Jorge Benavides

The Folin–Ciocalteu assay revisited: improvement of its specificity for total phenolic content determination

This study presents a review of the Folin–Ciocalteu (F–C) assay for total phenolic content (TPC) determinations and describes different approaches to improve its specificity. Phenolics are regarded as the molecules with the highest potential to neutralize free radicals. Therefore, their quantification is a common practice in different areas of food research. However, when determining TPC in plant food extracts, the presence of reducing interferants [ascorbic acid (AA)] produces inaccurate estimations of TPC values. Different methodologies have been proposed to improve the specificity of the F–C assay. These methodologies include: (i) the use of solid phase extraction (SPE) cartridges to separate interferants from phenolics; (ii) the calculation of a corrected TPC value based on the AA reducing activity present in the extract; and (iii) the pre-treatment of extracts with oxidative agents prior to TPC quantification. These methods are described in detail in the present study. Likewise, their advantages and disadvantages are discussed based on new experimental data. A simple modification of the F–C assay procedure is proposed to quantify both the TPC value and the AA reducing activity in plant food extracts. Values obtained by the modified F–C assay can be used to estimate a corrected TPC value.

Aqueous two-phase affinity partitioning systems: Current applications and trends ☆

Aqueous two-phase systems (ATPS) have been studied and used for product recovery and purification from diverse biological sources. ATPS are characterized by their versatility, easy scale up parameters, process integration capability and relative low cost. This technique is commonly regarded as a primary recovery stage mainly due to its low selectivity. However, the use of strategies involving the modification of ATPS with affinity ligands have resulted in significant increases in recovery yields and purification folds of biological products. The aim of this review is to highlight current applications, trends and challenges regarding affinity partitioning in aqueous two-phase systems for the fractionation, recovery and purification of biological products.

Plants as Biofactories: Glyphosate-Induced Production of Shikimic Acid and Phenolic Antioxidants in Wounded Carrot Tissue

The use of plants to produce chemical compounds with pharmaceutical and nutraceutical applications has intensified in recent years. In this regard, genetic engineering is the most commonly used tool to generate crop lines with enhanced concentrations of desirable chemicals. However, growing genetically modified plants is still limited because they are perceived as potential biological hazards that can create an ecological imbalance. The application of postharvest abiotic stresses on plants induces the accumulation of secondary metabolites and thus can be used as an alternative to genetic modification. The present project evaluated the feasibility of producing shikimic acid (SA) and phenolic compounds (PC) in wounded carrots ( Daucus carota ) treated with glyphosate. The spray application of a concentrated glyphosate solution on wounded carrot tissue increased the concentrations of SA and chlorogenic acid by ∼1735 and ∼5700%, respectively. The results presented herein demonstrate the potential of stressed carrot tissue as a biofactory of SA and PC.

Recovery of crocins from saffron stigmas (Crocus sativus) in aqueous two-phase systems

Crocins are carotenoid derivates that have recently attracted the interest of the scientific community due to their nutraceutical properties. Saffron (dry Crocus sativus stigmas) is one of the main known sources of crocins. In this study the potential use of aqueous two-phase system (ATPS) for the extraction of crocins from C. sativus stigmas was evaluated. The partitioning behavior of crocins in different types of ATPS (polymer-polymer, polymer-salt, alcohol-salt and ionic liquid-salt) was evaluated. Ethanol-potassium phosphate ATPS were selected based on their high top phase recovery yield and low cost of system constituents. The evaluation and optimization of system parameters rendered conditions (V(R)=3.2, ethanol 19.8% (w/w), potassium phosphate 16.5% (w/w), TLL of 25% (w/w), 0.1M NaCl and 2% (w/w) of sample load) under which more than 75% of total crocins were recovered in the top (ethanol rich) phase, whereas the wasted stigmas accumulated in the bottom phase. Lastly, a comparison between an optimized solid-liquid extraction using ethanol:water as solvent and ATPS was conducted demonstrating that similar yields are achieved with both strategies (76.89 ± 18% and 79.27 ± 1.6%, respectively). However, ATPS rendered a higher extraction selectivity of 1.3 ± 0.04 mg of crocins for each mg of phenolic compound, whereas ethanolic extraction showed a selectivity of 0.87 ± 0.01. The results reported herein demonstrate the potential application of ATPS, particularly ethanol-potassium phosphate systems, for the recovery of crocins from C. sativus stigmas.

Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot.

The application of postharvest abiotic stresses is an effective strategy to activate the primary and secondary metabolism of plants inducing the accumulation of antioxidant phenolic compounds. In the present study, the effect of water stress applied alone and in combination with wounding stress on the activation of primary (shikimic acid) and secondary (phenylpropanoid) metabolic pathways related with the accumulation of phenolic compound in plants was evaluated. Carrot (Daucus carota) was used as model system for this study, and the effect of abiotic stresses was evaluated at the gene expression level and on the accumulation of metabolites. As control of the study, whole carrots were stored under the same conditions. Results demonstrated that water stress activated the primary and secondary metabolism of carrots, favoring the lignification process. Likewise, wounding stress induced higher activation of the primary and secondary metabolism of carrots as compared to water stress alone, leading to higher accumulation of shikimic acid, phenolic compounds, and lignin. Additional water stress applied on wounded carrots exerted a synergistic effect on the wound-response at the gene expression level. For instance, when wounded carrots were treated with water stress, the tissue showed 20- and 14-fold increases in the relative expression of 3-deoxy-D-arabino-heptulosanate synthase and phenylalanine ammonia-lyase genes, respectively. However, since lignification was increased, lower accumulation of phenolic compounds was detected. Indicatively, at 48 h of storage, wounded carrots treated with water stress showed ~31% lower levels of phenolic compounds and ~23% higher lignin content as compared with wounded controls. In the present study, it was demonstrated that water stress is one of the pivotal mechanism of the wound-response in carrot. Results allowed the elucidation of strategies to induce the accumulation of specific primary or secondary metabolites when plants are treated with water stress alone or when additional water stress is applied on wounded tissue. If the accumulation of a specific primary or secondary metabolite were desirable, it would be recommended to apply both stresses to accelerate their biosynthesis. However, strategies such as the use of enzymatic inhibitors to block the carbon flux and enhance the accumulation of specific compounds should be designed.

Advances and trends in the design, analysis, and characterization of polymer-protein conjugates for "PEGylaided" bioprocesses

In addition to their use as therapeutics and because of their enhanced properties, PEGylated proteins have potential application in fields such as bioprocessing. However, the use of PEGylated conjugates to improve the performance of bioprocess has not been widely explored. This limited additional industrial use of PEG–protein conjugates can be attributed to the fact that PEGylation reactions, separation of the products, and final characterization of the structure and activity of the resulting species are not trivial tasks. The development of bioprocessing operations based on PEGylated proteins relies heavily in the use of analytical tools that must sometimes be adapted from the strategies used in pharmaceutical conjugate development. For instance, to evaluate conjugate performance in bioprocessing operations, both chromatographic and non-chromatographic steps must be used to separate and quantify the resulting reaction species. Characterization of the conjugates by mass spectrometry, circular dichroism, and specific activity assays, among other adapted techniques, is then required to evaluate the feasibility of using the conjugates in any operation. Correct selection of the technical and analytical methods in each of the steps from design of the PEGylation reaction to its final engineering application will ensure success in implementing a “PEGylaided” process. In this context, the objective of this review is to describe technological and analytical trends in developing successful applications of PEGylated conjugates in bioprocesses and to describe potential fields in which these proteins can be exploited.

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two‐phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt, and ionic liquid (IL)–salt). The systems composed of PEG 3350‐potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1‐fold purification) and t‐butanol‐20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8‐fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG‐salt ATPS for the potential recovery of SOD.

UVA, UVB Light Doses and Harvesting Time Differentially Tailor Glucosinolate and Phenolic Profiles in Broccoli Sprouts

Broccoli sprouts contain health-promoting glucosinolate and phenolic compounds that can be enhanced by applying ultraviolet light (UV). Here, the effect of UVA or UVB radiation on glucosinolate and phenolic profiles was assessed in broccoli sprouts. Sprouts were exposed for 120 min to low intensity and high intensity UVA (UVAL, UVAH) or UVB (UVBL, UVBH) with UV intensity values of 3.16, 4.05, 2.28 and 3.34 W/m2, respectively. Harvest occurred 2 or 24 h post-treatment; and methanol/water or ethanol/water (70%, v/v) extracts were prepared. Seven glucosinolates and 22 phenolics were identified. Ethanol extracts showed higher levels of certain glucosinolates such as glucoraphanin, whereas methanol extracts showed slight higher levels of phenolics. The highest glucosinolate accumulation occurred 24 h after UVBH treatment, increasing 4-methoxy-glucobrassicin, glucobrassicin and glucoraphanin by ~170, 78 and 73%, respectively. Furthermore, UVAL radiation and harvest 2 h afterwards accumulated gallic acid hexoside I (~14%), 4-O-caffeoylquinic acid (~42%), gallic acid derivative (~48%) and 1-sinapoyl-2,2-diferulolyl-gentiobiose (~61%). Increases in sinapoyl malate (~12%), gallotannic acid (~48%) and 5-sinapoyl-quinic acid (~121%) were observed with UVBH Results indicate that UV-irradiated broccoli sprouts could be exploited as a functional food for fresh consumption or as a source of bioactive phytochemicals with potential industrial applications.

Quantification of RNase A and Its PEGylated Conjugates on Polymer-Salt Rich Environments Using UV Spectrophotometry

Ribonuclease A (RNase A) from bovine pancreas and its PEGylated conjugates has proven to have potential therapeutic applications. Aqueous Two-Phase Systems (ATPS) is a promising primary recovery strategy for the fractionation of proteins and their PEGylated conjugates. However, in order to characterize the partition behavior of these molecules in ATPS, an easy-to-implement method is needed to estimate protein concentration in each phase. This paper presents a novel methodology based on UV absorbance to quantify RNase A and its PEGylated conjugates on polymer (polyethylene glycol) and salt (potassium phosphate) rich environments, simulating conditions found on polymer-salt ATPS.

Application of Aqueous Two-Phase Systems for the Recovery of Bioactive Low-Molecular Weight Compounds

The partition behavior of small compounds in aqueous two-phase systems (ATPS) is less characterized (compared to biomacromolecules), since the liquid-liquid fractionation of such compounds is commonly done on organic-aqueous biphasic systems. In this study, five standard compounds (iohexol, salicin, chlorogenic acid, epicatechin, and gallic acid) were selected to evaluate the application of four types of ATPS for the fractionation of low-molecular weight compounds. Polyethylene glycol-salt, 1-ethyl-3-methylimidazolium acetate-salt, and ethanol-potassium phosphate ATPS achieved recovery yields higher than 75% in most cases. This study proves that ATPS can be applied efficiently for the recovery of small bioactive molecules of commercial interest.