Metabolomic analysis and ecofriendly enrichment of sunflower meal extract.

BACKGROUND: The presence of phenolic compounds in sunflower is well reported in the literature; however, knowledge is scarce when it comes to the composition of other secondary metabolites in this species and their by-products. This work evaluated, for the first time, the phytochemical composition of sunflower meal produced in Brazil. A combination of mixture design and central composite rotatable design 23 models was then applied to maximize the recovery of bioactive compounds using ecologically friendly solvents and concentrating by applying activated carbon, a sustainable adsorbent. The product of this extraction-concentration was also evaluated by an untargeted metabolomic approach using ultra-performance liquid chromatography coupled to mass spectrometry. RESULTS: A diverse and abundant profile of phenolic compounds was obtained from Brazilian sunflower meal: in total, 51 natural products were tentatively identified, 35 of which for the first time in sunflower. The sorption capacity of the activated charcoal, in the optimized process conditions, was effective in the separation and concentration of minority secondary metabolites. The ecofriendly extract proved to be enriched in plumberoside, p-coumaric acid, and alkaloids. CONCLUSIONS: Investigation of the phytochemical profile of sunflower meal produced in Brazil pointed to several secondary metabolites reported for the first time in sunflower samples, including phenolic compounds, alkaloids, and terpenes. The use of activated charcoal in an alkaline medium as an adsorbent for the concentration of these phytochemicals, from an aqueous extract, generated a potentially cost-effective, ecofriendly extract, enriched in minor metabolites, indicating a possible innovative way to selectively obtain these compounds from sunflower meal. © 2022 Society of Chemical Industry.

Pectinases Secretion by Saccharomyces cerevisiae: Optimization in Solid-State Fermentation and Identification by a Shotgun Proteomics Approach.

A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL-1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin-lyase and pectate-lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL-1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin-lyases, one pectate-lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.

Vanilla from Brazilian Atlantic Forest: In vitro and in silico toxicity assessment and high-resolution metabolomic analysis of Vanilla spp. ethanolic extracts.

The natural vanilla market, which generates millions annually, is predominantly dependent on Vanilla planifolia, a species characterized by low genetic variability and susceptibility to pathogens. There is an increasing demand for natural vanilla, prized for its complex, authentic, and superior quality compared to artificial counterparts. Therefore, there is a necessity for innovative production alternatives to ensure a consistent and stable supply of vanilla flavors. In this context, vanilla crop wild relatives (WRs) emerge as promising natural sources of the spice. However, these novel species must undergo toxicity assessments to evaluate potential risks and ensure safety for consumption. This study aimed to assess the non-mutagenic and non-carcinogenic properties of ethanolic extracts from V. bahiana, V. chamissonis, V. cribbiana, and V. planifolia through integrated metabolomic profiling, in vitro toxicity assays, and in silico analyses. The integrated approach of metabolomics, in vitro assays, and in silico analyses has highlighted the need for further safety assessments of Vanilla cribbiana ethanolic extract. While the extracts of V. bahiana, V. chamissonis, and V. planifolia generally demonstrated non-mutagenic properties in the Ames assay, V. cribbiana exhibited mutagenicity at high concentrations (5000 µg/plate) in the TA98 strain without metabolic activation. This finding, coupled with the dose-dependent cytotoxicity observed in WST-1 (Water Soluble Tetrazolium) assays, a colorimetric method that assesses the viability of cells exposed to a test substance, underscores the importance of concentration in the safety evaluation of these extracts. Kaempferol and pyrogallol, identified with higher intensity in V. cribbiana, are potential candidates for in vitro mutagenicity. Although the results are not conclusive, they suggest the safety of these extracts at low concentrations. This study emphasizes the value of an integrated approach in providing a nuanced understanding of the safety profiles of natural products, advocating for cautious use and further research into V. cribbiana mutagenicity.

Critical Review for the Production of Antidiabetic Peptides by a Bibliometric Approach.

The current bibliometric review evaluated recent papers that researched dietary protein sources to generate antidiabetic bioactive peptides/hydrolysates for the management of diabetes. Scopus and PubMed databases were searched to extract bibliometric data and, after a systematic four-step process was performed to select the articles, 75 papers were included in this review. The countries of origin of the authors who published the most were China (67%); Ireland (59%); and Spain (37%). The journals that published most articles on the subject were Food Chemistry (n = 12); Food & Function (n = 8); and Food Research International (n = 6). The most used keywords were 'bioactive peptides' (occurrence 28) and 'antidiabetic' (occurrence 10). The most used enzymes were Alcalase® (17%), Trypsin (17%), Pepsin, and Flavourzyme® (15% each). It was found that different sources of protein have been used to generate dipeptidyl peptidase IV (DPP-IV), α-amylase, and α-glucosidase inhibitory peptides. In addition to antidiabetic properties, some articles (n = 30) carried out studies on multifunctional bioactive peptides, and the most cited were reported to have antioxidant and antihypertensive activities (n = 19 and 17, respectively). The present review intended to offer bibliometric data on the most recent research on the production of antidiabetic peptides from dietary proteins to those interested in their obtention to act as hypoglycemic functional ingredients. The studies available in this period, compiled, are not yet enough to point out the best strategies for the production of antidiabetic peptides from food proteins and a more systematic effort in this direction is necessary to allow a future scale-up for the production of these possible functional ingredients.

