Renewing the potential of rice crop residues as value-added products in the cosmetics industry.

Purpose of this study is to explore the extraction of potentially valuable cosmetic ingredients from rice crop residues, aiming to mitigate their environmental impact. Methods: We employed AOAC methods to analyze the fat, protein, ash, fiber, soluble, and insoluble carbohydrate content in these residues. To identify sugars rich in galactose and acidic sugars, a total soluble carbohydrate extraction was performed. Cellulose, as part of the insoluble carbohydrates, was isolated through alkaline and acid hydrolysis, while sodium silicate was derived from the ash. Characterization of insoluble cellulose and silicate involved techniques like FTIR, DSC, PXRD, microphotography, porosity assessments, and water absorption studies. For proteins, alkaline solubilization and precipitation at the isoelectric point were utilized, with quantification via BCA and amino acid profiling through gas chromatography. Evaluation of radical scavenging capacity using DPPH led to the calculation of apparent molecular weight via SDS-PAGE. Results: The results revealed low levels of gum, mucilage, and pectin in both residues, contrasting with a high concentration of insoluble polysaccharides. Among these, Iß cellulose displayed potential attributes for cosmetic applications due to its oil and water adsorption characteristics. However, silicates obtained from the ashes did not exhibit direct use potential. In terms of protein extraction, we observed antioxidant properties, with enhanced performance through enzymatic hydrolysis, achieving a hydrolysis degree of 30.41% and a DPPH radical absorption rate exceeding 70%. Conclusion: Rice residues, particularly husk and straw, shown valuable substances suitable for potential cosmetic applications, encompassing cellulose, hydrolyzed proteins, and ash as a silicate precursor.

Heterologous Expression of an Insecticidal Peptide Obtained from the Transcriptome of the Colombian Spider Phoneutria depilate.

Spider venoms are composed, among other substances, of peptide toxins whose selectivity for certain physiological targets has made them powerful tools for applications such as bioinsecticides, analgesics, antiarrhythmics, antibacterials, antifungals and antimalarials, among others. Bioinsecticides are an environmentally friendly alternative to conventional agrochemicals. In this paper, the primary structure of an insecticidal peptide was obtained from the venom gland transcriptome of the ctenid spider Phoneutria depilata (Transcript ID PhdNtxNav24). The peptide contains 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. Using the amino acid sequence of such peptide, a synthetic gene was constructed de novo by overlapping PCRs and cloned into an expression vector. A recombinant peptide, named delta-ctenitoxin (rCtx-4), was obtained. It was expressed, folded, purified and validated using mass spectrometry (7994.61 Da). The insecticidal activity of rCtx-4 was demonstrated through intrathoracic injection in crickets (LD50 1.2 µg/g insect) and it was not toxic to mice. rCtx-4 is a potential bioinsecticide that could have a broad spectrum of applications in agriculture.

Expression in Pichia pastoris of human antibody fragments that neutralize venoms of Mexican scorpions.

The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.

Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp.

Background: Scorpion neurotoxins such as those that modify the mammalian voltage-gated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes - one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 - and another non-native toxin - named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions - were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.

Effect of the Concentration, pH, and Ca2+ Ions on the Rheological Properties of Concentrate Proteins from Quinoa, Lentil, and Black Bean.

Given consumer trends propelling a movement toward using plant protein in the food industry and searching for alternative protein ingredients by the industry, this study aimed to assess the influence of factors such as protein concentration, medium pH, and the presence of a divalent ion (Ca2+) upon the rheological properties such as viscosity change and gel formation of dispersion proteins extracted from quinoa, black beans, and lentils. A solution of each protein was prepared by varying its concentration (2.5%, 5.0%, and 10%), the pH (5.0, 7.0, and 9.0), and the incorporation of calcium chloride (0.0% and 1.0%). Each obtained solution was subjected to rheological tests to determine the parameters: consistency index (K), flow behavior (n), the storage (G') and loss (G'') modules, and the phase shift angle (δ). The results demonstrate that the incorporation of Ca2+, the shift in protein levels, and the decrease in pH modified the rheological behaviors of proteins, which were also influenced by the structural characteristics of each protein studied. However, thermal treatment and protein concentrations caused the most significant impact on proteins' rheological behavior, forming gels independently of other conditions. It was possible to study and interpret the studied proteins' rheological variations according to the environment's conditions.

