Cruciferous Plant Extracts, Their Isothyocianate or Indol Derivatives, and Their Effect on Cellular Viability of Breast Cancer Cell Lines.

Brassicaceaes are rich in glucosinolates (GSL), whose derivatives, the isothyocianates sulforaphane (SFN), iberine (IB), or indole derivatives as indole-3-carbinol (I3C), have anticancer activities. We evaluated the effects of a broccoli sprout (Brassica oleracea var italica) and red cabbage (B. oleracea L. var capitata f. rubra) extracts and their GSL derivatives on breast cancer cells. Broccoli sprout aqueous extract (BSE) and red cabbage aqueous (RCA) or ethanolic (RCE) extracts were high in SFN, IB, and/or I3C. BSE and RCA decreased proliferation at doses of 2.5-5 mg/mL but induced proliferation at lower doses. RCE decreased proliferation starting at 10 µg/mL with selectivity toward cancer cells. SFN, IB, or I3C alone or in combination did not decrease proliferation similarly, suggesting synergistic effects with other phytochemicals in the extract. RCE showed selectivity toward breast cancer cells, but the effect of the individual metabolites or their combination did not reduce proliferation to the same extent. It will be important to determine the combination responsible for this effect to characterize their use for breast cancer treatment.

The Effect of Broccoli Glucosinolates Hydrolysis Products on Botrytis cinerea: A Potential New Antifungal Agent.

The present study investigates the interactions between eight glucosinolate hydrolysis products (GHPs) sourced from broccoli by-products and the detoxifying enzymes of Botrytis cinerea, namely eburicol 14-alpha-demethylase (CYP51) and glutathione-S-transferase (GST), through in silico analysis. Additionally, in vitro assays were conducted to explore the impact of these compounds on fungal growth. Our findings reveal that GHPs exhibit greater efficacy in inhibiting conidia germination compared to mycelium growth. Furthermore, the results demonstrate the antifungal activity of glucosinolate hydrolysis products derived from various parts of the broccoli plant, including inflorescences, leaves, and stems, against B. cinerea. Importantly, the results suggest that these hydrolysis products interact with the detoxifying enzymes of the fungus, potentially contributing to their antifungal properties. Extracts rich in GHPs, particularly iberin and indole-GHPs, derived from broccoli by-products emerge as promising candidates for biofungicidal applications, offering a sustainable and novel approach to plant protection by harnessing bioactive compounds from agricultural residues.

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 µm in length, 7.1-17.2 µm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

Effect of foliar application of bio-stimulants on growth, yield and nutritional quality of broccoli / Efeito da aplicação foliar de bioestimulantes no crescimento, produtividade e qualidade nutricional de brócolis

Broccoli is one of important cole crop grown all over the world due to its unique nutritional profile consumed fresh as well as processed. It contains a wide range of nutrients, vitamins, minerals and specific anti-cancer compounds such as glucosinolates. Broccoli cultivation in Pakistan is increasing rapidly, however, till now there is no standardized cropping technology for broccoli cultivation under local climate. Considering research gap (lack of suitable varieties, poor growth, and unavailability of optimized crop technology), trial was conducted at Vegetable research area, Institute of Horticultural Sciences, University of Agriculture, Faisalabad to evaluate the impact of bio-stimulants on different broccoli cultivars under local climatic conditions. The set of experimental treatments was laid out in Randomized Complete Block Design (RCBD) with three replications. Pre-harvest application of Isabion and Seaweed extract significantly enhanced the plant height (11%), dry weight (4%), leaf area (7%), and yield plant-1 (5%). Moreover, Isabion and seaweed extract application led to the increase in antioxidant enzymes i.e., superoxide dismutase (18%), peroxidase (38%) and catalase (12%). In crux, the foliar application of bio-stimulants (Isabion and seaweed extract) on broccoli enhanced the growth, yield, and contents of antioxidant enzymes.

