Genome-guided purification of high amounts of the siderophore ornibactin and detection of potentially novel burkholdine derivatives produced by Burkholderia catarinensis 89T.

AIM: The increased availability of genome sequences has enabled the development of valuable tools for the prediction and identification of bacterial natural products. Burkholderia catarinensis 89T produces siderophores and an unknown potent antifungal metabolite. The aim of this work was to identify and purify natural products of B. catarinensis 89T through a genome-guided approach. MATERIALS AND METHODS: The analysis of B. catarinensis 89T genome revealed 16 clusters putatively related to secondary metabolism and antibiotics production. Of particular note was the identification of a nonribosomal peptide synthetase (NRPS) cluster related to the production of the siderophore ornibactin, a hybrid NRPS-polyketide synthase Type 1 cluster for the production of the antifungal glycolipopeptide burkholdine, and a gene cluster encoding homoserine lactones (HSL), probably involved in the regulation of both metabolites. We were able to purify high amounts of the ornibactin derivatives D/C6 and F/C8, while also detecting the derivative B/C4 in mass spectrometry investigations. A group of metabolites with molecular masses ranging from 1188 to 1272 Da could be detected in MS experiments, which we postulate to be new burkholdine analogs produced by B. catarinensis. The comparison of B. catarinensis BGCs with other Bcc members corroborates the hypothesis that this bacterium could produce new derivatives of these metabolites. Moreover, the quorum sensing metabolites C6-HSL, C8-HSL, and 3OH-C8-HSL were observed in LC-MS/MS analysis. CONCLUSION: The new species B. catarinensis is a potential source of new bioactive secondary metabolites. Our results highlight the importance of genome-guided purification and identification of metabolites of biotechnological importance.

Unveiling Microbial Chemical Interactions Based on Metabolomics Approaches.

Microorganisms are ubiquitous in diverse habitats and studying their chemical interactions with the environment and comprehend its complex relations with both hosts and environment, are crucial for the development of strategies to control microbial diseases. This chapter discusses the importance of studying microorganisms with agricultural benefits, using specialized metabolites as examples. Herein we highlight the challenges and opportunities in utilizing microorganisms as alternatives to synthetic pesticides and fertilizers in agriculture. Genome-guided investigations and improved analytical methodologies are necessary to characterize diverse and complex biomolecules produced by microorganisms. Predicting and isolating bioproducts based on genetic information have become a focus for researchers, aided by tools like antiSMASH, BiG-SCAPE, PRISM, and others. However, translating genomic data into practical applications can be complex. Therefore, integrating genomics, transcriptomics, and metabolomics enhances chemical characterization, aiding in discovering new metabolic pathways and specialized metabolites. Additionally, elicitation is one promising strategy to enhance beneficial metabolite production. Finally, identify and characterize microbial secondary metabolites remain challenging due to their low production, complex chemical structure characterization and different environmental factors necessary for metabolite in vitro production.

UHPLC-ESI-QTOF-MS/MS Profiling of Phytochemicals from Araticum Fruit (Annona crassiflora Mart.) and Its Antioxidant Activity.

Araticum is a native species of the Brazilian Cerrado with a high potential for exploitation. Several studies have stated that araticum is a rich source of phytochemicals with multifaceted biological actions. However, little information is available regarding the characterization of phytochemicals found in the pulp of this fruit. In this context, this study aimed to carry out a comprehensive characterization of phytochemicals present in the araticum pulp using ultra-high-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer (UHPLC-ESI-QTOF-MS/MS). The antioxidant potential of araticum pulp was also evaluated. UHPLC-ESI-QTOF-MS/MS profiling of the phytochemicals allowed for the identification and annotation of 139 phytochemicals, including organic acids, jasmonates, iridoids, phenolic compounds, alkaloids, annonaceous acetogenins, fatty acid derivatives, and other compounds. Among them, 116 compounds have been found for the first time in araticum pulp. Phenolic compounds and their derivatives represented about 59% of the phytochemicals identified in the extract. Moreover, araticum pulp showed high total phenolic compound content and antioxidant activity. The majority of identified phytochemicals have been associated with key roles in the plant's defense mechanisms against biotic and abiotic stress factors in the Cerrado environment. Furthermore, many of these phytochemicals found in the araticum pulp are already widely recognized for their beneficial effects on human health. Our findings showed that the araticum fruit contains different classes of phytochemicals that exert various biological activities, both in the plant itself and in humans.

Transcriptional Control of the Production of Aspergillus fumigatus Conidia-Borne Secondary Metabolite Fumiquinazoline C Important for Phagocytosis Protection.

Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.

Impact of ripening on the health-promoting components from fruta-do-lobo (Solanum lycocarpum St. Hill).

Fruta-do-lobo (Solanum lycocarpum St. Hill) is an underutilized native fruit commonly found in the Brazilian Cerrado, very known due to the presence of glycoalkaloids. In this work we evaluated the biochemical changes on carbohydrates, phenolic and alkaloids during ripening of fruta-do-lobo using chromatographic and spectrometric techniques. During ripening, we observed an increase in glucose, fructose and sucrose, while oligosaccharides levels varied. Chlorogenic acid isomers represented 80% of the identified phenolic compounds in unripe stage, but they reduced during ripening, resulting in predominance of p-coumaroylquinic acid (peel and pulp) and 1-O-sinapoyl-glucoside (seeds). Statistical analysis shows that the unripe fractions were richer in alkaloids compounds, which were the most important for antioxidant activity. Molecular network analysis summarizes the compound changes during ripening, especially regarding the alkaloid compounds, with a reduction of around 85% of solamargine abundance. These data show that fruta-do-lobo can presents different chemical compositions due their ripening stage providing support for future research aimed to the application of these compounds in glycemia control or uses of their extracts with higher content of alkaloids compounds.

Genipap (Genipa americana L.) fruit extract as a source of antioxidant and antiproliferative iridoids.

Iridoid blue-based pigments can be found in fruits of genipap (Genipa americana L.). Besides being a potential source of natural blue colorant in the food industry, they have also been associated with pharmacological effects. Therefore, the recovery of iridoids by ultrasound-assisted extraction from both unripe and ripe fruits was analysed by UPLC-DAD-ESI-(-)-QTOF-MS/MS. Nine iridoids were identified from their exact masses and fragmentation pattern, namely geniposidic acid, gardenoside, genipin-1-ß-gentiobioside, geniposide, 6''-O-p-coumaroyl-1-ß-gentiobioside geniposidic acid, 6''-O-p-coumaroylgenipin-gentiobioside, genipin, 6'-O-p-coumaroyl-geniposidic acid and 6'-O-feruloyl-geniposidic acid. Among them, genipin (60.77 mg/g fdw) was found to be the most abundant iridoid in unripe genipap extract, while the ripe genipap extract mainly contained geniposide and geniposidic acid (89.48 and 25.04 mg/g fdw, respectively). It was also observed that the iridoids of the unripe genipap extract are able to scavenger DPPH, ABTS and peroxyl radicals as well as exerting a cytostatic effect against both glioma and breast cancer cell lines. This study provided information about the properties of unripe and ripe genipap extracts which can be used as a reference for further studies focusing on the potential application of G. americana L. in commercial products containing natural blue colorant with functional claims.

Evaluation of fruta-do-lobo (Solanum lycocarpum St. Hill) starch on the growth of probiotic strains.

