilliermondii. The influence of different culture media and fermentation conditions were evaluated on the production of xylitol from oil palm empty fruit bunch using C guilliermondii. The acid hydrolyzated oil palm empty fruit bunch supplemented with4g yeast extract/L, 3g (NH4)2SO4/L, 0.5g MgSO4.7H2O/L y 0.1g CaCl2.2H2O/L, was the best culture medium for the yeast growth in submerged culture because it had the highest values, statistically significant, of the specific growth rate 0.12h-1 and biomass production 5.5g/L (p<0,05). The suitable fermentation conditions were obtained with hydrolyzated oil palm empty fruit bunch supplemented at pH 5.5, initial xylose concentration of 17g/L and an inoculum of 3g/L. Microaerobic condition is an important factor on the fermentation process, with a volume of 40mL in a flask of 100mL, that produces the highest concentrations of xylitol (6.7g/L) (p<0,05).

Optimization of technique for add itional liquid fermentation of Poria cocos and comparison on chemical constituents from different sources of Poria / 中草药

Objective To optimize the technique for additional liquid fermentation of Poriacocos in fermentor and to compare the chemical constituents in natural Poria, fermented Poria, medicinal fermented Poria, and compound medicinal fermented Poria. Methods The additional liquid fermentations of Poriacocos in fermentor had been carried out among different initial fermentation media, such as nutrition medium, medicinal medium, and compound medicinal medium. An optimum initial fermentation medium had been screened and an optimum time of additional liquid fermentation had been determined. The chemical constituents fromthedifferent sources of Poria had been isolated and detected according to their physicochemical properties. Results The compound medicinal medium with Coicis Semen and Lycii Fructus was an optimum initial fermentation medium, and 120 h was an optimum time of additional liquid fermentation. The total saccharides, polysaccharides, amino acids, trace elements, and ashes in thedifferent sources of Poria had been detected. Conclusion There are considerable influences on liquid fermentation of Poriacocos in different initial fermentation media. There are rather big differences of chemical constituents from the different sources of Poria.

Estudio de las condiciones de mezclado en fermentador para la producción de blastosporas de Beauveria bassiana / Study of the mixing conditions in bioreactor for blastospores production of Beauveria bassiana

Se caracterizaron tres fermentadores: New Brunswick M-19 de 14 litros, Applikon Biocontroller 1035 de 7 litros y New Brunswick Bioflo III de 7 litros, determinando el coeficiente volumétrico de transferencia de oxígeno (KLa), la retención de gas (RG) y el tiempo de mezclado (tM). El fermentador New Brunswick Bioflo III tuvo los mejores valores con una relación de diámetro del impulsor/diámetro del tanque (DI/DT) de 0.43, KLa = 9.5-208 h-1 y tM = 1.0-3.0 s, por lo que fue seleccionado para realizar la producción de blastosporas de Beauveria bassiana, utilizando melaza como fuente de carbono. Se estudiaron las condiciones de mezclado, utilizando diferentes combinaciones de impulsores tipo Rushton, Maxflo y Lightnin, bajo un diseño experimental factorial 32. El tiempo de propagación fue de 4 días, el volumen de trabajo 4 litros, 10% de inóculo (1x106 blastosporas/ml), temperatura 30°C, agitación de 400-500 rpm, aireación de 0.5-1.0 vvm, y pH de 5.4.El hongo se desarrolló mejor utilizando la combinación de impulsores Rushton-Maxflo a 400 rpm y 1.0 vvm (F = 10.324, p ≤ 0.0123) (DMS=0.585), obteniendo una concentración de 1.2x109 blastosporas/ml, 2.2 g/l de biomasa y 2.48 g/l de consumo de sustrato (Y x/s=0.89). Las condiciones de mezclado y los parámetros obtenidos pueden ser aplicados en otros fermentadores para optimizar la producción de blastosporas de B. bassiana en la elaboración experimental de bioinsecticidas.

In this work three fermenters were characterized: New Brunswick M-19 of 14 liters, Biocontroller Applikon 1035 of 7 liters and New Brunswick Bioflo III of 7 liters, determining the volumetric coefficient oxygen transfer (KLa), gas hold up (GH) and the mixing time (tM). The fermenter New Brunswick Bioflo III had the best values with a ratio of diameter of the impeller/vessel (DI/DT) of 0.43, KLa = 9.5-208 h-1 and tM = 1.0-3.0 s, so it was selected for the production of blastospores of Beauveria bassiana, using molasses as carbon source. We studied the mixing conditions, using different combinations of impellers, type Rushton, Lightnin and Maxflo under a factorial experimental design 32. The propagation time was of 4 days, working volume 4l, 10% inoculum (1x106 blastospores/ml), temperature 30°C, agitation of 400-500 rpm, aeration 0.5-1.0 vvm, and pH of 5.4. The fungus growth better using a combination of impellers Rushton-Maxflo at 400 rpm and 1.0 vvm (F = 10.324, p ≤ 0.0123) (LSD = 0.585) obtaining a concentration of 1.2x109 blastospores/ml, 2.2 g/l biomass and 2.48 g/l of substrate consumption (Y x/s = 0.89). The mixing conditions and the parameters obtained can be applied to optimize the blastospore productions of B. bassiana to fermenter level in the experimental production of bioinsecticides.

