A monolithic stationary phase with dendritic nanostructures for the separation of PEGylated proteins.

Separation of PEGylated protein mixtures into individual species is a challenging procedure, and many efforts have been focused on creating novel chromatographic supports for this purpose. In this study, a new monolithic stationary phase with hyperbranched nanostructures was chemically synthesized. For this, monoliths with a support matrix of poly (glycidyl methacrylate-co-ethylene dimethacrylate) and ethylenediamine chemistry were modified with third-generation dendrons with butyl-end groups. The new monolith was analyzed by infrared spectroscopy, confirming the dendron with butyl ligands and exhibited low mass transfer resistance as observed by breakthrough frontal analysis. This support was able to separate mono-PEG ribonuclease A from the PEGylation mixture, indicated by a single band (∼30 kDa) in the electrophoretic analysis. Moreover, the separation of mono-PEGylated positional isomers was probably observed, as the protein with ∼30 kDa was found in two separate peaks. Interestingly, the dendronized monolith allowed the separation of the reaction mixture into individual PEGylated species when using high ammonium sulfate concentrations (2 M). A correlation between the PEGylation degree and the strength of the hydrophobic interactions on the monolith was observed. This chromatographic approach combines the natural branched architecture of dendrons and the higher capabilities of the monoliths enhancing the hydrophobic surface area, and therefore the interaction between the PEGylated proteins and ligands. Thus, the novel support represents a novel platform for the purification of PEGylated from non-PEGylated proteins with biotechnological applications.

Proteome wide evaluation of the separation ability of hydrophobic interaction chromatography by fluorescent dye binding analysis.

Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity--as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S(0)) for each fraction; a unique correlation between S(0) and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how--and in which extent--the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies.

Changes in the Chemical Composition of Edible Grasshoppers (Sphenarium purpurascens) Fed Exclusively with Soy Sprouts or Maize Leaves.

In recent times, insects have gained attention because of their nutritional characteristics as well as the environmental advantages of their production. In this research, the effect of the diet of grasshoppers (Sphenarium purpurascens) under controlled conditions on their chemical and nutritional content was studied. The insects were divided into two groups: maize leaf-fed grasshoppers (MFG) and soy sprout-fed grasshoppers (SFG). To evaluate the changes in composition, chemical analysis (protein, fiber, fat, ashes, and chitin) was carried out in triplicate according to AOAC procedures, and a Student's t-test was used to determine any significant differences. The results showed a higher content of crude protein, in vitro protein digestibility percentage, and sum of non-essential amino acids (NEAAs) in the MFG samples compared with the SFG samples. The total dietary fiber, insoluble dietary fiber, soluble dietary fiber, sum of the EAA, non-essential amino acid percentage (EAA%), and biological value percentage (BV%) were higher in the SFG than the MFG, while in the amino acid profile and chitin content, no significant differences were obtained, although an increase in oleic acid in the SFG was observed. In FTIR, a ß-sheet appeared in the SFG, which could be related to the low in vitro protein digestibility. The use of a soy sprout diet caused changes in the chemical composition and nutritional content of grasshoppers. This represents an opportunity to improve their nutritional value for commercial interests.

Biological activity of extracts and hydrolysates from early- and adult-stage edible grasshopper Sphenarium purpurascens.

Edible insects have become a promising food source because they are rich in protein, fatty acids, minerals, among others. In recent years, edible insects have been proposed to be used as innovative functional ingredients in terms of biological activity. The present study aimed to determine and compare biological activities of the extracts and hydrolysates obtained from early- and adult-stage edible grasshoppers Sphenarium purpurascens to evaluate their potential as a source of bioactive compounds. Proximal analyses showed that in adult grasshoppers (AGs), the percentage of protein (48.9% ± 1.2), crude fat (13.1% ± 0.09), and chitin (15.6% ± 0.81) was significantly higher than early grasshoppers (EGs) (42.2% ± 0.55, 9.35% ± 0.08, and 10.5% ± 0.15, respectively). Total phenolic compounds, 2,2-diphenyl-1-picrylhydrazyl (DPPH•), and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) free radical scavenging were analyzed and reported. Enzymatic hydrolysis increased the concentration of total phenolic compounds and higher antioxidant capacity (up to 252.78 mM trolox). Once fractionated by ultrafiltration, the fraction that presented the highest antioxidant activity against DPPH• and ABTS•+ was that with molecules ≤ 10 kDa. Furthermore, the bioaccessibility of the samples was analyzed by in vitro protein digestion using a multienzymatic method, and a recovery index (RI) was reported. Extracts and hydrolysates were analyzed by UPLC-MS, and this allowed the identification of phenolic acids and flavonoids. The results obtained in this work suggest that the grasshopper can be used as a possible source of bioactive compounds that can be used in the food or pharmaceutical industry.

Benefits and Challenges in the Incorporation of Insects in Food Products.

Edible insects are being accepted by a growing number of consumers in recent years not only as a snack but also as a side dish or an ingredient to produce other foods. Most of the edible insects belong to one of these groups of insects such as caterpillars, butterflies, moths, wasps, beetles, crickets, grasshoppers, bees, and ants. Insect properties are analyzed and reported in the articles reviewed here, and one common feature is nutrimental content, which is one of the most important characteristics mentioned, especially proteins, lipids, fiber, and minerals. On the other hand, insects can be used as a substitute for flour of cereals for the enrichment of snacks because of their high content of proteins, lipids, and fiber. Technological properties are not altered when these insects-derived ingredients are added and sensorial analysis is satisfactory, and only in some cases, change in color takes place. Insects can be used as substitute ingredients in meat products; the products obtained have higher mineral content than traditional ones, and some texture properties (like elasticity) can be improved. In extruded products, insects are an alternative source of proteins to feed livestock, showing desirable characteristics. Isolates of proteins of insects have demonstrated bioactive activity, and these can be used to improve food formulations. Bioactive compounds, as antioxidant agents, insulin regulators, and anti-inflammatory peptides, are high-value products that can be obtained from insects. Fatty acids that play a significant role in human health and lipids from insects have showed positive impacts on coronary disease, inflammation, and cancer. Insects can be a vector for foodborne microbial contamination, but the application of good manufacturing practices and effective preservation techniques jointly with the development of appropriate safety regulations will decrease the appearance of such risks. However, allergens presented in some insects are a hazard that must be analyzed and taken into account. Despite all the favorable health-promoting characteristics present in insects and insects-derived ingredients, willingness to consume them has yet to be generalized.