In vitro evaluation of nanocomposites of linseed mucilage and k-carrageenan loaded with Achyrocline satureioides nanoemulsion: a gradual-release candidate of antimicrobials for the treatment of bovine mastitis.

This research paper presents the development and evaluation of pioneering nanocomposites (NCs) based on the combination of k-carrageenan and linseed mucilage. When loaded with macela extract nanoemulsion they present an innovative approach for the sustained release of antimicrobial herbal constituents, specifically tailored for bovine mastitis treatment. The NCs, encompassing various ratios of k-carrageenan and linseed mucilage polymers (8:2, 7:3, and 5:5 w/w) with 1.25 mg of macela extract/g of gel, underwent in vitro assessment, emphasizing viscosity, degradation speed, release of herbal actives from macela nanoemulsion and antimicrobial activity. The NCs exhibited thermoreversible characteristics, transitioning from liquid at 60°C to a gel at 25°C. NCs allowed a gradual release of phenolic compounds, reaching approximately 80% of total phenolics release (w/v) within 72 h. NCs inhibited the growth of MRSA (ATCC 33592) until 8 h of incubation. No toxic effect in vitro of NCs was found on MAC-T cells. Thus, the developed materials are relevant for the treatment of bovine mastitis, especially in the dry period, and the data support future evaluations in vivo.

Protection of bovine mammary epithelial cells by a nanoemulsion of the medicinal herb Achyrocline satureioides (Lam.) DC and its capacity of permeation through mammary epithelium.

The low levels of toxicity and cytoprotective effect attributed to Achyrocline satureioides (Lam.) DC, a medicinal plant native to South America, are of interest for bovine mastitis therapy. This research paper reports the hypothesis that a nanoemulsion of macela extract (Achyrocline satureioides) exerts protective effects on bovine mammary alveolar cells -T (MAC-T) and increases the permeation of flavonoid compounds through mammary epithelium. Extract-loaded nanoemulsions (2.5 mg/ml) (NE-ML) (n = 4) were prepared using high-pressure homogenization with varying concentrations of flaxseed oil and Tween 80. Permeation and retention of free and nanoencapsulated quercetin, 3-O-methylquercetin and luteolin were performed on mammary glandular epithelium using Franz diffusion cells. The cell viability was evaluated on mammary epithelial cells (MAC-T lineage) using the MTT method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) after exposure to loaded and blank nanoemulsions (NE-ML and NE-BL). Necrotic or apoptotic cell death was evaluated by flow cytometry after exposure to nanoemulsions (NE-ML and NE-BL). Subsequently, the cell death was assessed by previously treating MAC-T cells with NE-ML for 23 h, followed by exposure to H2O2 (2 mM) for 1 h. Higher permeation of quercetin and 3-O-methylquercetin in NE-ML was found compared to that of free extract with a final permeated amount of 50.7 ± 3.2 and 111.2 ± 0.6 µg/cm2 compared to 35.0 ± 0.6 and 48.9 ± 1.2, respectively. For NE-BL, the IC50 was at least 1.3% (v/v), while for the NE-ML, it was at least 2.6% (v/v). After exposure to NE-ML (5 and 1.2%, v/v), the percentage of apoptotic cells was reduced (±30%). For the H2O2 assay, the percentage of cells in necrosis was reduced by 40% after exposure to NE-ML1% (v/v) + H2O2 2 mM. The protective effects and increased permeation of macela nanoemulsion make this a promising new candidate for bovine mastitis therapy.

Cow milk enriched with nanoencapsulated phenolic extract of jaboticaba (Plinia peruviana).

This study evaluated the total phenolic content (TPC) and the antioxidant activity (AA) of cow's milk enriched with phenolic compounds extracted from jaboticaba peel, either by adding jaboticaba crude extract or a jaboticaba-loaded nanoemulsion. Three nanoemulsions with 5, 10 and 15% of jaboticaba extract were prepared. Average particle diameter (166.7-181.7 nm), polydispersity index (0.138-0.156) and zeta potential (ranging from - 35.30 to - 38.60 mV) were measured for the three different colloidal systems. The nanoemulsion with 15% of jaboticaba extract (J15-NE) was chosen for milk enrichment. J15-NE showed an encapsulation efficiency of 85.6% and remained stable for 60 days at 8 °C. Transmission electron microscopy of J15-NE displayed nanoparticles with a well-defined spherical shape. Reference milk, milk enriched with jaboticaba extract and milk enriched with J15-NE were characterised by a TPC of 93, 171 and 161 µg/ml GAE (gallic acid equivalent), respectively, and an AA of 0.04, 0.17 and 0.14 µg/ml TEAC (trolox equivalent antioxidant capacity), respectively. Thus, this study showed that nanoemulsion with jaboticaba peel extract could be exploited as an ingredient to enrich the properties of milk.