Unraveling potential enzymes and their functional role in fine cocoa beans fermentation using temporal shotgun metagenomics.

Cocoa beans fermentation is a spontaneous process, essential for the generation of quality starting material for fine chocolate production. The understanding of this process has been studied by the application of high-throughput sequencing technologies, which grants a better assessment of the different microbial taxa and their genes involved in this microbial succession. The present study used shotgun metagenomics to determine the enzyme-coding genes of the microbiota found in two different groups of cocoa beans varieties during the fermentation process. The statistical evaluation of the most abundant genes in each group and time studied allowed us to identify the potential metabolic pathways involved in the success of the different microorganisms. The results showed that, albeit the distinction between the initial (0 h) microbiota of each varietal group was clear, throughout fermentation (24-144 h) this difference disappeared, indicating the existence of selection pressures. Changes in the microbiota enzyme-coding genes over time pointed to the distinct ordering of fermentation at 24-48 h (T1), 72-96 h (T2), and 120-144 h (T3). At T1, the significantly more abundant enzyme-coding genes were related to threonine metabolism and those genes related to the glycolytic pathway, explained by the abundance of sugars in the medium. At T2, the genes linked to the metabolism of ceramides and hopanoids lipids were clearly dominant, which are associated with the resistance of microbial species to extreme temperatures and pH values. In T3, genes linked to trehalose metabolism, related to the response to heat stress, dominated. The results obtained in this study provided insights into the potential functionality of microbial community succession correlated to gene function, which could improve cocoa processing practices to ensure the production of more stable quality end products.

Artisan minas cheese of Serro: proteolysis during ripening.

The Artisan Minas Cheese (AMC) is the oldest and most traditional Brazilian cheese, it is produced in several regions of the state of Minas Gerais, such as the Serro region. The most striking features of the AMC-Serro are the use of raw milk and natural bacteria from the whey, popularly known as pingo, as well as the use of the rind washing process. The aim of the present study was to evaluate the proteolysis of the AMC-Serro from three different producers, during 60 days of maturation, and to relate the proteolysis to the producing farms, the production season and the rind washing during ripening. For this purpose, TRICINE-SDS-PAGE, proteolysis extension and depth indices, moisture, and texture (firmness) were evaluated. It was concluded that the temperature and moisture of the cheeses, that were determined by the location of the ripening room, the production season and the rind washing, were the most important factors. The degree of proteolysis also had an impact on the water loss during ripening, with effect on cheese safety. The results obtained in this study may be used to better understand the transformations during ripening of AMC-Serro and help the small traditional farmers to improve their product's quality and stability.

Vanilla bahiana, a contribution from the Atlantic Forest biodiversity for the production of vanilla: A proteomic approach through high-definition nanoLC/MS.

Only a few cultivated species of Vanilla are used to produce vanilla, despite the high demand, predatory exploitation, and low genetic variability that threaten the production of natural vanilla. Vanilla bahiana pods from the Atlantic Forest may be an alternative source of natural vanilla. This study applied bottom-up and shotgun proteomics analysis to identify proteins related to flowering, fruiting, and vanilla-flavor production. Extraction solutions, including Tris-HCl buffer, ß-mercaptoethanol and SDS, were assayed. SDS proved to be feasible for extraction of Vanilla fruit proteins and could be an alternative to the phenol method of protein extraction. Progenesis QI for Proteomics (QIP) software loaded with an Orchidaceae database identified 2326 proteins in our samples. Among these, 75 were highlighted as useful for the synthesis of compounds related to vanilla flavor, such as vanillin synthase, which was successfully extracted with 1% SDS, which also improved the variety of the extracted proteins. The proteins identified in V. bahiana pods indicate the enzymatic potential of this species, as further validated by quantifying the vanilla in the samples.

Trametes villosa Lignin Peroxidase (TvLiP): Genetic and Molecular Characterization.

White-rot basidiomycetes are the organisms that decompose lignin most efficiently, and Trametes villosa is a promising species for ligninolytic enzyme production. There are several publications on T. villosa applications for lignin degradation regarding the expression and secretion of laccase and manganese peroxidase (MnP) but no reports on the identification and characterization of lignin peroxidase (LiP), a relevant enzyme for the efficient breakdown of lignin. The object of this study was to identify and partially characterize, for the first time, gDNA, mRNA, and the corresponding lignin peroxidase (TvLiP) protein from T. villosa strain CCMB561 from the Brazilian semiarid region. The presence of ligninolytic enzymes produced by this strain grown in inducer media was qualitatively and quantitatively analyzed by spectrophotometry, qPCR, and dye fading using Remazol Brilliant Blue R. The spectrophotometric analysis showed that LiP activity was higher than that of MnP. The greatest LiP expression as measured by qPCR occurred on the 7th day, and the ABSA medium (agar, sugarcane bagasse, and ammonium sulfate) was the best that favored LiP expression. The amplification of the TvLiP gene median region covering approximately 50% of the T. versicolor LPGIV gene (87% identity); the presence of Trp199, Leu115, Asp193, Trp199, and Ala203 in the translated amplicon of the T. villosa mRNA; and the close phylogenetic relationship between TvLiP and T. versicolor LiP all indicate that the target enzyme is a lignin peroxidase. Therefore, T. villosa CCMB561 has great potential for use as a LiP, MnP, and Lac producer for industrial applications.

Production of Basidiomata and Ligninolytic Enzymes by the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Licuri (Syagrus coronata) Wastes in Brazil.

Ganoderma lucidum is a medicinal mushroom with different forms of bioactivity that has been used in popular medicine for centuries. This study aimed to test the application of agricultural wastes (fruit shells, leaves, and bracts) from the endemic Brazilian palm tree Syagrus coronata (licuri) as substrates for the production of G. lucidum basidiomata and ligninolytic enzymes via solid-state fermentation. The best culture conditions were the same for all substrates (pH 6.5, carbon-to-nitrogen ratio = 40, and temperature 30°C) and were established from preliminary assays. The yield was not significantly different for bracts (33.53 g/kg) and leaves (37.48 g/kg), nor for the biological efficiency in these same substrates: bracts, 3.35%; leaves, 3.75%. The highest laccase (13.80 U/L) and manganese peroxidase (14.92 U/L) activities were achieved after 14 and 28 days of incubation, respectively, using bracts as the substrate. Licuri residues are then potential substrates to be used in the bioconversion process for mycelia, basidiomata, and ligninolytic enzyme production by G. lucidum.

Antioxidant Activity and Cytotoxicity Effect of Cocoa Beans Subjected to Different Processing Conditions in Human Lung Carcinoma Cells.

Lung cancer is a common malignancy in men and the second leading cause of cancer-related mortality in men in the western world. Phenolic cocoa ingredients have a strong antioxidative activity and the potential to have a protective effect against cancer. In the present study, we have evaluated the influence of cocoa beans subjected to different processing conditions on cell viability and apoptosis of human lung cancer cells (A549). We measured the viability of lung cells treated with cocoa beans, unroasted slates (US), roasted slates (RS), unroasted well fermented (UWF) cocoa, and roasted well fermented (RWF) cocoa for 24 h. Using an MTT assay, we observed a decrease in the viability of A549 cells after treatment with cocoa bean extracts. Flow cytometer analysis revealed that cocoa beans increased the percentage of cells in sub-G1 phase and promoted up to twofold increase of apoptotic cells when compared to the control group. Taken together, the present study suggests that cocoa beans may have a protective effect against lung cancer.

Lytic enzyme production optimization using low-cost substrates and its application in the clarification of xanthan gum culture broth.

Lytic enzymes are widely used in industrial biotechnology as they are able to hydrolyze the bacterial cell wall. One application of these enzymes is the clarification of the culture broth for the production of xanthan gum, because of its viability in viscous media and high specificity. The screening process for filamentous fungi producing lytic enzymes, the optimization of production of these enzymes by the selected microorganism, and the optimization of the application of the enzymes produced in the clarification of culture broth are presented in this article. Eleven fungal isolates were tested for their ability to produce enzymes able to increase the transmittance of the culture broth containing cells of Xanthomonas campestris. To optimize the secretion of lytic enzymes by the selected microorganism the following variables were tested: solid substrate, initial pH, incubation temperature, and addition of inducer (gelatin). Thereafter, secretion of the enzymes over time of incubation was assessed. To optimize the clarification process a central composite rotational design was applied in which the pH of the reaction medium, the dilution of the broth, and the reaction temperature were evaluated. The isolate identified as Aspergillus tamarii was selected for increasing the transmittance of the broth from 2.1% to 54.8%. The best conditions for cultivation of this microorganism were: use of coconut husk as solid substrate, with 90% moisture, at 30°C for 20 days. The lytic enzymes produced thereby were able to increase the transmittance of the culture broth from 2.1% to 70.6% at 65°C, without dilution and without pH adjustment.

Improving chocolate flavor in poor-quality cocoa almonds by enzymatic treatment.

