Production of a Fungal Punicalagin-Degrading Enzyme by Solid-State Fermentation: Studies of Purification and Characterization.

The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by Aspergillus niger GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained Km values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid.

Successive Fermentation of Aguamiel and Molasses by Aspergillus oryzae and Saccharomyces cerevisiae to Obtain High Purity Fructooligosaccharides.

Fructooligosaccharides (FOS) are usually synthesized with pure enzymes using highly concentrated sucrose solutions. In this work, low-cost aguamiel and molasses were explored as sucrose alternatives to produce FOS, via whole-cell fermentation, with an Aspergillus oryzae DIA-MF strain. FOS production process was optimized through a central composite experimental design, with two independent variables: initial sucrose concentration in a medium composed of aguamiel and molasses (AgMe), and inoculum concentration. The optimized process-165 g/L initial sucrose in AgMe (adjusted with concentrated molasses) and 1 × 107 spores/mL inoculum concentration-resulted in an FOS production of 119 ± 12 g/L and a yield of 0.64 ± 0.05 g FOS/g GFi. Among the FOSs produced were kestose, nystose, 1-fructofuranosyl-nystose, and potentially a novel trisaccharide produced by this strain. To reduce the content of mono- and disaccharides in the mixture, run a successive fermentation was run with two Saccharomyces cerevisiae strains. Fermentations run with S. cerevisiae S227 improved FOS purity in the mixture from 39 ± 3% to 61.0 ± 0.6% (w/w) after 16 h of fermentation. This study showed that agro-industrial wastes such as molasses with aguamiel are excellent alternatives as substrate sources for the production of prebiotic FOS, resulting in a lower-cost process.

Recovery of Bioactive Ellagitannins by Ultrasound/Microwave-Assisted Extraction from Mexican Rambutan Peel (Nephelium lappaceum L.).

Rambutan (Nephelium lappaceum L.) is a tropical fruit from Asia which has become the main target of many studies involving polyphenolic analysis. Mexico produces over 8 million tons per year of rambutan, generating a huge amount of agro-industrial waste since only the pulp is used and the peel, which comprises around 45% of the fruit's weight, is left behind. This waste can later be used in the recovery of polyphenolic fractions. In this work, emerging technologies such as microwave, ultrasound, and the hybridization of both were tested in the extraction of phenolic compounds from Mexican rambutan peel. The results show that the hybrid technology extraction yielded the highest polyphenolic content (176.38 mg GAE/g of dry rambutan peel). The HPLC/MS/ESI analysis revealed three majoritarian compounds: geraniin, corilagin, and ellagic acid. These compounds explain the excellent results for the biological assays, namely antioxidant activity evaluated by the DPPH, ABTS, and LOI (Lipid oxidation inhibition) assays that exhibited great antioxidant capacity with IC50 values of 0.098, 0.335, and 0.034 mg/mL respectively, as well as prebiotic activity demonstrated by a µMax (maximum growth) of 0.203 for Lactobacillus paracasei. Lastly, these compounds have shown no hemolytic activity, opening the door for the elaboration of different products in the food, cosmetic, and pharmaceutical industries.

Antioxidant Dietary Fiber Sourced from Agroindustrial Byproducts and Its Applications.

Agroindustrial activities generate various residues or byproducts which are inefficiently utilized, impacting the environment and increasing production costs. These byproducts contain significant amounts of bioactive compounds, including dietary fiber with associated phenolic compounds, known as antioxidant dietary fiber (ADF). Phenolic compounds are related to the prevention of diseases related to oxidative stress, such as neurodegenerative and cardiovascular diseases. The mechanism of ADF depends on its chemical structure and the interactions between the dietary fiber and associated phenolic compounds. This work describes ADF, the main byproducts considered sources of ADF, its mechanisms of action, and its potential use in the formulation of foods destined for human consumption. ADF responds to the demand for low-cost, functional ingredients with great health benefits. A higher intake of antioxidant dietary fiber contributes to reducing the risk of diseases such as type II diabetes, colon cancer, obesity, and kidney stones, and has bile-acid retention-excretion, gastrointestinal laxative, hypoglycemic, hypocholesterolemic, prebiotic, and cardioprotective effects. ADF is a functional, sustainable, and profitable ingredient with different applications in agroindustry; its use can improve the technofunctional and nutritional properties of food, helping to close the cycle following the premise of the circular economy.

Structural characterization of native and oxidized procyanidins (condensed tannins) from coffee pulp (Coffea arabica) using phloroglucinolysis and thioglycolysis-HPLC-ESI-MS.

Procyanidins from coffee pulp are responsible from the limited valorization of this by-product. Information about procyanidin structure is still scarce and imprecise. The aim of this work was to study the native and oxidized procyanidins from coffee pulp with respect to composition and structure. An aqueous acetone extract from coffee pulp was purified using Sephadex LH-20. Butanolysis, phloroglucinolysis and thioglycolysis coupled to HLPC-ESI-MS were applied for the characterization of the native and oxidized procyanidins. The purification allowed to recovery three fractions (aqueous, ethanolic and acetonic) and only acetone fraction showed a high concentration of procyanidins (98%, w/w). HPLC-ESI-MS of procyanidins-rich fraction without any reaction resulted in a UV-Vis chromatogram unresolved typical of the presence of procyanidins. The extracted ion chromatogram and MS2 analysis revealed the presence from dimers to pentamers of native procyanidins. Interestingly, by first time an A-type trimeric procyanidin (m/z of 863) was observed in coffee pulp. In our study, (-)-epicatechin was the constitutive unit of procyanidins with an aDP of 6.8 (oligomeric native procyanidins) according to the phloroglucinolysis assay. Two oxidation markers useful to characterization of oxidized procyanidins were observed in the procyanidins-rich fraction after thioglycolysis, a dimer A2-ext and a molecule that corresponds to a linkage between an extension and a terminal unit. Coffee pulp procyanidins were presented with only a minor class of oxidized procyanidins. As far as we know, this is the first study about characterization of the oxidized procyanidins from coffee pulp.

Functionality of Agave Bagasse as Supplement for the Development of Prebiotics-Enriched Foods.

Agave bagasse is a fibrous-like material obtained during aguamiel extraction, which is also in contact with indigenous microbiota of agave plant during aguamiel fermentation. This plant is a well-known carrier of the prebiotic fructan-type carbohydrates, which have multiple ascribable health benefits. In the present work, the potential of ashen and green agave bagasse as functional ingredients in supplemented cookies was studied. For its application, the chemical, functional, properties of agave bagasses and formulated cookies were evaluated, as well as the physical properties of cookies. Chemical characterization was carried out by the proximate analysis of both bagasses and cookies, besides, the analysis of oligosaccharides was made by thin-layer chromatography and high-performance anion-exchange chromatography. In the same way, functional properties such as oil holding capacity, organic molecule absorption capacity, swelling capacity, and water holding capacity were analyzed in both agave bagasses and supplemented cookies. Finally, modifications in color and texture due to bagasse addition was studied through an analysis of total color difference and a penetrometric test, respectively. In this sense, ashen and green agave bagasses demonstrated chemical and functional properties for use in the food industry, since they increased oil holding capacity of cookies and transferred prebiotic fructooligosaccharides to both agave bagasse formulations, which remain active as a prebiotic ingredient in cookies after in vitro digestion and cookie manufacture, including thermal treatment. Hence, agave bagasse could be considered a valuable alternative for the addition of the nutritionally-relevant dietary fiber in healthier foods.

Purification and biochemical characterization of an Aspergillus niger phytase produced by solid-state fermentation using triticale residues as substrate.

In this study, an extracellular phytase produced by Aspergillus niger 7A-1, was biochemically characterized for possible industrial application. The enzyme was purified from a crude extract obtained by solid-state fermentation (SSF) of triticale waste. The extract was obtained by microfiltration, ultrafiltration (300, 100 and 30 kDa) and DEAE-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 89 kDa by SDS-PAGE. The purified enzyme was most active at pH 5.3 and 56 °C, and retained 50% activity over a wide pH range of 4 to 7. The enzymatic thermostability assay showed that the enzyme retained more than 70% activity at 80 °C for 60 s, 40% activity for 120 s and 9% after 300 s. The phytase showed broad substrate specificity, a Km value of 220 µM and Vmax of 25 µM/min. The purified phytase retained 50% of its activity with phosphorylated compounds such as phenyl phosphate, 1-Naphthyl phosphate, 2-Naphthyl phosphate, p-Nitrophenyl phosphate and Glycerol-2-phosphate. The inhibition of phytase activity by metal ions was observed to be drastically inhibited (50%) by Ca++ and was slightly inhibited (10%) by Ni++, K+, and Na+, at 10 and 20 mM concentrations. A positive effect was obtained with Mg++, Mn++, Cu++, Cd++ and Ba++ at 25 and 35% with stimulatory effect on the phytase activity.

