The complete biodegradation pathway of ellagitannins by Aspergillus niger in solid-state fermentation.

Our research group has found preliminary evidences of the fungal biodegradation pathway of ellagitannins, revealing first the existence of an enzyme responsible for ellagitannins degradation, which hydrolyzes pomegranate ellagitannins and it was called ellagitannase or elagitannin acyl hydrolase. However, it is necessary to generate new and clear information in order to understand the ellagitannin degradation mechanisms. This work describes the distinctive and unique features of ellagitannin metabolism in fungi. In this study, hydrolysis of pomegranate ellagitannins by Aspergillus niger GH1 was studied by solid-state culture using polyurethane foam as support and pomegranate ellagitannins as substrate. The experiment was performed during 36 h. Results showed that ellagitannin biodegradation started after 6 h of fermentation, reaching the maximal biodegradation value at 18 h. It was observed that ellagitannase activity appeared after 6 h of culture, then, the enzymatic activity was maintained up to 24 h of culture reaching 390.15 U/L, after this period the enzymatic activity decreased. Electrophoretic band for ellagitannase was observed at 18 h. A band obtained using non-denaturing electrophoresis was identified as ellagitannase, then, a tandem analysis to reveal the ellagitannase activity was performed using Petri plate with pomegranate ellagitannins. The extracts were analyzed by HPLC/MS to evaluate ellagitannins degradation. Punicalin, gallagic acid, and ellagic acid were obtained from punicalagin. HPLC/MS analysis identified the gallagic acid as an intermediate molecule and immediate precursor of ellagic acid. The potential application of catabolic metabolism of ellagitannin hydrolysis for ellagic acid production is outlined.

Effect of different polyphenol sources on the efficiency of ellagic acid release by Aspergillus niger.

Fungal hydrolysis of ellagitannins produces hexahydroxydiphenic acid, which is considered an intermediate molecule in ellagic acid release. Ellagic acid has important and desirable beneficial health properties. The aim of this work was to identify the effect of different sources of ellagitannins on the efficiency of ellagic acid release by Aspergillus niger. Three strains of A. niger (GH1, PSH and HT4) were assessed for ellagic acid release from different polyphenol sources: cranberry, creosote bush, and pomegranate used as substrate. Polyurethane foam was used as support for solid-state culture in column reactors. Ellagitannase activity was measured for each of the treatments. Ellagic acid was quantified by high performance liquid chromatography. When pomegranate polyphenols were used, a maximum value of ellagic acid (350.21 mg/g) was reached with A. niger HT4 in solid-state culture. The highest amount of ellagitannase (5176.81 U/l) was obtained at 8h of culture when cranberry polyphenols and strain A. niger PSH were used. Results demonstrated the effect of different polyphenol sources and A. niger strains on ellagic acid release. It was observed that the best source for releasing ellagic acid was pomegranate polyphenols and A. niger HT4 strain, which has the ability to degrade these compounds for obtaining a potent bioactive molecule such as ellagic acid.

Enzymatic Biotransformation of Pomegranate Ellagitannins: Initial Approach to Reaction Conditions.

BACKGROUND: Ellagitannase (Ellagitannin acyl hydrolase) is an inducible enzyme with great potential use in food industry since allows the ellagic acid release from ellagitannins. OBJECTIVE: In this work, ellagitannase was produced by the fungus Aspergillus niger GH1 in solid state fermentation using polyurethane foam as solid support and pomegranate husk ellagitannins as sole carbon source and ellagitannase inducer and an initial approach to the enzymatic reaction conditions was reached. MATERIALS AND METHODS: Ellagitannase was produced by Aspergillus niger GH1 in solid state fermentation and the ideal reaction conditions for ellagitannase activity based on ellagic acid quantification as ellagitannins biotransformation product by high performance liquid chromatographic are reported. RESULTS: The enzyme ideal reaction conditions were substrate concentration of 1 mg.mL-1, 60 °C and pH 5.0, during 10 min of reaction. The kinetic enzyme constants (V max = 30.34 mM.mL-1.min-1 and K m = 1.48 x 103 mM) using punicalagin assubstrate were determined. CONCLUSION: The assay was completed in a short time and may find application in future studies of ellagic acid production.

