Nanocomposite films based on chia (Salvia hispanica L.) flour seeds incorporating antioxidant chitosan nanoparticles.

Chia (Salvia hispanica L.) flour seeds produce films with good barrier properties against water vapor and could be used as food packaging; however, their mechanical properties are poor, which limits their application. The incorporation of nanoparticles into natural polymers is a strategy used to improve the properties of films to increase their applications. Furthermore, nanoparticles can encapsulate antioxidant agents and generate active films. The objective of this study was to evaluate the influence of chia flour (4%-7%), glycerol (15%-25%), and chia extract-loaded chitosan nanoparticles (ChCNp) (0%-0.75%) on the physical, mechanical, barrier, structural and antioxidant properties of chia flour nanocomposite films. Chitosan nanoparticles loaded with antioxidant chia extract were synthesized by ionic gelation and incorporated into the films. The thickness, water vapor permeability, tensile strength, and antioxidant properties of the films were evaluated using a Box-Behnken experimental design. Structural analysis was conducted using the FTIR technique. The results of the ANOVA of the responses were adjusted to second and third order polynomial models obtaining determination coefficients of 0.96-0.99. The water vapor permeability of the films was 3.89 × 10-8-1.68 × 10-7 g mm/Pa s m2, tensile strength was 0.67-3.59 MPa and antioxidant activity was 57.12%-67.84%. The variables presented different effects on the films. Increasing the chia seed flour concentration negatively affected the water vapor permeability but improved the tensile strength and the antioxidant capacity of the films. The increase in glycerol concentration caused the films to become brittle. The nanoparticles had a significant effect on the thickness of the films and improved their mechanical and antioxidant properties. However, they did not show an effect on barrier properties. The results demonstrate that it is possible to obtain nanocomposite films with antioxidant capacity from chia seed flour and with the incorporation of chitosan nanoparticles loaded with antioxidants.

Supercritical CO2 Treatment to Modify Techno-Functional Properties of Proteins Extracted from Tomato Seeds.

Tomato seeds are a rich source of protein that can be utilized for various industrial food purposes. This study delves into the effects of using supercritical CO2 (scCO2) on the structure and techno-functional properties of proteins extracted from defatted tomato seeds. The defatted meal was obtained using hexane (TSMH) and scCO2 (TSMC), and proteins were extracted using water (PEWH and PEWC) and saline solution (PESH and PESC). The results showed that scCO2 treatment significantly improved the techno-functional properties of protein extracts, such as oil-holding capacity and foaming capacity (especially for PEWC). Moreover, emulsifying capacity and stability were enhanced for PEWC and PESC, ranging between 4.8 and 46.7% and 11.3 and 96.3%, respectively. This was made possible by the changes in helix structure content induced by scCO2 treatment, which increased for PEWC (5.2%) and decreased for PESC (8.0%). Additionally, 2D electrophoresis revealed that scCO2 hydrolyzed alkaline proteins in the extracts. These findings demonstrate the potential of scCO2 treatment in producing modified proteins for food applications.

Hernandulcin Production in Elicited Hairy Roots of Phyla scaberrima: Toward Sustainable Production of a Non-Caloric Sweetener with Nutraceutical Properties.

Herein we report on the generation of hairy root lines of P. scaberrima able to produce hernandulcin (HE), a non-caloric sweetener with nutraceutical properties. From ten different lines analyzed, three synthesized up to 100 mg ⋅ L-1 HE under the batch culture conditions standardized in this investigation. Adding elicitors (salicylic acid, chitin, Glucanex, polyethylene glycol) and biosynthetic precursors (farnesol and (+)-epi-alpha-bisabolol) significantly altered HE accumulation. Chitin and Glucanex enhanced HE production from 130 to 160 mg ⋅ L-1 , whereas farnesol and (+)-epi-alpha-bisabolol from 165 to 200 mg ⋅ L-1 without dependence on biomass accumulation. Improved batch cultures containing liquid Murashige & Skoog medium (MS; pH 7), added with 4 % sucrose, 0.5 mg ⋅ L-1 naphthaleneacetic acid, 100 mg ⋅ L-1 Glucanex, 150 mg ⋅ L-1 chitin, 250 mg ⋅ L-1 farnesol, and 150 mg ⋅ L-1 (+)-epi-alpha-bisabolol at 25 °C (12 h light/12 h darkness), triggered HE accumulation to 250 mg ⋅ L-1 in 25 days. The efficiency of each recombinant line is discussed.

Moringa straw as cellulase production inducer and cellulolytic fungi source / Residuo de moringa como inductor de la producción de celulasas y como fuente de hongos celulolíticos

Abstract Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and puri-fied 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606 U/l) and exoglucanase (205 U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of p-glucosidase activity (664 U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and p-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum. © 2019 Asociación Argentina de Microbiología. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Resumen Actualmente, la valorización de los residuos agroindustriales es de gran interés. En este trabajo se emplearon residuos de madera blanda de Moringa oleifera para la producción de celulasas de bajo costo. El objetivo fue aislar, identificar y caracterizar microorganismos con actividad celulolítica en diferentes fuentes de carbono. A partir de la biomasa de M. oleifera, se aislaron e identificaron 42 microorganismos productores de celulasas. Los hongos que presentaron los mayores halos de hidrólisis en carboximetilcelulosa como sustrato fueron identificados molecularmente como Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) y Cladosporium cladosporioides (FC2). Mediante fermentación sumergida, se evaluó la capacidad de estas cepas en la producción de celulasas utilizando celulosa cristalina (Avicel) y amorfa (CMC) como fuentes de carbono. P. funiculosum y C. cladosporioides presentaron las mayores actividades de endoglucanasa (606 U/l) y exoglucanasa (205 U/l) en medio Avicel, mientras que F. verticillioides mostró la mayor actividad de p-glucosidasa (664 U/l) en medio CMC. Además, se evaluó el efecto de tres medios de cultivo (A, B y C) sobre la producción de celulasas en P. funiculosum empleando residuos de moringa como fuente de carbono. Los resultados mostraron que en el medio C, la productividad volumétrica de celulasas se incrementó en 2,77; 8,26 y 2,30 veces para las actividades de endoglucanasa, exoglucanasa y p-glucosidasa, respectivamente. Los extractos enzimáticos producidos tienen gran potencial para su utilización biotecnológica, especialmente en la sacarificación de residuos de moringa y la producción de bioetanol de segunda generación. Este es el primer estudio del uso de la biomasa de M. oleifera para inducir la producción de diversas celulasas en P. funiculosum.

The insertion of bioactive peptides at the C-terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity

BACKGROUND: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. RESULTS: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a ß/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. CONCLUSION: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.

Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit.