Comparative study between Salkowski reagent and chromatographic method for auxins quantification from bacterial production.

Introduction: The Salkowski reagent method is a colorimetric technique used to determine auxin production, specifically as indole-3-acetic acid (IAA). It was developed to determine indoles rapidly; however, it does not follow Beer's law at high concentrations of IAA. Thus, there could be an overestimation of IAA with the Salkowski technique due to the detection of other indole compounds. Methods: This study aims to compare the Salkowski colorimetric method versus a chromatographic method to evidence the imprecision or overestimation obtained when auxins, such as indole-acetic acid (IAA), are determined as traits from promoting growth plant bacteria (PGPB), using ten different strains from three different isolation sources. The analysis used the same bacterial culture to compare the Salkowski colorimetric and chromatographic results. Each bacterium was cultivated in the modified TSA without or with tryptophan for 96 h. The same supernatant culture was used in both methods: Salkowski reagent and ultra-performance liquid chromatography coupled with a Mass Spectrometer (LC-MS/MS). Results: The first method indicated 5.4 to 27.4 mg L-1 without tryptophan in ten evaluated strains. When tryptophan was used as an inductor of auxin production, an increase was observed with an interval from 4.4 to 160 mg L-1. The principal auxin produced by all strains was IAA from that evaluated by the LC-MS/MS method, with significantly higher concentration with tryptophan addition than without. Strains belonging to the Kocuria genus were highlighted by high IAA production. The indole-3-propionic acid (IPA) was detected in all the bacterial cultures without tryptophan and only in K. turfanensis As05 with tryptophan, while it was not detected in other strains. In addition, indole-3-butyric acid (IBA) was detected at trace levels (13-16 µg L-1). Conclusions: The Salkowski reagent overestimates the IAA concentration with an interval of 41-1042 folds without tryptophan and 7-16330 folds with tryptophan as inductor. In future works, it will be necessary to determine IAA or other auxins using more suitable sensitive techniques and methodologies.

Hydrogel Based on Chitosan/Gelatin/Poly(Vinyl Alcohol) for In Vitro Human Auricular Chondrocyte Culture.

Three-dimensional (3D) hydrogels provide tissue-like complexities and allow for the spatial orientation of cells, leading to more realistic cellular responses in pathophysiological environments. There is a growing interest in developing multifunctional hydrogels using ternary mixtures for biomedical applications. This study examined the biocompatibility and suitability of human auricular chondrocytes from microtia cultured onto steam-sterilized 3D Chitosan/Gelatin/Poly(Vinyl Alcohol) (CS/Gel/PVA) hydrogels as scaffolds for tissue engineering applications. Hydrogels were prepared in a polymer ratio (1:1:1) through freezing/thawing and freeze-drying and were sterilized by autoclaving. The macrostructure of the resulting hydrogels was investigated by scanning electron microscopy (SEM), showing a heterogeneous macroporous structure with a pore size between 50 and 500 µm. Fourier-transform infrared (FTIR) spectra showed that the three polymers interacted through hydrogen bonding between the amino and hydroxyl moieties. The profile of amino acids present in the gelatin and the hydrogel was determined by ultra-performance liquid chromatography (UPLC), suggesting that the majority of amino acids interacted during the formation of the hydrogel. The cytocompatibility, viability, cell growth and formation of extracellular matrix (ECM) proteins were evaluated to demonstrate the suitability and functionality of the 3D hydrogels for the culture of auricular chondrocytes. The cytocompatibility of the 3D hydrogels was confirmed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reaching 100% viability after 72 h. Chondrocyte viability showed a high affinity of chondrocytes for the hydrogel after 14 days, using the Live/Dead assay. The chondrocyte attachment onto the 3D hydrogels and the formation of an ECM were observed using SEM. Immunofluorescence confirmed the expression of elastin, aggrecan and type II collagen, three of the main components found in an elastic cartilage extracellular matrix. These results demonstrate the suitability and functionality of a CS/Gel/PVA hydrogel as a 3D support for the auricular chondrocytes culture, suggesting that these hydrogels are a potential biomaterial for cartilage tissue engineering applications, aimed at the regeneration of elastic cartilage.

Proteome and immunogenicity differences in BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c during planktonic growth in 7H9 and Proskauer Beck media.

Bacillus Calmette-Guèrin (BCG) remains as the only vaccine employed to prevent tuberculosis (TB) during childhood. Among factors likely contributing to the variable efficacy of BCG is the modification in its antigenic repertoire that may arise from in vitro growth conditions. Our vaccine candidate, BCGΔBCG1419c, improved protection against TB in mice and guinea pigs with bacteria grown in either 7H9 OADC Tween 80 or in Proskauer Beck Tween 80 media in independent studies. Here, we compared the proteomes of planktonic cultures of BCG and BCGΔBCG1419c, grown in both media. Further to this, we compared systemic immunogenicity ex vivo elicited by both types of BCG strains and cultures when used to vaccinate BALB/c mice. Both the parental strain BCG Pasteur ATCC 35734, and its isogenic mutant BCGΔBCG1419c, had several medium-dependent changes. Moreover, ex vivo immune responses to a multiantigenic (PPD) or a single antigenic (Ag85A) stimulus were also medium-dependent. Then, not only the presence or absence of the BCG1419c gene in our strains under study affected the proteome produced in vitro but also that this was affected by culture medium, potentially leading to changes in the capacity to induce ex vivo immune responses.

Slow-release microencapsulates containing nanoliposomes for bioremediation of soil hydrocarbons contaminated.

Encapsulation and nutrient addition in bacterial formulations have disadvantages concerning cell viability during release, storage, and under field conditions. Then, the objective of this work was to encapsulate a bacterial consortium with hydrocarbon-degrading capacities in different matrices composed of cross-linked alginate/ polyvinyl alcohol /halloysite beads (M1, M2, and M3) containing nanoliposomes loaded with or without nutrients and evaluate their viability and release in a liquid medium, and soil (microcosmos). Also, evaluate their capacity to remove total petroleum hydrocarbons (TPH) for 165 days and matrices characterization. The encapsulate consortium showed a quick adaptation to contaminated soil and a percentage of removal (PR) of TPH up to 30% after seven days. All the matrices displayed a PR of up to 90% after 165 days. The matrix M2 displayed significant resistance to degradation and higher cell viability with a PR of 94%. This result supports the encapsulation of bacteria in a sustainable matrix supplemented with nutrients as a well-looked strategy for improving viability and survival and, therefore, enhancing their effectiveness in the remediation of hydrocarbon-contaminated soils.

Forastero and Criollo cocoa beans, differences on the profile of volatile and non-volatile compounds in the process from fermentation to liquor.

Cocoa bean fermentation is an important process because during this process, aroma compounds are produced, the astringency decreases, and the embryo dies. The fermentation processes of the Criollo and Forastero types have been studied separately without comparing them at the same time and in the same place. The aim of this work was to determine differences in the profile of volatile and nonvolatile compounds of Criollo and Forastero cocoa from the fermentation process to the final stage of obtaining the liquor. The experiments were carried out at the same time in the Maya region. Volatile compounds were determined by HS-SPME GC-MS (headspace solid phase-microextraction with gas chromatography-mass spectrometry). Sugars, organic acids, and alkaloids were determined by ultrahigh-performance liquid chromatography (UHPLC-PDA/UV). Criollo cocoa liquor was defined by the volatile and nonvolatile compounds such as acetic acid, phenylethyl alcohol, benzaldehyde, 2-phenylethyl acetate, acetophenone and 3-methylbutanal., which are associated with sour, honey, almond, flowery and chocolate aroma. Forastero cocoa liquor was represented with a significant difference by acetic acid, isobutyl acetate, 2,3-diethyl-5-methylpyrazine and ethyl octanoate and these could provide aroma descriptors such as sour, fruity and nutty. This study characterized for the first time the dynamics of volatile compounds during the fermentation, drying, and roasting stages and in the final cocoa liquor of Criollo and Forastero from cocoa beans of the same origin.

Thermal Treatment to Obtain 5-Hydroxymethyl Furfural (5-HMF), Furfural and Phenolic Compounds from Vinasse Waste from Agave.

Vinasses represent important final disposal problems due to their physical-chemical composition. This work analyzed the composition of tequila vinasses and increased 5-hydroxymethylfurfural, furfural, and phenolic compounds using thermal hydrolysis with hydrogen peroxide as a catalyst. A statistical Taguchi design was used, and a UPLC-MS (XEVO TQS Micro) analysis determined the presence and increase of the components. The treatment at 130 °C, 40 min, and 0.5% of catalyst presented the highest increase for 5-HMF (127 mg/L), furfural (3.07 mg/L), and phenol compounds as chlorogenic (0.36 mg/L), and vanillic acid (2.75 mg/L). Additionally, the highest removal of total sugars (57.3%), sucrose (99.3%), and COD (32.9%). For the treatment T130:30m:0P the syringic (0.74 mg/L) and coumaric (0.013 mg/L) acids obtained the highest increase, and the treatment T120:30m:1P increased 3-hydroxybenzoic (1.30 mg/L) and sinapic (0.06 mg/L) acid. The revaluation of vinasses through thermal treatments provides guidelines to reduce the impact generated on the environment.

Identifying Bixa orellana L. New Carotenoid Cleavage Dioxygenases 1 and 4 Potentially Involved in Bixin Biosynthesis.

Carotene cleavage dioxygenases (CCDs) are a large family of Fe2+ dependent enzymes responsible for the production of a wide variety of apocarotenoids, such as bixin. Among the natural apocarotenoids, bixin is second in economic importance. It has a red-orange color and is produced mainly in the seeds of B. orellana. The biosynthesis of bixin aldehyde from the oxidative cleavage of lycopene at 5,6/5',6' bonds by a CCD is considered the first step of bixin biosynthesis. Eight BoCCD (BoCCD1-1, BoCCD1-3, BoCCD1-4, CCD4-1, BoCCD4-2, BoCCD4-3 and BoCCD4-4) genes potentially involved in the first step of B. orellana bixin biosynthesis have been identified. However, the cleavage activity upon lycopene to produce bixin aldehyde has only been demonstrated for BoCCD1-1 and BoCCD4-3. Using in vivo (Escherichia coli) and in vitro approaches, we determined that the other identified BoCCDs enzymes (BoCCD1-3, BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4) also participate in the biosynthesis of bixin aldehyde from lycopene. The LC-ESI-QTOF-MS/MS analysis showed a peak corresponding to bixin aldehyde (m/z 349.1) in pACCRT-EIB E. coli cells that express the BoCCD1 and BoCCD4 proteins, which was confirmed by in vitro enzymatic assay. Interestingly, in the in vivo assay of BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4, bixin aldehyde was oxidized to norbixin (m/z 380.2), the second product of the bixin biosynthesis pathway. In silico analysis also showed that BoCCD1 and BoCCD4 proteins encode functional dioxygenases that can use lycopene as substrate. The production of bixin aldehyde and norbixin was corroborated based on their ion fragmentation pattern, as well as by Fourier transform infrared (FTIR) spectroscopy. This work made it possible to clarify at the same time the first and second steps of the bixin biosynthesis pathway that had not been evaluated for a long time.

Thalassobacillus, a genus of extreme to moderate environmental halophiles with biotechnological potential.

Thalassobacillus is a moderately halophilic genus that has been isolated from several sites worldwide, such as hypersaline lakes, saline soils, salt flats, and volcanic mud. Halophilic bacteria have provided functional stable biomolecules in harsh conditions for industrial purposes. Despite its potential biotechnological applications, Thalassobacillus has not been fully characterized yet. This review describes the Thalassobacillus genus, with the few species reported, pointing out its possible applications in enzymes (amylases, cellulases, xylanases, and others), biosurfactants, bioactive compounds, biofuels production, bioremediation, and plant growth promotion. The Thalassobacillus genus represents a little-explored biological resource but with a high potential.

Proteomic characterization of a second-generation version of the BCGΔBCG1419c vaccine candidate by means of electrospray-ionization quadrupole time-of-flight mass spectrometry.

Tuberculosis (TB) is the most important infectious disease worldwide, based on the number of new cases and deaths reported by the World Health Organization. Several vaccine candidates against TB have been characterized at preclinical and clinical levels. The BCGΔBCG1419c vaccine candidate, which lacks the BCG1419c gene that encodes for a c-di-GMP phosphodiesterase, provides improved efficacy against chronic TB, reactivation from latent-like infection and against chronic TB in the presence of type 2 diabetes in murine models. We previously reported that compared with wild type BCG, BCGΔBCG1419c changed levels of several proteins. Here, using a label-free proteomic approach, we confirmed that a novel, second-generation version of BCGΔBCG1419c maintains changes in antigenic proteins already reported, and here we further found differences in secreted proteins, as well as that this new BCGΔBCG1419c version modifies its production of proteins involved in redox and nitrogen/protein metabolism compared with wild type BCG. This work contributes to the proteomic characterization of a novel vaccine candidate that is more effective against TB than parental BCG in diverse murine models.

Transcriptional and Mycolic Acid Profiling in Mycobacterium bovis BCG In Vitro Show an Effect for c-di-GMP and Overlap between Dormancy and Biofilms.