Bioactive Protecting Coating of Guar Gum with Thyme Oil to Extend Shelf Life of Tilapia (Oreoschromis niloticus) Fillets.

Edible coatings are safe, legal, and sensory acceptable for food applications and they can be incorporated as natural additives due to their antimicrobial activity, thickening capacity, nutrient content, and bioactive agents for protecting seafood from physical, chemical, and microbiological damage that affects its shelf-life. This study aimed to evaluate the effect of the guar gum bioactive coating with thyme oil on the quality of tilapia fish fillets for 15 days of storage at 4 °C, as a means to extend shelf-life. pH, moisture, ash, fat, color, thiobarbituric acid reactive substances (TBARS), total volatile basic nitrogen (TVB-N), microbiological, and sensory examinations were investigated, and the results were analyzed by analysis of variance. The treatments were control (uncoated, UC), GGC (coated with guar gum, GGC), and guar gum combined with thyme oil (GGCTH). Tilapia fillets were stored at 4 °C, the safe temperature for refrigerated storage for 15 days. GGCTH had a slower increase of pH after 15 days of storage in comparison with GGC and UC (p < 0.05). GGC and GGCTH resulted in lower and lowest lightness (L*; p < 0.05) values, lower and lowest redness (a*; p < 0.01) values, and greater and greatest yellowness (b*; p < 0.05) values compared to UC, respectively. UC reduced shear force at 5 (0.37 kgf), 10 (0.32 kgf), and 15 (0.30 kgf) days post-storage in comparison with GGC (0.43, 0.43, and 0.43 kgf) and GGCTH (0.43, 0.44, and 0.44 kgf), respectively. There was less (p < 0.05) deterioration, as well as differences in textural and sensorial variables between uncoated and coated fish fillets. The microbiological analyses demonstrated that there was greater microbial growth in the uncoated fillets than in the coated ones. It was concluded that this bioactive coating with thyme oil retards microbial colonization of fish and reduces degradability of quality variables, therefore, it is a reliable and effective alternative to extend the shelf-life of tilapia fillets.

Solid bioprocess of tarbush (Flourensia cernua) leaves for ß-glucosidase production by Aspergillus niger: initial approach to fiber-glycoside interaction for enzyme induction.

Commercial cellulase production has increased in recent years and consistent research has been carried out to improve levels of ß-glucosidase. Bioprocesses have been successfully adapted to produce this enzyme, with solid-state fermentations as the best-suited technique involving fungi. The aim of this study was to use leaves of tarbush (Flourensia cernua), an abundant shrub of the Chihuahuan Desert, as a carbon source for ß-glucosidase production by Aspergillus niger. During the solid bioprocess, this enzyme reached its peak production at 36 h of culture with 3876.6 U/L. There is a particular interest in the substrate composition because of the possibility of phenolic glycosides having an important role in ß-glucosidase production. HPLC-MS analyses showed that glycosides were present with the highest accumulation at 36 h of fungal culture. Luteolin and apigenin glycosides [1.8 and 2.4 absorbance units, respectively] were also detected and showed their highest point of detection alongside the highest ß-glucosidase activity. No apparent changes in cellulose were observed, while hemicellulose content decreased, which could be related to production and activity of ß-glucosidase. This study shows that leaves of F. cernua are an important raw material for ß-glucosidase production and give a source of compounds of added value which also may have an important role for ß-glucosidase production.

Effect of growth conditions on ß-glucosidase production using Flourensia cernua leaves in a solid-state fungal bioprocess.

Flourensia cernua foliage was used in a solid-state fungal bioprocess to identify factors that could affect ß-glucosidase production such as growth medium components and partial identification of molecules from the plant material. Under an exploratory experimental design, each variable had their distinctive result on conditions, which affects and could further improve ß-glucosidase production. Under the experimental design, 1482 U/L of ß-glucosidase were detected, which marks an improvement in production compared to levels obtained in a control treatment with an activity of 1092 U/L. It was shown that inoculum, water content and pH were the factors with the greater effect on ß-glucosidase production. Polyphenolic content and cellulosic fiber in the form of raw fiber were measured to assess compound degradation of the plant material. Although fiber content was apparently unaffected, polyphenolic content decreased; ß-glucosidase was produced by A. niger GH1. This behavior could be associated with fiber level and polyphenolic content because molecules of this type can be hydrolyzed by ß-glucosidase. According to our results, F. cernua biomass can be used as a carbon source for ß-glucosidase production in a short culture time.

Polyphenolic content, in vitro antioxidant activity and chemical composition of extract from Nephelium lappaceum L. (Mexican rambutan) husk.

OBJECTIVE: To determinate the recovery of total polyphenolic compounds content, in vitro antioxidant activity and HPLC/ESI/MS characterization of extract from Nephelium lappaceum L. (Mexican rambutan). METHODS: The rambutan husk extract was obtained by aqueous extraction and a polyphenolic fraction was recovered using Amberlite XAD-16. The total polyphenolic compounds content was determined by the Folin Ciocalteu and butanol-HCI methods. In vitro antioxidant activity was performed using ABTS and ferric reducing antioxidant power methods. RESULTS: Mexican rambutan husk showed a total polyphenolic content of 582 mg/g and an evident antioxidant activity by ABTS and ferric reducing antioxidant power analysis. The HPLC/ESI/MS assay allowed the identification of 13 compounds, most of which belong to ellagitannins. Geraniin, corilagin and ellagic acid were present in the sample; the mineral composition was also evaluated. CONCLUSIONS: Rambutan husk cultivated in Mexico is a promising source for the recovery of added value bioactive compounds with antioxidant activity, which have potential applications as bioactive antioxidant agents for the treatment of diseases.

Antifungal ellagitannin isolated from Euphorbia antisyphilitica Zucc.

OBJECTIVE: To study antifungal activity of a new ellagitannin isolated from the plant residues of Euphorbia antisyphilitica (E. antisyphilitica) Zucc in the wax extraction process. METHODS: An extract was prepared from dehydrated and pulverized residues and fractionated by liquid chromatography on Amberilte XAD-16, until obtained an ellagitannin-rich ethanolic fraction which was treated by rotaevaporation to recover the ellagitannin as fine powder. An aqueous solution was prepared and treated through ionic exchange liquid chromatography (Q XL) and gel permeation chromatography (G 25). The ellagitannin-rich fraction was thermogravimetrically evaluated (TGA and DTA) to test the thermo-stability of ellagic acid (monomeric unit). Then ellagitannin powder was analyzed by infrared spectrospcopy to determinate the functional groups and, also mass spectroscopy was used to determine the molecular ion. RESULTS: The principal functional groups of ellagitannin were determined, the molecular weight was 860.7 g/mol; and an effective antifungal activity against phytopathogenic fungi was demonstrated. CONCLUSIONS: It can be concluded that the new ellagitannin (860.7 g/mol) isolated from E. antisyphilitica Zucc is an effective antifungal agent against Alternaria alternata, Fusarium oxyzporum, Colletotrichum gloeosporoides and Rhizoctnia solani.

Efecto de las diferentes fuentes de polifenoles sobre la eficiencia de liberación de ácido elágico por Aspergillus niger / Effect of different polyphenol sources on the efficiency of ellagic acid release by Aspergillus niger