Crotoxin B: Heterologous Expression, Protein Folding, Immunogenic Properties, and Irregular Presence in Crotalid Venoms.

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

Transcriptomic Analysis of the Venom Gland and Enzymatic Characterization of the Venom of Phoneutria depilata (Ctenidae) from Colombia.

The transcriptome of the venom glands of the Phoneutria depilata spider was analyzed using RNA-seq with an Illumina protocol, which yielded 86,424 assembled transcripts. A total of 682 transcripts were identified as potentially coding for venom components. Most of the transcripts found were neurotoxins (156) that commonly act on sodium and calcium channels. Nevertheless, transcripts coding for some enzymes (239), growth factors (48), clotting factors (6), and a diuretic hormone (1) were found, which have not been described in this spider genus. Furthermore, an enzymatic characterization of the venom of P. depilata was performed, and the proteomic analysis showed a correlation between active protein bands and protein sequences found in the transcriptome. The transcriptomic analysis of P. depilata venom glands show a deeper description of its protein components, allowing the identification of novel molecules that could lead to the treatment of human diseases, or could be models for developing bioinsecticides.

Antimicrobial and Immunomodulatory Effects of Selected Chemokine and Antimicrobial Peptide on Cytokine Profile during Salmonella Typhimurium Infection in Mouse.

The antimicrobial and immunomodulatory capacities of the peptide Css54 and the chemokine MCP-1 were tested. The first, a peptide isolated from the venom of the scorpion Centruroides suffusus suffusus was synthesized chemically. In contrast, the second is a monocyte chemoattractant expressed as a recombinant protein in our lab. It was observed in vitro that Css54 inhibited the growth of Salmonella enterica serovar Typhimurium (6.2 µg/mL). At high concentrations, it was toxic to macrophages (25 µg/mL), activated macrophage phagocytosis (1.5 µg/mL), and bound Salmonella LPS (3 µg/mL). On the other hand, the recombinant MCP-1 neither inhibited the growth of Salmonella Typhimurium nor was it toxic to macrophages (up to 25 µg/mL), nor activated macrophage phagocytosis or bound Salmonella LPS (up to 3 µg/mL). Although it was observed in vivo in mice Balb/C that both Css54 and MCP-1 did not resolve the intraperitoneal infection by S. Typhimurium, Css54 decreased the expression of IL-6 and increased IL-10, IL-12p70, and TNF-α levels; meanwhile, MCP-1 decreased the expression of IFN-γ and increased IL-12p70 and TNF-α. It was also observed that the combination of both molecules Css54 and MCP-1 increased the expression of IL-10 and TNF-α.

Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp

Background: Scorpion neurotoxins such as those that modify the mammalian voltagegated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes ­ one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 ­ and another non-native toxin ­ named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions ­ were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.(AU)

Use of Plant Proteins as Microencapsulating Agents of Bioactive Compounds Extracted from Annatto Seeds (Bixa orellana L.).

This study aimed to assess the thermal stability of the bioactive compounds from annatto seed extract, encapsulated by ionic gelation using quinoa proteins, lentil proteins, soy proteins, and sodium caseinate as carrying materials. The 10.0% aqueous dispersions of the different proteins (carriers) were prepared and mixed with the annatto seed extract. The dispersions were then extruded into a calcium chloride solution to induce the extract encapsulation. The capsules were characterized by encapsulation efficiency, particle size, infrared transmission spectroscopy, confocal microscopy, and scanning electron microscopy (SEM). The antioxidant and antimicrobial activities, the polyphenol compounds, and bixin content from the free and encapsulated extract were assessed once stored for 12 d at different temperatures (4 °C, 25 °C, and 65 °C). The results demonstrated the ability of the proteins to encapsulate the annatto extract with encapsulation efficiencies ranging from 58% to 80%, where the protein structure and amino acid content were the relevant factors to obtain high encapsulation efficiencies. The free extracts stored at 65 °C for 12 d experienced a degradation of bixin and polyphenol compounds, respectively. Conversely, the encapsulated extract had degradations from ~34.00% to ~4.05% for polyphenol compounds and ~20.0% for bixin, respectively. These proteins have a potential encapsulation capacity of annatto extract by ionic gelation.