Fruta-do-lobo (Solanum lycocarpum St. Hill) is a native fruit commonly used in Brazilian folk medicine as a hypoglycemic agent. These properties are attributed to their starch, mainly its resistant fraction. Resistant starch has shown to increases the growth of Bifidobacterium and Lactobacillus in the gut, even though not being selective for these strains. In this scenario, this study aimed to investigate the potential prebiotic activity of fruta-do-lobo starch (FLS). FLS showed around 30% of resistant starch and their prebiotic potential was evaluated with five probiotic strains L. acidophilus (LA3 and LA5), L. casei (LC01) and B. animalis (BB12) and B. lactis (BLC1) in a concentration range of 1.0-2.0% of starch. In a preliminary screening, we evaluated, during 48 h, the viability of the starch with promoting growth agent. An increase in the growth of the probiotic strains tested was observed. We also evaluated the microorganism's metabolic activity by assessing the short-chain fatty acid (SCFA) production, using the best starch growth promotion conditions (2% of FLS and strains BLC1, LA5, and LC01). As expected, MRS and lactose were preferentially metabolized by BLC1, with the highest growth rates: 0.231 and 0.224 h-1, respectively. However, for this strain, the FLS growth rate (0.222 h-1) was 65% higher than FOS (0.144 h-1). Also, for LA5 FLS promoted higher growth (0.150 h-1) than FOS (0.135 h-1). Additionally, FLS promoted acetate production. These data are promising and indicate that FLS may have prebiotic potential and more studies need to be done with pathogenic microorganisms.

Optimization of Eugenia punicifolia (Kunth) D. C. leaf extraction using a simplex centroid design focused on extracting phenolics with antioxidant and antiproliferative activities.

Eugenia punicifolia (Kunth) D. C. (Myrtaceae) has been showing interesting biological activities in the literature which was correlated to its phenolic compounds. In the sense of a better recovering of phenolics with the best antioxidant and antiproliferative activities, an extraction, based on multivariate analytical approach, was developed from E. punicifolia leaves. The different extractor solvents (ethanol, methanol and water) and their binary and ternary combinations were evaluated using a simplex-centroid mixture design and surface response methodology. The optimized crude extracts were investigated for phenol and flavonoid content and compared to their antioxidant (EC50) and antiproliferative properties against HEp-2 (cell line derived from the oropharyngeal carcinoma) and mononuclear viability cells. Ethanolic extracts showed the best phenolic content with the highest antioxidant activity and moderated activity antiproliferative to HEp-2. ESI-QTOF-MS revealed the presence of quercetin and myricetin derivatives, which was correlated to activities tested. Then, simplex-centroid design allowed us to correlate the Eugenia punicifolia biological activities with the extracts obtained from solvent different polarity mixtures.

In vitro bioactivity approach of unripe genipap (Genipa americana L., Rubiaceae) fruit extract and its solid lipid microparticle.

Growing awareness in favor of innovative and healthier alternatives is creating a noticeable shift from synthetic colorants to natural additives. And, such a swing in the consumer market is growing slowly but noticeably. In this context, genipap (Genipa americana L.) fruit represents an emerging source of blue colorants in Latin America with extensive application possibilities. This is despite the fact that there are few studies concerning its toxicity predictive factors. In this early-stage study we propose to investigate safety issues around genipap extract (IBBP); we also attempt to identify fingerprint profiling of both IBBP extract and solid lipid microparticles containing IBBP extract (SLM-IBBP) using in vitro assays. The main compounds identified were genipin, and genipin 1-ß-gentiobioside. Results indicated that IBBP extract, at 25 µg/mL, was able to promote DNA damage in CHO-K1 cells, suggesting a genotoxic effect. On the other hand, the SLM-IBBP inhibited almost all cancer cell lines with GI50 ranging from 0.25 µg/mL to 43.5 µg/mL. Also, IBBP-SLM seems to exert a desirable apoptosis induction (at 25 µg/mL dosage). The next steps for our work, therefore, will focus on other nanoparticle formulation approaches, in particular with the use of natural Brazilian starch. An evaluation of the metabolism and distribution of microparticles, and their safety for food and pharmaceutical purposes, are also required.

Antifungal potential of secondary metabolites involved in the interaction between citrus pathogens.