Metabolic Profiling and Classification of Propolis Samples from Southern Brazil: An NMR-Based Platform Coupled with Machine Learning.

The chemical composition of propolis is affected by environmental factors and harvest season, making it difficult to standardize its extracts for medicinal usage. By detecting a typical chemical profile associated with propolis from a specific production region or season, certain types of propolis may be used to obtain a specific pharmacological activity. In this study, propolis from three agroecological regions (plain, plateau, and highlands) from southern Brazil, collected over the four seasons of 2010, were investigated through a novel NMR-based metabolomics data analysis workflow. Chemometrics and machine learning algorithms (PLS-DA and RF), including methods to estimate variable importance in classification, were used in this study. The machine learning and feature selection methods permitted construction of models for propolis sample classification with high accuracy (>75%, reaching ∼90% in the best case), better discriminating samples regarding their collection seasons comparatively to the harvest regions. PLS-DA and RF allowed the identification of biomarkers for sample discrimination, expanding the set of discriminating features and adding relevant information for the identification of the class-determining metabolites. The NMR-based metabolomics analytical platform, coupled to bioinformatic tools, allowed characterization and classification of Brazilian propolis samples regarding the metabolite signature of important compounds, i.e., chemical fingerprint, harvest seasons, and production regions.

The effects of Brazilian propolis on etiological agents of mastitis and the viability of bovine mammary gland explants.

The objective of this study was to evaluate in vitro the antimicrobial activity of Brazilian propolis from Urupema, São Joaquim, and Agua Doce (Santa Catarina State) and green propolis from Minas Gerais State, and the effects of propolis on bovine mammary gland explant viability. The propolis samples differed in flavonoid content and antioxidant activity. Green propolis showed the highest content of flavonoids, followed by the sample from São Joaquim. The propolis from Urupema showed the lowest flavonoid content along with the lowest antioxidant activity. The total phenolics were similar across all studied samples. Despite phytochemical differences, the propolis samples from Minas Gerais, São Joaquim, and Urupema presented the same level of antimicrobial activity against Staphylococcus aureus strains. The reduction in S. aureus growth was, on average, 1.5 and 4 log10 times at 200 and 500 µg/mL, respectively. At concentrations of 1,000 µg/mL, all propolis reduced bacterial growth to zero. On the other hand, when the propolis were tested against strains of Escherichia coli, the samples presented weak antimicrobial activity. Mammary explants were maintained in culture for 96h without a loss in viability, demonstrating the applicability of the model in evaluating the toxicity of propolis. The origin and chemical composition of the propolis had an effect on mammary explant viability. We encountered inhibitory concentrations of 272.4, 171.8, 63.85, and 13.26 µg/mL for the propolis from Água Doce, Urupema, São Joaquim, and Mina Gerais, respectively. A clear association between greater antimicrobial activity and toxicity for mammary explants was observed. Of all propolis tested, the Urupema sample was noteworthy, as it showed antimicrobial activity at less toxic concentrations than the other samples, reducing bacterial growth to an average of 9.3 × 10(2) cfu/mL after 6h of contact using 200 µg/mL of extract. The results demonstrate the potential for Brazilian propolis in the treatment of mastitis, although effectiveness is dependent on geographical origin and concentration. The results from the mammary gland explant assays are promising for the investigation of other natural products with antimicrobial and anti-inflammatory properties that can be used in the intramammary treatment of subclinical mastitis and during dry cow therapy.

Phenolic content and ferric reducing-antioxidant power of cow's milk produced in different pasture-based production systems in southern Brazil.

BACKGROUND: Phenolic compounds are widely present in forage. However, few studies have been carried out to investigate the presence of these compounds in animal products such as milk. In this study, the total phenolic content (TPC) and ferric reducing-antioxidant power (FRAP) of milk produced under agroecological and conventional management systems in southern Brazil were determined. Three systems were compared: (a) agroecological, with intensive rotational grazing, also known as Voisin grazing (ECO); (b) semi-intensive conventional (SIC); and (c) conventional grazing (CGR) (n = 8 per group). Pastures with distinct and more diverse botanical composition were observed on the ECO farms. RESULTS: Significantly (P < 0.05) lower amounts of TPC were found in agroecologically produced milk in autumn and summer and the CGR system showed the highest FRAP value (P < 0.05) for the samples collected in autumn. Positive correlations where found between the TPC and FRAP values obtained for the milk samples (0.198, P < 0.05), milk FRAP and forage TPC values (0.344, P < 0.05), and the TPC and FRAP values obtained for the forage (0.70, p < 0.01). PCA applied to the UV spectra dataset (200-350 nm) clearly distinguished the samples collected from the ECO system in the winter. CONCLUSION: Our results revealed that, under the conditions prevalent in southern Brazil, since the cows were allowed to graze in all of the systems, the TPC and antioxidant capacity of the milk samples showed minimal variation. However, since the forage TPC and FRAP values for the milk were correlated, TPC appears to be a promising variable for the purpose of monitoring forage prior to its selection aimed at enhancing the antioxidant activity of milk.

Carotenoid and anthocyanin contents of grains of Brazilian maize landraces.

BACKGROUND: Carotenoid and anthocyanin contents of 26 maize landraces cultivated in southern Brazil were determined to evaluate their potential as natural colorants or functional food ingredients. RESULTS: The major carotenoids detected in the whole grain flour were zeaxanthin and lutein. Anthocyanins of landraces with purple starchy endosperm (Lingua de Papagaio and Mato Grosso Palha Roxa) were more extractable in methanol-HCl (1%, v/v), exhibiting 2.45 and 0.94 g kg(-1) of whole grains flour, respectively. In contrast, butanol-HCl (30%, v/v) was more effective for the extraction of anthocyanins from the purple-colored landraces Roxo 29 and Roxo 41; genotypes with pigments localized in the outer parts (pericarp) of grains (2.60 and 2.19 g kg(-1)). The Roxo 41 landrace showed the highest concentration of pigments, e.g. 11.72 10(-3) g kg(-1) of total carotenoids and 2.16 g kg(-1) of total anthocyanins. Similarly, the yellow-colored MPA 1 and the purple-colored Roxo 29 landraces showed prominent amounts of carotenoids (10.86 10(-3) g kg(-1)) and anthocyanins (2.60 g kg(-1)), respectively. CONCLUSION: Our findings suggest that the colored grains of maize landraces studied may hold promise for the development of grain-based functional foods or natural colorants regarding their carotenoid and anthocyanin contents and as genetic resource in breeding programs.

Exploratory and discriminant analysis of plant phenolic profiles obtained by UV-vis scanning spectroscopy.

Some species of cover crops produce phenolic compounds with allelopathic potential. The use of math, statistical and computational tools to analyze data obtained with spectrophotometry can assist in the chemical profile discrimination to choose which species and cultivation are the best for weed management purposes. The aim of this study was to perform exploratory and discriminant analysis using R package specmine on the phenolic profile of Secale cereale L., Avena strigosa L. and Raphanus sativus L. shoots obtained by UV-vis scanning spectrophotometry. Plants were collected at 60, 80 and 100 days after sowing and at 15 and 30 days after rolling in experiment in Brazil. Exploratory and discriminant analysis, namely principal component analysis, hierarchical clustering analysis, t-test, fold-change, analysis of variance and supervised machine learning analysis were performed. Results showed a stronger tendency to cluster phenolic profiles according to plant species rather than crop management system, period of sampling or plant phenologic stage. PCA analysis showed a strong distinction of S. cereale L. and A. strigosa L. 30 days after rolling. Due to the fast analysis and friendly use, the R package specmine can be recommended as a supporting tool to exploratory and discriminatory analysis of multivariate data.

Identification and antimicrobial susceptibility of milk pathogen isolated from dairy production systems.

Livestock has been recognized as a reservoir of antibiotic-resistant bacteria. Prevalence of resistance has been associated with herd size and intensification of animal production systems. Brazil is one of the emergent hotspots of bacterial resistance, which is also associated with animal husbandry. This study aimed to evaluate the resistance profile of pathogens that cause subclinical mastitis and the relationship between resistance status at farm level and different production systems. Milk samples from cows diagnosed with subclinical mastitis were collected from farms that adopt different husbandry systems with different production intensities, i.e., agroecological, low input, high input, Free-Stall and Compost-bedded pack barn. Etiological agents were isolated and microbiologically identified, and antibiotic susceptibility testing was conducted, using the disk diffusion method. The main isolated agents were Streptococcus spp. (n = 54, 30.5 %) and coagulase-positive Staphylococcus (CPS) (n = 54; 30.5 %). The recovered isolates displayed high antibiotic resistance against Sulfamethazine (80.2 %), Gentamicin (29.37 %), Penicillin (29.37 %), Oxacillin (28.82 %) and Ampicillin (26 %). Multidrug resistance was found for all agents and in all farming systems (39.54 %). Neither production systems (p = 0.26) nor farming systems (p = 0.24) significantly affected the resistance rates of samples. Therefore, intensive production systems may not be a root cause of increased rates of antimicrobial resistance in the milk production chain, suggesting that other environmental factors should be investigated. It is noteworthy that high levels of multidrug resistance were even found in bacteria earlier considered as minor pathogens. This development can be taken as a warning that environmental bacteria are potential transmitters of resistance genes to the environment.

Nanotechnology: A Promising Tool Towards Wound Healing.

Nanotechnology is an exciting emerging field with multiple applications in skin regeneration. Nanofibers have gained special attention in skin regeneration based on their structural similarity to the extracellular matrix. A wide variety of polymeric nanofibers with distinct properties have been developed and tested as scaffolds for skin regeneration. Besides providing support for tissue repair, nanofibrous materials can act as delivery systems for drugs, proteins, growth factors, and other molecules. Moreover, the morphology, biodegradability, and other functionalities of nanofibrous materials can be controlled towards specific conditions of wound healing. Other nanostructured drug delivery systems, such as nanoparticles, micelles, nanoemulsions, and liposomes, have been used to improve wound healing at different stages. These nanoscale delivery systems have demonstrated several benefits for the wound healing process, including reduced cytotoxicity of drugs, administration of poorly water-soluble drugs, improved skin penetration, controlled release properties, antimicrobial activity, and protection of drugs against light, temperature, enzymes or pH degradation, as well as stimulation of fibroblast proliferation and reduced inflammation.

Farm Management in Organic and Conventional Dairy Production Systems Based on Pasture in Southern Brazil and Its Consequences on Production and Milk Quality.

Pasture-based dairy production is used widely on family dairy farms in Southern Brazil. This study investigates conventional high input (C-HI), conventional low input (C-LI), and organic low input (O-LI) pasture-based systems and their effects on quantity and quality of the milk produced. We conducted technical site visits and interviews monthly over one year on 24 family farms (n = 8 per type). C-HI farms had the greatest total area (28.9 ha), greatest percentage of area with annual pasture (38.7%), largest number of lactating animals (26.2) and greatest milk yield per cow (22.8 kg·day(-1)). O-LI farms had the largest perennial pasture area (52.3%), with the greatest botanical richness during all seasons. Area of perennial pasture was positively correlated with number of species consumed by the animals (R² = 0.74). Milk from O-LI farms had higher levels of fat and total solids only during the winter. Hygienic and microbiological quality of the milk was poor for all farms and need to be improved. C-HI farms had high milk yield related to high input, C-LI had intermediate characteristics and O-LI utilized a year round perennial pasture as a strategy to diminish the use of supplements in animal diets, which is an important aspect in ensuring production sustainability.

Seasonal variation of propolis from southern Brazil: phytochemical screening, antimicrobial activity, and effects on bovine mammary epithelial cells / Variação sazonal da própolis do Sul do Brasil: screening fitoquímico, atividade antimicrobiana e efeitos em células epiteliais mamária bovina

Previous studies have reported on the phytotherapeutic potential of propolis from southern Brazil (Urupema, Santa Catarina State), in particular, its efficacy in the treatment of bovine mastitis. The present study evaluates the effect of seasonal variation on the chemical composition of propolis from southern Brazil and its resultant antimicrobial and cytotoxic activities in the context of treating bovine mastitis. Antimicrobial activity against Staphylococcus aureus was assessed, along with the cytotoxicity and induction of apoptosis in MAC-T bovine mammary epithelial cells. With the exception of spring propolis, the flavonoid quercetin was the main compound present in all samples. The minimum inhibitory concentration (MIC) of propolis against S. aureus from mastitic milk was 140 µg/mL for samples collected during the spring, autumn, and winter seasons, but 280 µg/mL for samples collected in summer. For MAC-T cells, the spring propolis extract was more toxic with an IC50 of 120 µg/mL. However, for 120 µg/mL of spring propolis extract, only 0.77% of necrotic and 37% of apoptotic MAC-T cells were found, respectively. Nonetheless, the induction of cell death through apoptosis by propolis extract suggests less severe damage to bovine mammary glands. Moreover, only negligible seasonal variation was found in the chemical composition of propolis from southern Brazil, and no biological activities were determined to be harmful. Therefore, this propolis shows promise as an alternative to commercial antimicrobials in the control of bovine mastitis, offering support for organic milk production.(AU)