On-line monitoring of Aspergillus niger GH1 growth in a bioprocess for the production of ellagic acid and ellagitannase by solid-state fermentation.

The present work describes the monitoring of CO2 production by Aspergillus niger GH1 in a bioprocess for the production of ellagitannase (EAH) and ellagic acid by solid state fermentation. Pomegranate ellagitannins, mainly punicalagin, were used as carbon source and EAH inducer. A second condition, using ellagitannins and maltose as growth promoting carbon source, was tested. The ellagic acid production was quantified and the EAH activity was assayed. The accumulated metabolites were identified by HPLC-ESI-MS/MS. Higher CO2 production (7.79mg/grams of dry material) was reached in media supplemented with maltose. Short-time lag phase (7.79h) and exponential phase (10.42h) were obtained using only ellagitannins, despite its lower CO2 production (3.79mg/grams of dry material). Without the use of maltose lower ellagic acid (11.85mg/L/h) and EAH (21.80U/L/h) productivities were reached. The use of maltose enhances the productivity of EA (33.18mg/L/h) and EAH (33.70U/L/h). Besides of punicalin and ellagic acid, two unknown compounds with mass weight of 702 and 290g/mol (ions 701 and 289m/z in negative mode, respectively) were identified and characterized by HPLC-ESI-MS/MS analysis.

Ellagitannins: Bioavailability, Purification and Biotechnological Degradation.

The ellagitannins are a group of phenolic compounds with biological activities. Ellagic acid is the product obtained from hydrolysis of ellagitannins. Information related to the biosynthesis of ellagitannins still been scarce and confused. The ellagitannins are obtained from plants and their purification process implies mainly the use of chromatographic techniques. The ellagitannin acyl hydrolase (EAH) also known as ellagitannase is an enzyme capable of hydrolyzing the ester bonds of ellagitannins and the consequent releasing of ellagic acid. Information about the EAH is not clear because the enzyme had showed different activities due to the low purity or complexity of substrates and there is no available information about the biochemical, physicochemical and molecular characteristics of EAH. The present review describes information related to the sources, biosynthesis and the purification of ellagitannins and a current assessment on the production of ellagitannase.

Pectin from Husk Tomato (Physalis ixocarpa Brot.): Rheological behavior at different extraction conditions.

A rheological study was carried out to evaluate formulations of test dispersions and gels of high methoxyl pectins (HTHMP) obtained at different conditions from husk tomato waste (Physalis ixocarpa Brot.). The effect of extraction agent (hydrochloric acid or citric acid), blanching time (10 or 15min) and extraction time (15, 20 or 25min) on the rheology of the tested samples was evaluated. Flow behavior and activation energy were evaluated on the test dispersions, while (Ea) frequency sweeps, temperature sweep, creep-recovery test and penetration test were performed on the gels. HTHMP dispersions showed shear thinning flow behavior, while showing a good fit to Cross model. Extraction agent, blanching time and extraction time did not have effect on Cross parameters (ηz, η∞, C, and m). Ea decreased as blanching time and extraction time increased. Frequency sweeps revealed high dependence on frequency for both G' and G", while temperature sweeps (25- 95°C) showed thermostable husk tomato pectin gels. Hydrocloric acid (HCl) extracted pectin gels showed stronger structure than citric acid (CA) gels.

Characterisation of Pomegranate-Husk Polyphenols and Semi-Preparative Fractionation of Punicalagin.