O brócolis é uma das importantes culturas de repolho cultivadas em todo o mundo devido ao seu perfil nutricional único consumido fresco e processado. Contém uma ampla gama de nutrientes, vitaminas, minerais e compostos anticancerígenos específicos, como glucosinolatos. O cultivo de brócolis no Paquistão está aumentando rapidamente, no entanto, até agora não há tecnologia de cultivo padronizada para o seu cultivo sob o clima local. Considerando a lacuna de pesquisa, como a falta de variedades adequadas, o baixo crescimento e a indisponibilidade de tecnologia de cultivo otimizada, o teste foi realizado na área de pesquisa de vegetais, no Instituto de Ciências Hortícolas na Universidade de Agricultura, em Faisalabad, Paquistão, para avaliar o impacto de bioestimulantes em diferentes cultivares de brócolis nas condições climáticas locais. O conjunto de tratamentos experimentais foi disposto em Randomized Complete Block Design (RCBD) com três repetições. A aplicação pré-colheita de Isabion e extrato de algas marinhas aumentou significativamente a altura da planta (11%), peso seco (4%), área foliar (7%) e rendimento planta (5%). Além disso, a aplicação de Isabion e extrato de algas marinhas levou ao aumento das enzimas antioxidantes, ou seja, superóxido dismutase (18%), peroxidase (38%) e catalase (12%). No crux, a aplicação foliar de bioestimulantes (Isabion e extrato de algas marinhas) melhorou o crescimento, rendimento e conteúdo de enzimas antioxidantes do vegetal

Valor nutricional de hojas y tallos de brócoli, apio y betarraga disponibles en un mercado mayorista de Santiago de Chile

La ingesta diaria de hortalizas se relaciona con menor riesgo de cáncer, diabetes, enfermedades coronarias y obesidad, pero partes de estos alimentos como cáscaras, tallos y hojas no son reutilizadas, aumentando los desperdicios e impactando negativamente al medio ambiente. El objetivo de este trabajo fue establecer el valor nutricional de hojas y tallos de apio, betarraga y brócoli del Mercado Mayorista Lo Valledor en Santiago de Chile. Un total de 6 muestras se recolectaron desde puntos de ventas del Mercado Mayorista, 3 de hojas de hortalizas (apio, betarraga y brócoli) y 3 de tallos (apio, betarraga y brócoli). La recolección, preparación y entrega de las mismas se realizaron de acuerdo a protocolos indicados por el laboratorio encargado de realizar los análisis nutricionales. Las hojas de betarraga presentaron mayor contenido de proteínas (2,6 g), fibra dietética total (4,5 g) y fibra dietética insoluble (3,3 g) que los tallos, pero estos aportan más carbohidratos (7 g). Las hojas de apio tienen más calorías (20,4 kcal), proteínas (2,4 g), fibra dietética total (7,4 g), fibra dietética soluble (1,3 g) y fibra dietética insoluble (5,3 g) aunque los tallos presentan un contenido superior de carbohidratos (4,1 g). Finalmente, las hojas de brócoli destacan por su contenido de carbohidratos (9,0 g), proteínas (2,5 g), fibra dietética total (10,4 g) y fibra dietética insoluble (7,9 g). De acuerdo al valor nutricional obtenido, estos excedentes son una opción de alimento en la dieta diaria de las personas y su valorización debe ser contemplada.

The daily intake of vegetables is related to a lower risk of cancer, diabetes, coronary diseases and obesity, but parts of them such as peels, stems and leaves are not reused, increasing waste and negatively impacting the environment. The objective of this work was to establish the nutritional value of celery, beetroot and broccoli leaves and stems from the Lo Valledor wholesale market in Santiago, Chile. A total of 6 samples were collected from sales points of the Lo Valledor wholesale market, 3 from vegetable leaves (celery, beetroot and broccoli) and 3 from stems (celery, beetroot and broccoli). The collection, preparation and delivery of them were carried out according to protocols indicated by the laboratory in charge of carrying out the nutritional analyzes. The beet leaves had higher protein content (2.6 g), total dietary fiber (4.5 g) and insoluble dietary fiber (3.3 g) than the stems, but they provide more carbohydrates (7 g). Celery leaves had more calories (20.4 kcal), protein (2.4 g), total dietary fiber (7.4 g), soluble dietary fiber (1.3 g), and insoluble dietary fiber (5.3 g) although the stems have a higher carbohydrate content (4.1 g). Finally, broccoli leaves stand out for their content of carbohydrates (9.0 g), proteins (2.5 g), total dietary fiber (10.4 g) and insoluble dietary fiber (7.9 g). According to the nutritional value obtained, these surpluses are a food option in the daily diet of people and their valorization should be contemplated.