UNLABELLED: This paper proposes a method to enzymatically treat poor-quality cocoa almonds (known as "slate") to ensure the formation of chocolate flavor precursors. The production of flavor precursors improves the quality of these almonds, which are usually responsible for the low quality of the liquor produced. Proteases and carboxypeptidases from different sources were tested under various conditions. The different treatments were evaluated by chemical analysis (hydrolysis efficiency) and sensory analysis of the treated material compared to good-quality cocoa almonds. The results show that it is possible, through the use of microbial enzymes, to generate the mixture of compounds that will release, after roasting, the characteristic chocolate flavor in poor-quality almonds. However, it is necessary to optimize the conditions of enzymatic treatment to obtain better results and thus establish a process that can be used for industrial purposes for manufacturing cocoa and chocolate. PRACTICAL APPLICATION: The basidiomycete Moniliophtora perniciosa is the causative agent of witches' broom disease (WBD) of the cocoa tree, whose seeds are the source of chocolate. It is the most important phytopathological problem of cocoa-producing areas of the American continent, and has decimated the Brazilian cocoa industry. In Bahia (Brazil), M. perniciosa was identified in 1989 and, as a consequence of its spreading, the annual production of cocoa almonds dropped from 450,000 to 90,000 tons within 12 y, reducing export values from an all-time high of about US$ 1 billion to 110 million. The high incidence of WBD incapacitates Brazil to produce enough cocoa almonds even for the internal market, leading the country to import low-quality cocoa almonds mainly from African countries. Our work proposes an enzymatic treatment to increase the quality of that cocoa almonds and, consequently, to improve the quality of the chocolate produced and consumed in the country.

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa.

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.

Influência da concentração de NaCl e pH na extração de ricina em torta de mamona (Ricinus communis L.) e sua caracterização por eletroforese / Influence of NaCl and pH concentration on the extraction of ricin in castor bean (Ricinus communis L.) and its characterization by electrophoresis

A mamoneira (Ricinus communis L.) é uma oleaginosa de alto valor econômico pelo fato de apresentar um mercado bem definido para o óleo extraído de suas sementes. A torta, que é um resíduo desta extração, se destaca pelo alto teor em proteínas. Dentre as proteínas encontradas na torta destaca-se a ricina, uma citotoxina, que inviabiliza sua utilização como fonte protéica alternativa para alimentação animal. O presente trabalho tem como objetivo identificar um melhor tratamento experimental para a extração de ricina da torta de mamona, visando futuros estudos de perda de integridade da ricina, o que garantiria a inocuidade do produto. Para tanto, buscou-se identificar a solução de maior capacidade de extração de proteínas, empregando a metodologia de superfície de resposta. Um delineamento composto central rotacional foi elaborado a fim de verificar o melhor pH e concentração de NaCl para a extração. Dos cinco diferentes valores de pH (4,0; 4,6; 6,0; 7,4; 8,0) e concentração de NaCl (0,0M; 0,3M; 1,0M; 1,7M; 2,0M) utilizados, o tratamento associando fosfato de potássio 0,2M/NaCl 1,7M pH 7,4 foi escolhido como o melhor. A concentração de proteína extraída neste tratamento chegou a valores quatro vezes maiores que o encontrado no de mínima extração de proteína. Pela evidenciação do gel de eletroforese não houve extração preferencial de ricina nos tratamentos testados, entretanto etapas de purificação usando diálise e precipitação com sulfato de amônio, permitiram uma evidenciação melhor das duas cadeias polipeptídicas de ricina.

Castor bean (Ricinus communis L.) is an oilseed crop of high economic value due to the fact of presenting a clearly defined market for the oil extracted from its seeds. Castor cake, which is a residue of oil extraction, is at the moment receiving special attention because of its high protein content. However, among the proteins found in this cake it is observed the presence of ricin, a cytotoxin, which turns this seed dangerous to be used as an alternative protein source for animal feed. The present research has the objective of identifying the best experimental treatment for extraction of ricin from castor cake, with the aim of future studies of loss of ricin integrity, which would ensure the safety of the product. With this aim, it was initiated the search for a buffer of higher capacity of proteins extraction, using the response surface methodology. A central composite design was developed in order to determine the best pH and NaCl concentration for extraction. Of the five different pH values (4.0, 4.6, 6.0, 7.4, 8.0) and NaCl (0.0M, 0.3M, 1.0M, 1.7M, 2.0M) used, the treatment containing 0.2M potassium phosphate / 1.7M NaCl pH 7.4 was chosen as the best. The amount of protein extracted in this treatment reached values four times larger than the minimum found in other treatment studied. The electrophoresis analysis showed that there was no preferential extraction of ricin in the treatments; however purification steps using dialysis and precipitation with ammonium sulfate led to a better resolution of the two polypeptide chains presents in ricin.