INTRODUCTION: Pomegranate-husk is the main by-product generated from the pomegranate industry. It is a potential source of compounds highly appreciated by different costumers. Punicalagin is the main compound present in pomegranate-husk. OBJECTIVE: To characterise the pomegranate-husk total polyphenols by HPLC-ESI-MS and to establish a method for the recovery of punicalagin using a medium pressure liquid chromatography (MPLC) system. MATERIALS AND METHODS: The characterisation of total pomegranate-husk polyphenols was carried out using liquid chromatography coupled to mass spectrometry. Thus, 200 mg of pomegranate-husk polyphenols were fractionated by MPLC. The isolated punicalagin was characterised by HPLC-MS and was tested as standard reagent for the measurement of its scavenging capacity reducing DPPH and ABTS radicals. RESULTS: Twenty peaks were identified by analytical HPLC-MS analysis from the pomegranate-husk polyphenols. The main compounds were the punicalagin anomers, punicalin and ellagic acid. The MPLC method allowed three fractions to be obtained. In fraction three 39.40 ± 8.06 mg of punicalagin anomers (purity > 97.9%) were recovered. The scavenging capacity of punicalagin showed an IC50 of 109.53 and 151.50 µg/mL for DPPH and ABTS radicals, respectively. CONCLUSION: The MPLC system was an excellent tool for the separation of the main ellagitannins from pomegranate husk and for the isolation of punicalagin anomers. Fraction three was rich in high purity punicalagin anomers. The IC50 was obtained for DPPH and ABTS radicals. Copyright © 2017 John Wiley & Sons, Ltd.

Study of enzymatic saccharification of Agave leaves biomass to yield fermentable sugars.

Agave is a good source of polysaccharides for the production of fermentable sugars as sustainable bioenergy feedstock solutions for semi-arid and arid lands. This plant grows in arid areas, which correspond to a large territory in northern Mexico. Having lignocellulose as the polysaccharide of interest, the information for the enzymatic saccharification of this kind of material is limited. Agave cell walls have a unique recalcitrant nature, but having a high cellulose content, makes this plant material an interesting research subject. In this work, acidic, alkaline and aqueous pretreatments were evaluated to generate a biomass rich in cellulose. The saccharification of pretreated Agave leaves-residue was evaluated under experimental designs to identify the most suitable conditions for enzymatic hydrolysis. Maximum value obtained was 31% glucose, which further increased to 41.4% at extended hydrolysis time of 96 h. The highest cellulose-saccharification reached was up to 61.81%, making Agave atrovirens an alternative for bioethanol production in its geographical area of cultivation.

Effect of ohmic heating processing conditions on color stability of fungal pigments.

The aim of this work was to analyze the effect of ohmic heating processing conditions on the color stability of a red pigment extract produced by Penicillium purpurogenum GH2 suspended in a buffer solution (pH 6) and in a beverage model system (pH 4). Color stability of pigmented extract was evaluated in the range of 60-90 ℃. The degradation pattern of pigments was well described by the first-order (fractional conversion) and Bigelow model. Degradation rate constants ranged between 0.009 and 0.088 min-1 in systems evaluated. Significant differences in the rate constant values of the ohmic heating-treated samples in comparison with conventional thermal treatment suggested a possible effect of the oscillating electric field generated during ohmic heating. The thermodynamic analysis also indicated differences in the color degradation mechanism during ohmic heating specifically when the pigment was suspended in the beverage model system. In general, red pigments produced by P. purpurogenum GH2 presented good thermal stability under the range of the evaluated experimental conditions, showing potential future applications in pasteurized food matrices using ohmic heating treatment.

Biotechnological production and application of fructooligosaccharides.

Currently, prebiotics are all carbohydrates of relatively short chain length. One important group is the fructooligosaccharides (FOS), a special kind of prebiotic associated to the selective stimulation of the activity of certain groups of colonic bacteria. They have a positive and beneficial effect on intestinal microbiota, reducing the incidence of gastrointestinal infections and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of FOS, such as solid-state fermentations utilizing various agro-industrial by-products. By optimizing the culture parameters, FOS yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of FOS. This article is an overview of the results considering recent studies on FOS biosynthesis, physicochemical properties, sources, biotechnological production and applications.

Agave atrovirens fibers as substrate and support for solid-state fermentation for cellulase production by Trichoderma asperellum.