Estudos prévios tem demonstrado o potencial terapêutico da propolis do Sul do Brasil (Urupema, Santa Catarina), em particular no tratamento da mastite bovina. O presente estudo tem como objetivo avaliar o efeito da variação sazonal sobre a composição química da própolis de Urupema do Sul do Brasil e suas atividades antimicrobiana e citotóxica visando o tratamento da mastite bovina. A atividade antimicrobiana contra Staphylococcus aureus foi avaliada, juntamente com a citotoxicidade e indução de apoptose em células epiteliais mamárias bovina da linhagem MAC-T. Com exceção da própolis da primavera, o flavonóide quercetina foi o composto majoritário em todas as amostras. A concentração inibitória mínima (CIM) da propolis contra S. aureus de leite mastítico foi 140 µg/mL para as amostras de primavera, outono e inverno e 280 µg/mL para a amostra coletada no verão. Para as células MAC-T, o extrato de própolis da primavera foi o mais tóxico, sendo a IC50 120 µg/mL. Entretanto, com 120 µg/mL do extrato de própolis primaveril, somente 0,77% de células MAC-T necróticas e 37% apoptóticas foram encontradas. Portanto, a indução da morte celular por apoptose pelo extrato de própolis sugere danos possivelmente menos graves a glândula mamária bovina. Além disso, somente uma pequena variação sazonal foi encontrada para a composição química da propolis do Sul do Brasil, a qual não prejudicou suas atividades biológicas. Portanto, esta própolis mostra como uma alternativa promissora ao uso de antimicrobianos comerciais no controle da mastite bovina e uma opção para subsidiar a produção orgânica de leite.Palavras-chave: Células MAC-T. Mastite bovina. Propolis brasileira. Staphylococcus aureus.(AU)

Productive and Economic Responses in Grazing Dairy Cows to Grain Supplementation on Family Farms in the South of Brazil.

Pasture-based dairy production has been a major source of income for most family farms in the south of Brazil. Increasing milk prices have spurred an increase in grain supplementation, which has been poorly implemented, resulting in low levels of efficiency. To evaluate the consequences of supplementation on milk production and composition, grazing behavior and economic return, the widely used grain management system (CC-commercial concentrate, containing 21% CP, offered at 1 kg per 3.7 L of milk) was compared with an energy supplement (GC-ground corn, with 9.5% CP, offered at 0.4% of live weight). Ten Holstein cows were paired into two groups, and subjected to the two treatments in a crossover design. The cows remained in the same grazing group, and the grain supplement was offered individually at milking time and consumed completely. Each experimental period lasted 14 days, with 10 days for diet adaptation and four days for data collection; individual milk production and samples were collected to determine levels of fat, protein, lactose, carotenoids, vitamin A and N-urea. Grazing behavior was observed (scans every 5 min) in the first 4 h after the morning milking, and chemical composition of hand plucked samples of forage were measured. The cost of the supplement and profitability per treatment were calculated. Cows supplemented with GC consumed herbage with higher crude protein (CP: 16.23 vs. 14.62%; p < 0.05), had higher biting rate (44.21 vs. 39.54 bites/min; p < 0.03) and grazing time (22.20 vs. 20.55 scans; p < 0.05) than when receiving CC. There were no differences in milk composition between treatments (p > 0.05). However, higher concentrations of ß-carotene and total carotenoids were detected in the milk of cows at 70-164 days of lactation, compared to <70 days of lactation (p < 0.05). Milk production was higher (13.19 vs. 11.59 kg/day; p < 0.05) when cows consumed CC, but resulted in lower profitability compared to GC (US$ 4.39 vs. US$ 4.83/cow per day). Our results show that higher productivity does not necessarily improve profitability. Cows receiving supplement with lower levels of protein were able to adjust their grazing behavior to meet their protein needs and this level of diet modification did not alter milk composition.

Metabolic fingerprint of Brazilian maize landraces silk (stigma/styles) using NMR spectroscopy and chemometric methods.

Aqueous extract from maize silks is used by traditional medicine for the treatment of several ailments, mainly related to the urinary system. This work focuses on the application of NMR spectroscopy and chemometric analysis for the determination of metabolic fingerprint and pattern recognition of silk extracts from seven maize landraces cultivated in southern Brazil. Principal component analysis (PCA) of the (1)H NMR data set showed clear discrimination among the maize varieties by PC1 and PC2, pointing out three distinct metabolic profiles. Target compounds analysis showed significant differences (p < 0.05) in the contents of protocatechuic acid, gallic acid, t-cinnamic acid, and anthocyanins, corroborating the discrimination of the genotypes in this study as revealed by PCA analysis. Thus the combination of (1)H NMR and PCA is a useful tool for the discrimination of maize silks in respect to their chemical composition, including rapid authentication of the raw material of current pharmacological interest.