Numerous postharvest diseases have been reported that cause substantial losses of citrus fruits worldwide. Penicillium digitatum is responsible for up to 90% of production losses, and represent a problem for worldwide economy. In order to control phytopathogens, chemical fungicides have been extensively used. Yet, the use of some artificial fungicides cause concerns about environmental risks and fungal resistance. Therefore, studies focusing on new approaches, such as the use of natural products, are getting attention. Co-culture strategy can be applied to discover new bioactive compounds and to understand microbial ecology. Mass Spectrometry Imaging (MSI) was used to screen for potential antifungal metabolites involved in the interaction between Penicillium digitatum and Penicillium citrinum. MSI revealed a chemical warfare between the fungi: two tetrapeptides, deoxycitrinadin A, citrinadin A, chrysogenamide A and tryptoquialanines are produced in the fungi confrontation zone. Antimicrobial assays confirmed the antifungal activity of the investigated metabolites. Also, tryptoquialanines inhibited sporulation of P. citrinum. The fungal metabolites reported here were never described as antimicrobials until this date, demonstrating that co-cultures involving phytopathogens that compete for the same host is a positive strategy to discover new antifungal agents. However, the use of these natural products on the environment, as a safer strategy, needs further investigation. This paper aimed to contribute to the protection of agriculture, considering health and ecological risks.

A comprehensive characterization of Solanum lycocarpum St. Hill and Solanum oocarpum Sendtn: Chemical composition and antioxidant properties.

In this study we evaluated the proximate composition of two Solanaceae fruits from Brazilian Cerrado, their mineral content, volatile organic compounds (VOCs), phenolic compounds profile, and antioxidant capacity employing Oxygen Radical Absorbance Capacity (ORAC) assay, for each part of the fruits (pulp, peel and seeds). Our results showed that the pulp has a high moisture content (74.62-85.40 g/100 g) and soluble fiber (1.29-2.06 g/100 g) content, and low fat, protein, and ash content. The peel exhibited high levels of carbohydrates and total fibers (6.55-11.39 and 12.35-13.12 g/100 g, respectively), while the seed presented high content of fat, protein, and insoluble fiber (10.14-12.62, 9.14-13.24 and 19.84-23.15 g/100 g). Potassium is the main mineral found in both fruits. It is the first time that the carbohydrate profile, volatile components, and phenolic compounds of the fruta-do-lobo and juá-açu are reported. 1-Kestose (GF2) and nystose (GF3) were found in both fruits. The main VOCs of juá-açu were esters, while in fruta-do-lobo, aldehydes were the major components. UPLC-Q-ToF fraction analysis of juá-açu and fruta-do-lobo revealed 24 phenolic compounds, most being hydroxycinnamic acids derivatives in juá-açu, and chlorogenic acids in fruta-do-lobo. The antioxidant capacity (ORAC) of the fruits ranged from 1.35 to 11.51 µmol TE/100 mL of extract. These results indicate that Solanum genus can be interesting for the Brazilian fruit market, and that it has potential to be exploited for agroindustry for diversification of fruit products.

Iridoid blue-based pigments of Genipa americana L. (Rubiaceae) extract: Influence of pH and temperature on color stability and antioxidant capacity during in vitro simulated digestion.

Iridoid blue-based pigments (IBBP) extract of Genipa americana L. represents a natural alternative additive for food applications and also exerts desirable biological effects on human health. In this study the iridoids present in the extract were identified, the influence of pH and temperature on color difference (ΔE) of IBBP was evaluated using a central composite design (CCD) and finally the antioxidant capacity was monitored before and after its in vitro digestion. Ten glucoside iridoids were detected and the main compounds identified were genipin, genipin 1-ß-gentiobioside and geniposide. It was also observed an increase of 17-18% of antioxidant capacity after the in vitro digestion, respectively. Among the conditions tested, the color of extract was more stable at 12-20 °C and low pH (3.0-4.0), suggesting that it is compatible for coloring acidic foods. Finally, the in vitro digestion also increased the antioxidant capacity (ORAC assay) by 39%.