Many efforts have been made to produce cellulase with better features and conditions, and filamentous fungi have played an important role in the bioprocess, growing in liquid and solid cultures with sugarcane bagasse, corn stover and others lignocellulosic materials. In the present study, Agave atrovirens fibers were partially characterized, thermal pretreated and used as support, substrate and inducer source for cellulolytic complex production by four strains of the genus Trichoderma, where T. asperellum was selected as the best option for this process after evaluating the enzyme activity and the invasion capacity on the pretreated Agave fibers. Fungi were able to grow on the Agave fibers secreting the complex cellulolytic enzyme. Results show Agave fibers as a good carbon source and support for T. asperellum for the production of the cellulolytic complex (endoglucanase 12,860.8 U/g; exoglucanase 3144.4 U/g; and ß-glucosidase 384.4 U/g). These results show the promising potential this material could have in the production of the active enzyme cellulase complex.

Total phenolic content, in vitro antioxidant activity and chemical composition of plant extracts from semiarid Mexican region.

OBJECTIVE: To determine the extraction suitable conditions of total phenolic content (TPC) by heat-reflux system, antioxidant activities and HPLC characterization of the aqueous-ethanolic extracts of Jatropha dioica (J. dioica) (Dragon's blood), Flourensia cernua (F. cernua) (Tar bush), Eucalyptus camaldulensis (E. camaldulensis) (Eucalyptus) and Turnera diffusa (T. diffusa) (Damiana). METHODS: TPC was evaluated by the well-known colorimetric assay using Folin-Ciocalteu reagent. The antioxidant activities were assayed by three methods based on scavenging of DPPH, ABTS and by lipid oxidation inhibition. The chemical composition of the extracts obtained was subject to HPLC analysis. RESULTS: TPC in the plant extracts ranged from 2.3 to 14.12 mg gallic acid equivalents/g for J. dioica and E. camaldulensis, respectively. The plant extracts of F. cernua, E. camaldulensis and T. diffusa showed similar strong antioxidant activities on scavenging of DPPH and lipid oxidation inhibition. In contrast, J. dioica extracts had lowest potential antioxidant in three assays used. HPLC assay showed the presence of several phenolic compounds in the extracts used. CONCLUSIONS: The results obtained suggest that F. cernua, E. camaldulensis and T. diffusa are potential sources to obtain bioactive phenolic compounds with high antioxidant properties which can be used in the factories as antioxidant agents or for treatments in diseases.

Comparative study of fungal strains for thermostable inulinase production.

Fructose and fructo-oligosaccharides (FOS) are important ingredients in the food industry. Fructose is considered an alternative sweetener to sucrose because it has higher sweetening capacity and increases iron absorption in children, and FOS's are a source of dietary fiber with a bifidogenic effect. Both compounds can be obtained by enzymatic hydrolysis of inulin. However, inulin presents limited solubility at room temperature, thus, fructose and FOS production is carried out at 60°C. Therefore, there is a growing interest to isolate and characterize thermostable inulinases. The aim of this work was to evaluate the capacity of different fungal strains to produce potential thermostable inulinases. A total of 27 fungal strains belonging to the genera Aspergillus, Penicillium, Rhizopus, Rhizomucor and Thermomyces were evaluated for production of inulinase under submerged culture using Czapek Dox medium with inulin as a sole carbon source. Strains were incubated at 37°C and 200 rpm for 96 h. Crude enzyme extract was obtained to evaluate inulinase and invertase activity. In order to select the fungal strain with the highest thermostable inulinase production, a selection criterion was established. It was possible to determine the highest inulinase activity for Rhizopus microsporus 13aIV (10.71 U/mL) at 36 h with an optimum temperature of inulinase of 70°C. After 6 h at 60°C, the enzyme did not show any significant loss of activity and retained about 87% activity, while it only retains 57% activity at 70°C. According to hydrolysis products, R. microsporus produced endo and exo-inulinase.

Optimization of tannase production by Aspergillus niger in solid-state packed-bed bioreactor.

Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a Plackett­Burman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature (30°C), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.

Novel strategies for upstream and downstream processing of tannin acyl hydrolase.

Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme.