Valorization of Prickly Pear Peel Residues (Opuntia ficus-indica) Using Solid-State Fermentation.

Prickly pear peel (Opuntia ficus-indica) residues can be used as a substrate in solid-state fermentation to obtain bioactive compounds. The kinetic growth of some Aspergillus strains was evaluated. A Box-Hunter and Hunter design to evaluate the independent factors was used. These factors were temperature (°C), inoculum (spores/g), humidity (%), pH, NaNO3 (g/L), MgSO4 (g/L), KCl (g/L), and KH2PO4 (g/L). The response factors were the amount of hydrolyzable and condensed tannins. The antioxidant and antimicrobial activity of fermentation extracts was evaluated. Aspergillus niger strains GH1 and HT3 were the best for accumulating tannins. The humidity, inoculum, and temperature affect the release of hydrolyzable and condensed tannins. Treatment 13 (low values for temperature, inoculum, NaNO3, MgSO4; and high values for humidity, pH, KCl, KH2PO4) resulted in 32.9 mg/g of condensed tannins being obtained; while treatment 16 (high values for all the factors evaluated) resulted in 3.5 mg/g of hydrolyzable tannins being obtained. In addition, the fermented extracts showed higher antioxidant activity compared to the unfermented extracts. Treatments 13 and 16 showed low inhibition of E. coli, Alternaria sp., and Botrytis spp. The solid-state fermentation process involving prickly pear peel residues favors the accumulation of condensed and hydrolyzable tannins, with antioxidant and antifungal activity.

Sorghum (Sorghum bicolor L.) as a potential source of bioactive substances and their biological properties.

Sorghum is the fifth cereal most produced in the world after wheat, rice, maize, and barley. In some regions, this crop is replacing maize, due to its high yield, resistance to drought and heat. There are several varieties of sorghum, whose coloration varies from cream, lemon-yellow, red, and even black. Pigmented sorghum grain is a rich source of antioxidants like polyphenols, mainly tannins, which have multiple benefits on human health such as, antiproliferative properties associated with the prevention of certain cancers, antioxidant activities related to the prevention of associated diseases to oxidative stress, antimicrobial and anti-inflammatory effects, it also improves glucose metabolism. Despite having these types of compounds, it is not possible to assimilate them, their use in the food industry has been limited, since sorghum is considered a food of low nutritional value, due to the presence of anti-nutritional factors such as strong tannins which form complexes with proteins and iron, thus reducing their digestibility. Based on these restrictions that this product has had as food for humans, the analysis of this review emphasizes the valorization of sorghum as a source of bioactive substances and the importance they confer on human health because of the biological potential it has.

Influence of culture conditions on ellagitannase expression and fungal ellagitannin degradation.

The role and mechanism of elagitannase is misunderstood because it exhibited 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 the enzyme. This study was aimed to obtain enzymatic extracts by Aspergillus niger GH1 in solid-state fermentation, using dextrose and ellagitannins as inducers of ellagitannase. Protein and bioinformatic analysis were performed to identify the protein sequence expressed in terms of culture conditions. The presence of ellagitannins increased ellagitannase activity 1143-fold compared to dextrose. The higher ellagitannase activity was found at 18 h of culture (1143.30 U g-1PE). Three groups of proteins were identified in both cultures: ß-glucosidase, phospholipase C, and triacylglycerol lipase. However, only phospholipase C was overexpressed with ellagitannins as inducers, showing the most spontaneous reaction with punicalagin (ΔG -8.56). These results suggest that phospholipase could be involved in ellagitannins biosynthesis.

Ellagic acid production using polyphenols from orange peel waste by submerged fermentation

Background: Biotechnological processes are part of modern industry as well as stricter environmental requirements. The need to reduce production costs and pollution demands for alternatives that involve the integral use of agro-industrial waste to produce bioactive compounds. The citrus industry generates large amounts of wastes due to the destruction of the fruits by microorganisms and insects together with the large amounts of orange waste generated during the production of juice and for sale fresh. The aim of this study was used orange wastes rich in polyphenolic compounds can be used as source carbon of Aspergillus fumigatus MUM 1603 to generate high added value compounds, for example, ellagic acid and other molecules of polyphenolic origin through submerged fermentation system. Results: The orange peel waste had a high concentration of polyphenols, 28% being condensed, 27% ellagitannins, 25% flavonoids and 20% gallotannins. The major polyphenolic compounds were catechin, EA and quercetin. The conditions, using an experimental design of central compounds, that allow the production of the maximum concentration of EA (18.68 mg/g) were found to be: temperature 30°C, inoculum 2 × 107 (spores/g) and orange peel polyphenols 6.2 (g/L). Conclusion: The submerged fermentation process is an effective methodology for the biotransformation of molecules present in orange waste to obtain high value-added as ellagic acid that can be used as powerful antioxidants, antibacterial and other applications.