Maximization of Sulforaphane Content in Broccoli Sprouts by Blanching.

Broccoli sprouts are a recognized source of health-promoting compounds, such as glucosinolates, glucoraphanin, and sulforaphane (SFN). Maximization of SFN content can be achieved by technological processing. We investigated the effect of blanching conditions to determine the optimal treatment that maximizes sulforaphane content in broccoli sprouts. Broccoli seeds (cv. Traditional) grown under controlled conditions were harvested after 11 days from germination and subjected to different blanching conditions based on a central composite design with temperature and time as experimental factors. Results were analyzed by ANOVA followed by a Tukey test. The optimum conditions were identified through response surface methodology. Blanching increased sulforaphane content compared with untreated sprouts, agreeing with a decrease in total glucosinolates and glucoraphanin content. Temperature significantly affected SFN content. Higher temperatures and shorter immersion times favor glucoraphanin hydrolysis, thus increasing SFN content. The optimum conditions were blanching at 61 °C for 4.8 min, resulting in 54.3 ± 0.20 µmol SFN/g dry weight, representing a 3.3-fold increase with respect to untreated sprouts. This is the highest SFN content reported for sprouts subjected to any treatment so far. The process described in this work may contribute to developing functional foods and nutraceuticals that provide sulforaphane as an active principle.

Broccoli Myrosinase cDNA Expression in Escherichia coli and Saccharomyces cerevisiae.

Myrosinases (EC 3.2.1.147) are enzymes known for the generation of hydrolysis products that have a potential beneficial effect on human health. Their reaction mechanisms are widely studied, in order to improve and optimize secondary metabolite production processes. In this work, kinetic and biochemical properties of the broccoli myrosinase enzyme produced from its cDNA cloned in Escherichia coli and Saccharomyces cerevisiae were investigated. The results revealed that the thermal stability of the enzyme produced in S. cerevisiae was slightly higher (30 to 60 °C) than that of myrosinase produced in E. coli (20 to 50 °C). The effect of pH on the enzymatic activity was similar in both enzymes, with pH 3 being the optimum value under the reaction conditions used. The kinetic behavior of both enzymes was adjusted to the Michaelis-Menten model. The catalytic efficiency was up to 4 times higher in myrosinase produced in S. cerevisiae, compared to myrosinase produced in E. coli. The glycosylations present in the enzyme would be related to the formation of a dimeric quaternary structure and would not play an essential role in enzymatic activity, since both enzymes were biologically active. These results will probably allow the development of strategies for the production of bioactive metabolites of medical interest.

Contribution of the carbonized rice husk added to the substrate in the production of vegetable seedlings

The carbonized rice husk (CRH) is an agro-industrial residue with great potential for use in mixtures with other substrates for the production of vegetable seedlings. Thus, the purpose of this study was to evaluate the effect of the addition of CRH to the commercial substrate for the production of greenhouse seedlings. The evaluated vegetables were lettuce, broccoli and mustard, cultivated in polystyrene foam (Styrofoam®) trays. Five treatments were evaluated: 100% of the commercial substrate (CS); 25% of CRH + 75% of CS; 50% of CRH + 50% of CS; 75% CRH + 25% CS; and 100% CRH, in a completely randomized design with twelve replicates. The substrates were evaluated for their physical and chemical characteristics. The morphometric evaluations were: number of leaves per plant, plant height, shoot fresh matter yield, root length, and shoot and root dry matter yield. The data were submitted to regression analysis, and the homogeneity of the variance between the experiments was confirmed by the Cochran test. With the addition of CRH to the substrate, there was an increase in total porosity and P content and a reduction in density, water holding capacity, cation exchange capacity, electrical conductivity, and N, K, S, Ca and Mg contents. The carbonized rice husk added to the commercial substrate is a viable alternative for the production of lettuce, broccoli and mustard seedlings. The quality of the vegetable seedlings was higher when there was a 25% CRH + 75% CS mixture.

Plasmodiophora brassicae in Mexico: From Anecdote to Fact.

For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been stated as a fact. However, an intensive search of the scientific literature in English and Spanish, as well as gray literature including theses and government reports, did not reveal any information about the actual detection of the pathogen, affected hosts, or areas with clubroot presence, or any information about clubroot (hernia de la col in Mexico). We followed a multistep process to confirm whether P. brassicae was indeed in Mexico. First, we identified agricultural communities with a history of cruciferous crop cultivation. Second, we asked growers if they had seen clubroot on their crops, using pictures of the characteristic root galls. Third, we collected soil from the locations where clubroot was reported and looked for clubroot/P. brassicae in the soil using several cruciferous bait plants. For the first time we confirm the presence of the clubroot pathogen P. brassicae in Mexico, through a bioassay, the presence of resting spores, and a P. brassicae-specific PCR assay. The identification of P. brassicae in Mexico will contribute to our understanding of the genetic diversity of this elusive and devastating plant pathogen in future studies.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phenylalanine ammonia lyase is more relevant than Chalcone synthase and Chalcone isomerase in the biosynthesis of flavonoids during postharvest senescence of broccoli.

Broccoli contains a high content of nutraceutical compounds, such as glucosinolates and flavonoids. In this work, the effect of different treatments that modulate postharvest senescence of broccoli was evaluated and flavonoid metabolism during postharvest storage was analyzed at 20°C. A decrease in hue angle (HUE°) and chlorophylls and an increase in flavonoid content were detected during senescence. It observed that most of the treatments that delayed senescence also decreased flavonoid content, except visible light and UV-C treatments. In all cases, a direct correlation between those treatments that increased flavonoid biosynthesis and BoPAL gene expression was detected. This response was not detected in the expression of the other two flavonoid synthesis relevant genes BoCHS and BoCHI, suggesting that BoPAL has a greater influence on the regulation of the via, during broccoli senescence. PRACTICAL APPLICATIONS: Broccoli is a vegetable with valuable nutritional properties. Because it is in full development at the time of harvest, it has a short shelf life. In this work, it is showed that visible light and UV-C treatments not only delayed the senescence of broccoli, but also increased flavonoid content. Our results suggest that the most important enzyme in the phenylpropanoid biosynthesis pathway during broccoli postharvest is phenylalanine ammonia lyase, and that this may be a key point in regulating the biosynthesis of these nutritionally valuable compounds.

Fertilization recommendation for Brassica oleraceausing Bulletin 100 is still valid after more than two decades / Recomendação de fertilização para Brassica oleraceausando o Boletim 100 ainda é válida após mais de duas décadas

The 4R Nutrient Stewardship is a concept that aims to optimizefertilization of agricultural crops, encompassing social, economic and environmental aspects. This concept involves 4 scientific principles (the right source of fertilizer, the right rate, the right timing and the right place) that should be defined basedon local conditions and knowledge. This studyaimed at determining best fertilization practices for broccoli in the region of Tatuí-SPin Brazil, using the 4Rnutrient stewardship principles. Four experiments were installed, each one referring to a 4R principle, comparing the standard fertilization applied in the region with different management options. The recommended rate bythefertilization bulletin outperformed other rates we tested.An increase or decrease by 25% in the bulletin recommended rate ledto a reduction in broccoli productivity. Mineral fertilizer sourcepromoted highest growth, followed by a combination of mineral and organic. In regard to timing, the standard practice applied in the region, which is applying 100% ofN and K at planting, did not differ from splitting the dose in 3 parcels. Lastly, applying the fertilizer in the planting rowpromotedbetter growth than broadcasting on the soil surface. The standard fertilization practiced in the region promoted the highest growth, although there are other possibilities in terms of timing and placement that also resulted in similargrowth, and the decision should be made according to the reality of each farmer.(AU)