EASI-IMS an expedite and secure technique to screen for 25I-NBOH in blotter papers.

The increasing number of new psychoactive substances (NPS) and their quick worldwide spreading, often only slightly modified in the form of new derivatives and analogues, have brought the need for fast, wide-ranging, and unequivocal identification methods in clinical and forensic investigations. Because it usually provides secure results, gas chromatography coupled to mass spectrometry (GC-MS) has been routinely employed as the standard technique for the detection of NPS in blotter papers. For 25I-NBOH (N-(2-hydroxybenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-aminium), however, GC-MS analysis of an blotter paper extract leads to incorrect results. In this work, we investigated whether easy ambient sonic-spray mass spectrometry imaging (EASI-IMS), and ambient ionization MS method can be applied directly to the surface of the sample requiring therefore no extraction or sample preparations, would serve as an efficient, sensitive, and secure alternative for 25I-NBOH screening.

Criegee mechanism as a safe pathway of color reduction in sugarcane juice by ozonation.

The production of crystal sugar is based on sugarcane juice clarification through sulphitation, that is, heat treatment with sulfur dioxide. The use of ozonation as an alternative to sulphitation aims to eliminate the disadvantageous presence of residual sulfite in crystal sugar. Both treatments are used to reduce color of sugarcane juice. The objective of this work was to evaluate two process parameters (temperature and pH) to reduce gallic acid, a low molecular weight pigment (MW 170gmol-1) widely found in sugarcane. Gallic acid was used as a model compound in sucrose solutions. The results showed that degradation of gallic acid was favored from pH 7.0 to 7.82 and temperature values between 50 and 70°C. The reaction mechanism was proposed for gallic acid degradation by ozone based on Criegee mechanism. Ozonation was an efficient method to reduce the potential low molecular weight pigment present in the sugarcane.

Eugenia aurata and Eugenia punicifolia HBK inhibit inflammatory response by reducing neutrophil adhesion, degranulation and NET release.

BACKGROUND: Eugenia spp. are used in popular medicine in the treatment of pain, diabetes, intestinal disorders and cough. The aim of the work is to evaluate, ex vivo and in vivo, the anti-inflammatory activity of the hydroethanolic extracts of the leaves of Eugenia aurata (EA) and Eugenia punicifolia HBK (EP) upon neutrophils. METHODS: Ex vivo, isolated human neutrophils were sensitized by Eugenia extracts (0.1-1000 µg/mL) and stimulated by PMA. In these conditions, different neutrophil activities related to inflammatory process were measured: adhesion, degranulation and NET release. Neutrophil viability and tumor line cells were monitored. In vivo, neutrophil influx was evaluated by peritonitis model performed in mice pretreated with different concentrations of Eugenia extracts. Phytochemical profile was assessed by mass spectrometry. RESULTS: Ex vivo, EA and EP (1000 µg/mL) reduced cell adhesion and degranulation, respectively. NET release was inhibited by EA and EP. Anti-inflammatory activities occurred in the absence of cytotoxicity. In vivo, both EA as EP inhibited neutrophil migration. The phytochemical profile revealed that EA contains myricitrin, rutin, quinic acid and quercetin derivatives. EP presents gallic acid, quercetin derivatives, syringic acid, ellagic acid, monogalloyl-glucose, glycosyringic acid, mudanoside B, HHDP glucose isomer and digalloylglucose isomer. EA and EP inhibit neutrophil migration by different pathways. CONCLUSION: Different chemical compositions may explain the anti-inflammatory effects described herein for EA and EP. Both extracts inhibit NET release but only EA reduces cell adhesion whereas EP decreases elastase secretion. This work contributes to the elucidation of cellular mechanisms related to the anti-inflammatory activity for leaves of E. aurata and E. punicifolia HBK.