Fungal hydrolysis of ellagitannins produces hexahydroxydiphenic acid, which is considered an intermediate molecule in ellagic acid release. Ellagic acid has important and desirable beneficial health properties. The aim of this work was to identify the effect of different sources of ellagitannins on the efficiency of ellagic acid release by Aspergillus niger. Three strains of A. niger (GH1, PSH and HT4) were assessed for ellagic acid release from different polyphenol sources: cranberry, creosote bush, and pomegranate used as substrate. Polyurethane foam was used as support for solid-state culture in column reactors. Ellagitannase activity was measured for each of the treatments. Ellagic acid was quantified by high performance liquid chromatography. When pomegranate polyphenols were used, a maximum value of ellagic acid (350.21 mg/g) was reached with A. niger HT4 in solid-state culture. The highest amount of ellagitannase (5176.81 U/l) was obtained at 8 h of culture when cranberry polyphenols and strain A. niger PSH were used. Results demonstrated the effect of different polyphenol sources and A. niger strains on ellagic acid release. It was observed that the best source for releasing ellagic acid was pomegranate polyphenols and A. niger HT4 strain, which has the ability to degrade these compounds for obtaining a potent bioactive molecule such as ellagic acid.

La hidrólisis fúngica de los elagitaninos produce ácido hexahidroxidifénico, considerado como una molécula intermedia en la liberación de ácido elágico. El ácido elágico tiene importantes y deseables propiedades benéficas para la salud humana. El objetivo de este trabajo fue identificar el efecto de la fuente de elagitaninos sobre la eficiente liberación de ácido elágico por Aspergillus niger. La liberación de ácido elágico se realizó con tres cepas de A. niger (GH1, PSH y HT4) en presencia de diferentes fuentes de polifenoles (arándano, gobernadora y granada), usadas como sustrato. Se empleó espuma de poliuretano como soporte para el cultivo en estado sólido en reactores en columna. Se midió la actividad elagitanasa a cada uno de los tratamientos. El ácido elágico liberado se cuantificó por cromatografía líquida de alta resolución. Cuando se utilizaron los polifenoles de granada, se alcanzó un valor máximo de 350,21 mg/g de ácido elágico con A. niger HT4 en cultivo en estado sólido. La mayor actividad elagitanasa (5176.81 U/l) se obtuvo a 8 h de cultivo cuando se usaron los polifenoles de arándano como sustrato y A. niger PSH. Los resultados demostraron el efecto que tiene la fuente de polifenoles y la cepa de A. niger en la liberación de ácido elágico. Se observó que la mejor fuente para la liberación de ácido elágico fueron los polifenoles de granada y que la cepa A. niger HT4 posee la habilidad de degradar estos compuestos para la obtención de potentes moléculas bioactivas, como el ácido elágico.

Microbial production of ellagic acid and biodegradation of ellagitannins.

In the last years, tannin biodegradation has been the subject of a lot of studies due to its commercial importance and scientific relevance. Tannins are molecules of low biodegradation and represent the main chemical group of natural anti-microbials occurring in the plants. Among the different kinds of tannins, ellagitannins represent the group less studied manly due to their diversity and chemical complexity. The general outline of this work includes information on tannins, their classification and properties, biodegradation, ellagic acid production, and potential applications. In addition, it describes molecular, catalytic, and functional information. Special attention has been focused on the biodegradation of ellagitannins describing the possible role of microbial enzymes in the production of ellagic acid.

Ellagic acid production by Aspergillus niger in solid state fermentation of pomegranate residues.

Two Aspergillus niger strains (GH1 and PSH) previously isolated from a semiarid region of Mexico were characterized for their effectiveness in converting pomegranate ellagitannins (ET) into ellagic acid (EA) in a solid state fermentation (SSF). Pomegranate seeds and husk were used as support for the SSF. Released EA was evaluated by liquid chromatography. Yields of 6.3 and 4.6 mg of EA per gram of dried pomegranate husk were obtained with A. niger GH1 and PSH, respectively. Total hydrolyzable polyphenols of pomegranate husk were degraded during the first 72 h of culture (71 and 61%, by GH1 and PSH strains, respectively). Tannin acyl hydrolase activity was not clearly associated with EA production. EA that accumulated in cultures of A. niger GH1 was remarkably pure after a simple extraction process. Pomegranate husk is a good support, and at the same time an excellent substrate in the production of high commercial interest metabolites like EA due the degradation of its ET content.