O Manejo de Nutrientes 4C é um conceito que visa otimizar a fertilização de culturas agrícolas, englobando aspectos sociais, econômicos e ambientais. Este conceito envolve 4 princípios científicos (a fonte certa de fertilizante, a dose certa, a época certa e o local certo) que devem ser definidos levando em conta condições e conhecimentos locais. Este estudo teve como objetivo determinar as melhores práticas de fertilização para brócolis na região de Tatuí-SPno Brasil, utilizando osprincípiosdomanejo de nutrientes 4C. Foram instalados quatro experimentos, cada um referindo-se a um princípio 4C, comparando a fertilização padrão aplicada na região com diferentesopções de manejo.A dose recomendada pelo boletim de fertilização superou as outras doses testadas.Um aumento ou diminuição de 25% na dose causou uma redução na produtividade dos brócolis. A fonte que promoveu o maior crescimento foi a mineral, seguida por uma combinação de mineral e orgânico. Em relação à época, a prática padrão aplicada na região, que é aplicar 100% de N e K no plantio, não diferiu da divisão da dose em 3 parcelas. Por fim, a aplicação do fertilizante na linha de plantio promoveu melhorcrescimento do que a aplicação na superfície do solo. A fertilização padrão praticada na região promoveu o maior crescimento, emboraexistam outras possibilidades em termos de época e local que também resultaram em crescimentosemelhante, e a decisão deveser tomada de acordo com a realidade de cada agricultor.(AU)

Optimization of an Extraction Process to Obtain a Food-Grade Sulforaphane-Rich Extract from Broccoli (Brassica oleracea var. italica).

Sulforaphane (SFN) is a powerful health-promoting compound found in broccoli in the form of its inactive precursor, glucoraphanin (GFN). SFN formation occurs through the enzymatic hydrolysis of glucoraphanin by myrosinase under specific chemical conditions. Its incorporation in food formulations has been hindered by the thermal instability of SFN and low concentration in Brassicaceae. Then, extracting SFN from broccoli at a temperature below 40 °C appears as an option to recover and stabilize SFN, aiming at delivering it as a nutraceutical. We studied an eco-friendly extraction process to obtain an SFN-rich extract from broccoli. The effect of the broccoli mass/solvent ratio, ethanol concentration in the extractant solution, and extraction time on the recovery of SFN, GFN, phenolic compounds, and antioxidant activity were studied through a Box-Behnken design. The regression models explained more than 70% of the variability in the responses, adequately representing the system. The experimental factors differently affected the bioactive compound recovery and antioxidant activity of the extracts. The extraction conditions that allowed the highest recovery of bioactive compounds and antioxidant activity were identified and experimentally validated. The results may provide the basis for the design of a process to produce a sulforaphane-rich food supplement or nutraceutical by using a GRAS extractant.

Physicochemical Properties and Antioxidant Activity of Spray-Dry Broccoli (Brassica oleracea var Italica) Stalk and Floret Juice Powders.

This research presents the microencapsulation and conservation of antioxidants of broccoli juice processed by spray drying, and proposes the use of a by-product as a technological application. Broccoli juice (BJ) extracted from two sources, stalks and florets, was spray-dried employing maltodextrin (MX) as a carrier agent at concentrations of 5, 7.5, and 10%, and inlet temperatures of 150 and 220 °C. The total phenolic content (TPC), and antioxidant activity (AA) of the BJ-MX powders were determined together with the physicochemical characteristics, including particle morphology, microstructure, and thermal properties. Based on the TPC and AA, the optimal processing conditions found were 5% of MX and a drying temperature of 220 °C. However, the florets showed higher TPC, while stalks presented higher AA under those processing conditions. The particles exhibited micrometric sizes and a mixture of spherical-shape particles and pseudo-spherical particles. The diffractograms indicated an amorphous microstructure in all samples. The glass transition temperature (Tg) was determined in the range of 50 °C for the samples dried at 150 °C and 55 °C for those dried at 220 °C. This suggested that powders might be stored at temperatures below the Tg without presenting any loss of antioxidants.

Expression of BoNOL and BoHCAR genes during postharvest senescence of broccoli heads.

BACKGROUND: Chlorophyll is the most abundant pigment on Earth, essential for the capture of light energy during photosynthesis. During senescence, chlorophyll degradation is highly regulated in order to diminish toxicity of the free chlorophyll molecule due to its photoactivity. The first step in the chlorophyll degradation pathway is the conversion of chlorophyll b to chlorophyll a by means of two consecutive reactions catalyzed by enzymes coded by NYC1 (NON-YELLOW COLORING 1), NOL (NYC1-LIKE) and HCAR. RESULTS: In this work, we studied the expression of NOL and HCAR genes during postharvest senescence of broccoli. We found that the expression of BoNOL increase during the first days of storage and then decrease. In the case of BoHCAR, its expression is maintained during the first days and then it also diminishes. Additionally, the effect of different postharvest treatments on the expression of these genes was also analyzed. It was observed that the expression of BoNOL is lower in the treatments performed with 1-methylcyclopropene (1-MCP), 6-benzylaminopurine (6-BAP) and modified atmospheres, while BoHCAR expression showed an increase in these same treatments, and a decrease in the treatment with ethylene. There were no variations in the expression of both genes in heat treatment, UV-C treatment and visible light treatment. CONCLUSIONS: These results suggest that both BoHCAR and BoNOL show a lower regulation of their expression than other genes involved in chlorophyll degradation during senescence. © 2020 Society of Chemical Industry.

Evaluation of bacillus megaterium strain AB4 as a potential biocontrol agent of alternaria japonica, a mycopathogen of Brassica oleracea var. italica.

Alternaria japonica causes annual losses of up to 25 % of the world broccoli crops, for this reason this research focused on the development of biopreparations containing Bacillus megaterium to prevent the outbreak of this disease caused by Alternaria japonica in the crop of Brassica oleracea var. italica. During the laboratory phase two types of biopreparations were evaluated, the first biopreparation was obtained by liquid fermentation composed of 40 g.L-1 of fava bean flour and 5 g.L-1 of ground brown sugar. This showed a maximum cell growth of 3.8 × 108 CFU. mL-1; while the second biopreparation was obtained by solid fermentation composed of wheat bran and it achieved a maximum cell growth of 4.7 × 109 CFU. g-1. In the fieldwork phase the aforementioned biopreparations were applied in an open-field crop. At the end of the cultivation period, the degree of the disease in leaves and in the inflorescences was measured and through the statistical analysis, a significant difference was evidenced (α = 0.05). On the broccoli leaves the disease index values ​​do not exceed 15.56 % and the disease index for postharvest florets was around 38 %. The evaluated variables showed a statistical similarity with the chemical treatment, thus determining the effective effect of the biopreparations.

Class I defensins (BraDef) from broccoli (Brassica oleracea var. italica) seeds and their antimicrobial activity.

The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.

Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones.

Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets.

Effect of broccoli residues fermented with probiotics on the growth performance and health status of broilers challenged with Clostridium perfringens