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.

Ellagic Acid Recovery by Solid State Fermentation of Pomegranate Wastes by Aspergillus niger and Saccharomyces cerevisiae: A Comparison.

Fermentation in solid state culture (SSC) has been the focus of increasing interest because of its potential for industrial applications. In previous studies SSC of pomegranate wastes by Aspergillus niger has been extensively developed and optimized for the recovery of ellagic acid (EA), a high value bioactive. In this study we comparatively investigated the SSC of powdered pomegranate husks by A. niger and Saccharomyces cerevisiae and evaluated the recovery yields of EA by an ultrasound and microwave-assisted 7:3 water/ethanol extraction. Surprisingly enough, the yields obtained by S. cerevisiae fermentation (4% w/w) were found 5-fold higher than those of the A. niger fermented material, with a 10-fold increase with respect to the unfermented material. The EA origin was traced by HPLC analysis that showed a significant decrease in the levels of punicalagin isomers and granatin B and formation of punicalin following fermentation. Other extraction conditions that could warrant a complete solubilization of EA were evaluated. Using a 1:100 solid to solvent ratio and DMSO as the solvent, EA was obtained in 4% yields from S. cerevisiae fermented husks at a high purity degree. Hydrolytic treatment of S. cerevisiae fermented pomegranate husks afforded a material freed of the polysaccharides components that gave recovery yields of EA up to 12% w/w.

Emerging strategies for the development of food industries.

Undoubtedly, the food industry is undergoing a dynamic process of transformation in its continual development in order to meet the requirements and solve the great problems represented by a constantly growing global population and food claimant in both quantity and quality. In this sense, it is necessary to evaluate the technological trends and advances that will change the landscape of the food processing industry, highlighting the latest requirements for equipment functionality. In particular, it is crucial to evaluate the influence of sustainable green biotechnology-based technologies to consolidate the food industry of the future, today, and it must be done by analyzing the mega-consumption trends that shape the future of industry, which range from local sourcing to on-the-go food, to an increase in organic foods and clean labels (understanding ingredients on food labels). While these things may seem alien to food manufacturing, they have a considerable influence on the way products are manufactured. This paper reviews in detail the conditions of the food industry, and particularly analyzes the application of emerging technologies in food preservation, extraction of bioactive compounds, bioengineering tools and other bio-based strategies for the development of the food industry.

The transcription fidelity factor GreA impedes DNA break repair.

Homologous recombination repairs DNA double-strand breaks and must function even on actively transcribed DNA. Because break repair prevents chromosome loss, the completion of repair is expected to outweigh the transcription of broken templates. However, the interplay between DNA break repair and transcription processivity is unclear. Here we show that the transcription factor GreA inhibits break repair in Escherichia coli. GreA restarts backtracked RNA polymerase and hence promotes transcription fidelity. We report that removal of GreA results in markedly enhanced break repair via the classic RecBCD-RecA pathway. Using a deep-sequencing method to measure chromosomal exonucleolytic degradation, we demonstrate that the absence of GreA limits RecBCD-mediated resection. Our findings suggest that increased RNA polymerase backtracking promotes break repair by instigating RecA loading by RecBCD, without the influence of canonical Chi signals. The idea that backtracked RNA polymerase can stimulate recombination presents a DNA transaction conundrum: a transcription fidelity factor that compromises genomic integrity.

Measurement of gene regulation in individual cells reveals rapid switching between promoter states.

In vivo mapping of transcription-factor binding to the transcriptional output of the regulated gene is hindered by probabilistic promoter occupancy, the presence of multiple gene copies, and cell-to-cell variability. We demonstrate how to overcome these obstacles in the lysogeny maintenance promoter of bacteriophage lambda, P(RM). We simultaneously measured the concentration of the lambda repressor CI and the number of messenger RNAs (mRNAs) from P(RM) in individual Escherichia coli cells, and used a theoretical model to identify the stochastic activity corresponding to different CI binding configurations. We found that switching between promoter configurations is faster than mRNA lifetime and that individual gene copies within the same cell act independently. The simultaneous quantification of transcription factor and promoter activity, followed by stochastic theoretical analysis, provides a tool that can be applied to other genetic circuits.

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.

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.

Combining protein and mRNA quantification to decipher transcriptional regulation.

We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.

Optimization of ellagitannase production by Aspergillus niger GH1 by solid-state fermentation.

Ellagic acid is one of the most bioactive antioxidants with important applications in pharmaceutical, cosmetic, and food industries. However, there are few biotechnological processes developed for its production, because it requires precursors (ellagitannins) and the corresponding biocatalyst (ellagitannase). The aim of this study was to optimize the culture conditions for ellagitannase production by Aspergillus niger in solid-state fermentation (SSF). The bioprocess was carried out into a column bioreactor packed with polyurethane foam impregnated with an ellagitannins solution as carbon source. Four strains of Aspergillus niger (PSH, GH1, HT4, and HC2) were evaluated for ellagitannase production. The study was performed in two experimental steps. A Plackett-Burman design was used to determine the influencing parameters on ellagitannase production. Ellagitannins concentration, KCl, and MgSO4 were determined to be the most significant parameters. Box-Behnken design was used to define the interaction of the selected parameters. The highest enzyme value was obtained by A. niger PSH at concentrations of 7.5 g/L ellagitannins, 3.04 g/L KCl, and 0.76 g/L MgSO4. The methodology followed here allowed increasing the ellagitannase activity 10 times over other researcher results (938.8 U/g ellagitannins). These results are significantly higher than those reported previously and represent an important contribution for the establishment of a new bioprocess for ellagic acid and ellagitannase production.

Fungal biodegradation of pomegranate ellagitannins.

Ellagitannins (ETs) are phytochemicals derived from secondary metabolism associated to defense system, with complex chemical structures, which have high participation during all stages of protection against microbial infection. In this study, we report the fungal biodegradation of a bioactive ET, named punicaline which was recovered and purified from pomegranate peels and used as carbon source in solid-state culture (SSC) using polyurethane as solid support. SSC was kinetically monitored during 36 h of incubation time. ETs and glycosides consumption were spectrophotometrically determined. Ellagic acid (EA) accumulation was analyzed by HPLC. Several enzymatic activities were assayed (cellulase, xylanase, ß-glucosydase, polyphenoloxidase, tannase, and ET hydrolyzing activities). The consumption levels of ETs and glycosides were 66 and 40%, while EA accumulation reached 42.02 mg g(-1). A differential pattern of enzymatic activities was found; evidence from our studies suggests that the ET hydrolyzing activity is directly associated to EA accumulation, and production of this enzyme may represent the most critical step to successfully develop a bioprocess for production of an important bioactive compound, the EA.

The maternal-to-zygotic transition targets actin to promote robustness during morphogenesis.

Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/α-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-α (Sry-α) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-α are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-α collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-α and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at all life stages.

Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization.

We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2-3 d typically required for image and data analysis.

Single-virus tracking reveals a spatial receptor-dependent search mechanism.

Viral infection begins with the binding of a virus to a specific target on the surface of the host cell, followed by viral genome delivery into the host and a continuation of the infection process. Before binding occurs, the virus must first find its receptor by a process whose details are largely unknown. We applied high-resolution fluorescence microscopy and single-particle tracking to elucidate the target-finding process in bacteriophage λ as it infects an Escherichia coli cell. By monitoring the motion of individual viruses through the early stages of infection, we identified a unique spatial focusing process that allows a virus to arrive from its initial random landing site to its destination at the cell pole. The search process is governed by the interaction between the virus and the LamB receptors, and by the spatial organization of the receptor network on the cell surface. Our findings allowed us to develop a theoretical model for the target-finding process that reproduces the key features observed in experiment. We discuss the possible implications of our findings for the process of viral receptor-finding in higher systems.

General properties of transcriptional time series in Escherichia coli.

Gene activity is described by the time series of discrete, stochastic mRNA production events. This transcriptional time series shows intermittent, bursty behavior. One consequence of this temporal intricacy is that gene expression can be tuned by varying different features of the time series. Here we quantify copy-number statistics of mRNA from 20 Escherichia coli promoters using single-molecule fluorescence in situ hybridization in order to characterize the general properties of these transcriptional time series. We find that the degree of burstiness is correlated with gene expression level but is largely independent of other parameters of gene regulation. The observed behavior can be explained by the underlying variation in the duration of bursting events. Using Shannon's mutual information function, we estimate the mutual information transmitted between an outside stimulus, such as the extracellular concentration of inducer molecules, and intracellular levels of mRNA. This suggests that the outside stimulus transmits information reflected in the properties of transcriptional time series.

Lysogen stability is determined by the frequency of activity bursts from the fate-determining gene.

The ability of living cells to maintain an inheritable memory of their gene-expression state is key to cellular differentiation. Bacterial lysogeny serves as a simple paradigm for long-term cellular memory. In this study, we address the following question: in the absence of external perturbation, how long will a cell stay in the lysogenic state before spontaneously switching away from that state? We show by direct measurement that lysogen stability exhibits a simple exponential dependence on the frequency of activity bursts from the fate-determining gene, cI. We quantify these gene-activity bursts using single-molecule-resolution mRNA measurements in individual cells, analyzed using a stochastic mathematical model of the gene-network kinetics. The quantitative relation between stability and gene activity is independent of the fine details of gene regulation, suggesting that a quantitative prediction of cell-state stability may also be possible in more complex systems.