EVALUACIÓN DEL ANTAGONISMO Y MULTIPLICACIÓN DE Trichodermasp. EN SUSTRATO DE PLÁTANO EN MEDIO LÍQUIDO ESTáTICO / Evaluation Of Antagonism And Multiplication The Trichodermasp. In The Middle Static Liquid Banana Substrate

Se evaluó la capacidad antagónica de nueve aislados del hongo Trichoderma sp. proveniente de la colección de cepas del Laboratorio de Fitopatología de la Universidad de Córdoba, Colombia y su producción de esporas en medios de cultivo líquido estático, a través de pruebas in vitro de antagonismo, utilizando dos hongos fitopatógenos: Fusarium sp. y Rhizoctonia sp.; esta prueba permitió seleccionar el aislado con la mayor actividad antagónica, para así ajustar la producción de esporas mediante un proceso de fermentación en estado líquido en condición estática mediante procesos de buferización y sin buferización con fosfato 0,2N, utilizándose como sustrato principal harina de plátano (HP) 5%, además de complementos nutricionales que constituyeron los tratamientos: HPM (solución de melaza 5%), HPL (solución de levadura granulada al 2%), HPU (solución de urea 0,1%), y como testigo PGL (papa glucosa líquida). En los ensayos de antagonismo se seleccionó el aislado C2 de Trichoderma sp. por presentar la mayor capacidad antagónica frente al hongo Fusarium sp., con un porcentaje de inhibición del 78,30% y reportando un índice 4 de antagonismo, en los procesos de producción de esporas el tratamiento HPL (harina de plátano más levadura sin buffer) se destacó por su favorable producción de conidias, con una concentración de 1,1 x 10(9) conidias/mL. Los procesos de buferización se diferenciaron significativamente, demostrando que la variable buffer influyó en la producción de conidias, los medios que se amortiguaron mostraron una producción de 1,01 x 10(9) conidias/mL, con respecto a los que no se amortiguaron 6,3 x 10(8) conidias/mL.

The antagonistisc ability of nine isolated natives fungi Trichoderma sp from the collection of strains at Laboratory of phytopathology at University of Cordoba, Colombia was assessed so as its production of spores in crop liquid static medium, cross the in vitro proof antagonism using two phytopathogenic fungi: Fusarium sp. and Rhizoctonia sp., this proof let select the isolated with the major antagonistic activity and adjust the production of spores through fermentation process in liquid static by means of buffer and without buffer with phosphate 0,2 N, being used as principal substrate plantain flour (HP) 5%, besides of nutritional supplements that formed the trataments: HPM (molasses solution 5%), HPL (granulated yeast solution at 2%), HPU (urea solution 0.1%), and as witness PGL (potato glucose liquid). In the test of antagonism was selected the isolated C2 of Trichoderma sp. by present the major antagonistic ability in front of the fungi Fusarium sp., with a inhibition percentage at 78,30% and reporting a index 4 of antagonism, in the process of spores production, the tratament HPL (plantain flour plus yeast without buffer) stressed for the favorable production of conidias, with a concentration of 1,1 x 10(9) conidias/mL. The buffer process were significant differentiated, showing that the variable buffer influenced in the production of conidias, the mediums that shocked in the production show 1,01 x 10(9) conidias/mL, Ïwith respect at don’t shocked 6,3 x 10(8) conidias/mL.

Optimización de un medio de cultivo para la producción de la levadura Pichia onychis (Lv027) / Optimising culture medium for producing the yeast Pichia onychis (Lv027)

La optimización de la producción masiva de la levadura Pichia onychis fue investigada usando varios sustratos y evaluando diferentes condiciones físico-químicas de fermentación líquida. Inicialmente se realizó un screening aplicando el diseño estadístico Plackett-Burman para evaluar tres fuentes de carbono y ocho fuentes de nitrógeno (tanto orgánicas como inorgánicas) con el fin de seleccionar los factores nutricionales más influyentes sobre el crecimiento de la levadura. Posteriormente, se evaluaron cuatro fuentes nutricionales y dos variables físico-químicas utilizando un diseño factorial fraccionado como punto de partida, para llevar a cabo después un proceso de optimización aplicando un diseño estadístico Central Compuesto Rotacional. Un modelo de regresión polinomial se desarrolló usando los datos experimentales; los resultados muestran que la producción de biomasa fue afectada significativamente por condiciones tanto nutricionales como físico-químicas del medio de cultivo; el máximo rendimiento obtenido fue de 8,95 XlO9 células/mL equivalentes a una biomasa seca de 6,30 g/L, el cual se logró con las siguientes condiciones: 43,42 g/L de fuente de carbono, 0,261 g/L de fuente de nitrógeno orgánica, agitación de 110 rpm, pH = 6,0 con un tiempo de fermentación de 48 horas.

Optimising Pichia onychis yeast biomass production was evaluated using different substrates and different physicochemical conditions for liquid fermentation. The Plackett-Burman statistical design was initially applied for screening the most important nutritional variables (three carbon sources and eight nitrogen sources) affecting yeast biomass production. Four nutritional sources and two physicochemical variables were subsequently evaluated using a factorial fractionated design as the starting point for optimising the process by applying a central composite rotational design. The results obtained from employing a polynomial regression model using the experimental data showed that biomass production was strongly affected bynutritional and physicochemical conditions. The highest yield was obtained in the following conditions: 43,42 g/L carbon source, 0,261 g/L nitrogen organic source, shaking at 110 rpm, 6,0 pH, 48 h total fermentation time during which 8,95 XlO9 cells/mL were obtained, equivalent to 6,30 g/L dry biomass.

The Food Chinese (Medicine) Uses the Fungus Fermentation Engineering Research Progress / 微生物学通报

By reviewing fresh (medicine) fungi production process of history,expounded the modern food (medicine) with fungal fermentation engineering including liquid fermentation and solid state fermentation research,and highlighted the diversity of new solid-state fermentation works for the founding and development of mankind can provide inexhaustible,unlimited access to the traditional production process valuable end products.