This study aimed at investigating the effects of broccoli residues fermented with probiotics (BF) on the growth performance, immunity, and gut health in broilers challenged with Clostridium perfringens (C. perfringens). A total of 600 broilers (one day old) were randomly allotted into five treatments with six replicates of 20 birds each and were reared until 42 days of age. The treatments included a positive control (PC, fed a basal diet and reared on uncontaminated litter), a negative control (NC, birds reared on litter contaminated with C. perfringens and fed a basal diet), and NC plus BF at 25, 50 or 75 g/kg of diet. The BF contained yeast 3.1 × 10 (7) cfu/g, lactic acid bacteria 9.5 × 10 (6) cfu/g and Bacillus subtilis 3.5 × 10 (6) cfu/g. Birds in the NC group showed lower (p<0.05) feed intake and body weight gain, whereas BF supplementation recovered (p<0.05) the growth performance to the levels of PC group. Dietary BF at 50and 75 g/kg reduced (p<0.05) broiler mortality. Similarly, compared to the NC group, BF increased (p<0.05) immune organ weights and serum immunoglobulins A, G, and M to the levels of PC group. The ileal populations of Escherichia coli and Gram-negative bacteria were decreased (p<0.05) by BF to the levels of PC, and C. perfringens was also decreased (p<0.05) by BF. The serum profiles of mono- and di-amine oxidase were decreased (p<0.05) by BF. BF at 75 g/kg reduced (p<0.05) monoamine oxidase compared with the other BF doses. The results suggest that broccoli residues fermented with probiotics can be a novel biological feed additive to protect the performance and health of broilers against C. perfringens infection.(AU)

Effect of broccoli residues fermented with probiotics on the growth performance and health status of broilers challenged with Clostridium perfringens

This study aimed at investigating the effects of broccoli residues fermented with probiotics (BF) on the growth performance, immunity, and gut health in broilers challenged with Clostridium perfringens (C. perfringens). A total of 600 broilers (one day old) were randomly allotted into five treatments with six replicates of 20 birds each and were reared until 42 days of age. The treatments included a positive control (PC, fed a basal diet and reared on uncontaminated litter), a negative control (NC, birds reared on litter contaminated with C. perfringens and fed a basal diet), and NC plus BF at 25, 50 or 75 g/kg of diet. The BF contained yeast 3.1 × 10 (7) cfu/g, lactic acid bacteria 9.5 × 10 (6) cfu/g and Bacillus subtilis 3.5 × 10 (6) cfu/g. Birds in the NC group showed lower (p<0.05) feed intake and body weight gain, whereas BF supplementation recovered (p<0.05) the growth performance to the levels of PC group. Dietary BF at 50and 75 g/kg reduced (p<0.05) broiler mortality. Similarly, compared to the NC group, BF increased (p<0.05) immune organ weights and serum immunoglobulins A, G, and M to the levels of PC group. The ileal populations of Escherichia coli and Gram-negative bacteria were decreased (p<0.05) by BF to the levels of PC, and C. perfringens was also decreased (p<0.05) by BF. The serum profiles of mono- and di-amine oxidase were decreased (p<0.05) by BF. BF at 75 g/kg reduced (p<0.05) monoamine oxidase compared with the other BF doses. The results suggest that broccoli residues fermented with probiotics can be a novel biological feed additive to protect the performance and health of broilers against C. perfringens infection.

Kinetic study of sulforaphane stability in blanched and un-blanched broccoli (Brassica oleracea var. italica) florets during storage at low temperatures.

Sulforaphane is a health-promoting compound found in broccoli. Given its high thermo-lability, its preservation through high-temperature processes seems inconvenient. Accordingly, storage at low temperature is an alternative. There are no studies about the evolution of sulforaphane content during storage at low temperatures. The change of sulforaphane content in blanched and un-blanched broccoli florets during storage at 10, - 1, - 21 and - 45 °C for 83 days was studied. In blanched broccoli, sulforaphane content followed a first-order degradation kinetics (R2 ≥ 0.95). A two-consecutive irreversible reactions model described adequately the evolution of sulforaphane content in un-blanched broccoli (R2 ≥ 0.94). Activation energies from Arrhenius equation resulted in 19.4 kJ/mol for blanched and 30 kJ/mol (formation) and 58 kJ/mol (degradation) for un-blanched broccoli. Storage of un-blanched broccoli at - 45 °C for 40 days maximized sulforaphane content. These results could be useful to propose broccoli storage conditions that preserve or maximize sulforaphane content.