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA, kasB, and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR, therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.

Analysis of volatile compounds of five varieties of Maya cocoa during fermentation and drying processes by Venn diagram and PCA.

Fermented cocoa beans can be described as a complex matrix that integrates the chemical history of beans, their processing, and environmental factors. This study presents an analysis that aims to identify volatile compounds of five varieties of fine-aroma cocoa types. The cocoa types studied were Carmelo, Rojo Samuel, Lagarto, Arcoiris, Regalo de Dios, that grow in the Maya lands of Chiapas, Mexico. Profile of volatile compounds was obtained from each cacao type during fermentation and drying process. This profile of volatile compounds also was compared with beans unfermented, using a statistical analysis of Venn diagram and a multivariate Analysis of Principal Components (PCA). One hundred nine different compounds were identified by SPME-HS GC-MS, these compounds mainly related to desirable aromatic notes generated by esters, aldehydes, ketones, and alcohols. The differences in chemical composition of the volatile compounds were associated mainly with the process and not to cocoa varieties. Fermented dry cocoa beans showed a higher content of esters, aldehydes, pyrazines, alcohols, some acids, and furans where Lagarto (CL), Rojo Samuel (CR), and Regalo de Dios (TRD) cocoas type showed a more interesting aromatic profile. On the other hand, as expected dry unfermented cocoas presented a few numbers of aroma compounds, in the five cacao types, where alcohols, ketones and hydrocarbons predominated.

Phenolic Profile, Antioxidant and Anti-Proliferative Activities of Methanolic Extracts from Asclepias linaria Cav. Leaves.

Asclepias linaria Cav. (Apocynaceae) is a shrubby plant endemic of Mexico which has been used in traditional medicine. However, the bioactive potential of this plant remains unexplored. In this study, the phenolic composition, antioxidant, and cytotoxic activities of A. linaria leaves were determined. In order to estimate the phenolic composition of the leaves, the total phenolic, flavonoid, and condensed tannins contents were determined. Furthermore, the antioxidant activity was measured by the scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS•+) radicals and the total antioxidant capacity. The phenolic compounds identified in the A. linaria leaves by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) include phenolic acids, such as p-coumaric and ferulic acid, as well as flavonoids, such as rutin and quercetin. The leaves' extracts of A. linaria showed a high scavenging activity of DPPH• and ABTS•+ radicals (IC50 0.12 ± 0.001 and 0.51 ± 0.003 µg/mL, respectively), high total antioxidant capacity values (99.77 ± 4.32 mg of ascorbic acid equivalents/g of dry tissue), and had a cytotoxic effect against K562 and HL60 hematologic neoplasia cells lines, but no toxicity towards the normal mononuclear cell line was observed. These results highlight the potential of A. linaria and could be considered as a possible alternative source of anticancer compounds.

Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto (Bixa orellana L.).

Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world's food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5',6'.

Use of agave bagasse for production of an organic fertilizer by pretreatment with Bjerkandera adusta and vermicomposting with Eisenia fetida.

Agave tequilana Weber is used in tequila and fructans production, with agave bagasse generated as a solid waste. The main use of bagasse is to produce compost in tequila factories with a long traditional composting that lasts 6-8 months. The aim of this study was to evaluate the degradation of agave bagasse by combining a pretreatment with fungi and vermicomposting. Experiments were carried out with fractionated or whole bagasse, sterilized or not, subjecting it to a pretreatment with Bjerkandera adusta alone or combined with native fungi, or only with native bagasse fungi (non-sterilized), for 45 days. This was followed by a vermicomposting with Eisenia fetida and sewage sludge, for another 45 days. Physicochemical parameters, lignocellulose degradation, stability and maturity changes were measured. The results indicated that up to 90% of the residual sugars in bagasse were eliminated after 30 days in all treatments. The highest degradation rate in pretreatment was observed in non-sterilized, fractionated bagasse with native fungi plus B. adusta (BNFns) (71% hemicellulose, 43% cellulose and 71% lignin) at 45 days. The highest total degradation rates after vermicomposting were in fractionated bagasse pre-treated with native fungi (94% hemicellulose, 86% cellulose and 91% lignin). However, the treatment BNFns showed better maturity and stability parameters compared to that reported for traditional composts. Thus, it seems that a process involving vermicomposting and pretreatment with B. adusta could reduce the degradation time of bagasse to 3 months, compared to the traditional composting process, which requires from 6 to 8 months.