Profile of lactic acid bacteria (MALDI-TOF-MS) and physico-chemical and microbiological characteristics of the raw milk and fresh artisanal cheese from Serra Geral, Minas Gerais, Brazil.

Artisanal cheese from Serra Geral, Minas Gerais, Brazil, stands out for its cultural asset and socio-economic relevance. However, standards of identity and quality and the peculiar terroir associated with the edaphoclimatic conditions have not been established. Therefore, the production flow diagram and the physico-chemical and microbiological quality of the raw milk, pingo (natural starter culture), production benches, water and fresh cheese were investigated for the first time. In addition, lactic acid bacteria (LAB) from cheese and its production environment were identified by MALDI-TOF. For that, 12 cheese making facilities were selected. The raw milk and pingo showed adequate physico-chemical characteristics for cheesemaking; however, high microbial counts were found. In the water, total and thermotolerant coliforms were also identified. The fresh cheeses were classified as 'high moisture and fat' and 'soft mass'. Most physico-chemical parameters were satisfactory; however, there were high counts of total coliforms, Staphylococcus spp. and coagulase-positive staphylococci. There were high counts of LAB in the raw milk, pingo, bench surface and fresh cheese. A total of 84 microbial biotypes from MRS agar were isolated. Lactococcus lactis was the predominant LAB, followed by Lactococcus garvieae. Leuconostoc mesenteroides (benches), Leuconostoc pseudomesenteroides (fresh cheese), and Enterococcus faecium (pingo) were identified sporadically. These results indicate the risks to public health associated with the consumption of the fresh cheese, and measures to improve its safety are needed.

Bioaccessibility of bioactive amines in dark chocolates made with different proportions of under-fermented and fermented cocoa beans.

The influence of under-fermented (UF) cocoa (0 to 65 %) on bioactive amines in chocolate and their in vitro bioaccessibility was investigated. The same amines were found in all treatments; however, treatments were divided into two groups regarding total amines [0 & 20 % UF (34 mg/kg) and 35 to 65 % UF (17 mg/kg)] and phenolic levels [lower and higher, respectively]. Serotonin, tyramine, putrescine, cadaverine, agmatine and phenylethylamine were higher in chocolate with ≤ 20 % UF cocoa. Histamine and spermidine were not affected. Digestibility studies indicated that low levels of amines were present in the oral phase. Gastric digestion was effective in releasing tyramine, spermidine and phenylethylamine from conjugates. Serotonin and agmatine were not detected after in vitro digestion of chocolate with ≥ 35 % UF cocoa. Histamine was released during in vitro intestinal digestion. By adding different proportions of UF cocoa during chocolate production, the levels and bioaccessibility of amines can be modulated.

Boron nitride nanotubes for extraction of angiotensin receptor blockers from human plasma.

The limitations of silica-based sorbents boosted the development of new extraction phases. In this study, boron nitride nanotubes functionalized with octadecyl groups were used for the first time as sorbent for extraction of losartan and valsartan, the most used angiotensin receptor blockers in the clinical practice, from human plasma. The nanotubes were synthesized using the chemical vapor deposition technique, purified by acid treatment, functionalized with octadecylamine in a microwave reactor, and characterized by different techniques. The functionalized nanotubes were packed in solid phase extraction cartridges. Extraction conditions were optimized by means of a 23 factorial design with center points. The separation was performed on a biphenyl core-shell (100 × 4.6 mm; 2.6 µm) column, using 0.1 % (v/v) triethylamine solution and methanol (pH 3.2) as mobile phase, at 0.7 mL/min, in gradient elution. The injection volume was 10 µL and fluorescence detection was performed at excitation and emission wavelengths of 250 and 375 nm, respectively. The developed method was validated according to Brazilian Health Regulatory Agency (ANVISA), United States Food and Drug Administration (US FDA) and European Medicines Agency (EMA) guidelines and presented selectivity, precision, accuracy, and linearity in the concentration ranges of 50-1200 ng/mL for losartan and 20-1700 ng/mL for valsartan. Recoveries higher than 80 % were obtained. The method was fit for the quantification of losartan in plasma samples from patients under antihypertensive therapy, being useful in therapeutic drug monitoring, pharmacokinetics and bioequivalence studies.

Vortex-assisted liquid-liquid microextraction combined with liquid chromatography tandem mass spectrometry for simultaneous determination of cardiovascular drugs in human plasma.

Hypertension and dyslipidemias are among the main risk factors for the development of cardiovascular diseases, which are responsible for the death of approximately 17 million people each year. There are several drugs available for the treatment of these diseases. Therefore, methods for the simultaneous analysis of several of these drugs are useful in a wide range of situations. In this context, this study aimed to develop a modern method for the simultaneous determination of eight cardiovascular drugs in human plasma. A vortex-assisted liquid-liquid microextraction (VALLME) procedure, combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Mass spectrometry conditions, chromatographic separation, and sample preparation were optimized. For VALLME optimization, pH, sodium chloride concentration, volume of buffer solution, extraction solvent (type and volume), and vortex stirring time were evaluated. The method proved to be simple, fast, and environmentally friendly since low volumes of organic solvent were employed. Furthermore, the VALLME procedure required small sample volume, which is desirable when large volumes are scarce. Suitable recoveries and lower limits of quantification were achieved with a chromatographic run of only 8 min. The method was validated, showing to be selective, precise, and accurate. Furthermore, the analytical curves were well fitted to the selected models and the matrix effect did not affect method reliability. The developed method was successfully applied for the analysis of plasma samples obtained from volunteers attending a hospital service.

FTIR and PLS-regression in the evaluation of bioactive amines, total phenolic compounds and antioxidant potential of dark chocolates.

Cloning techniques are used to improve agronomical traits and answer to the demand for fine chocolate. The objective of this study was to predict the concentrations of bioactive amines, phenolic compounds, and the antioxidant potential of dark monoclonal chocolate from nine fine cocoa varieties by FTIR analysis and conventional techniques. Total phenolic compounds, bioactive amines and antioxidant activity varied significantly among chocolates. The antioxidant activity was also affected by the analytical method (DPPH vs. Rancimat). Chemometric models based on FTIR data provided satisfactory predictions of the concentrations of the amines: spermidine (R2 = 0.92; RMSEP = 0.39; RMSEC = 0.21), tryptamine (R2 = 0.92; RMSEP = 0.41; RMSEC = 0.20), cadaverine (R2 = 0.82; RMSEP = 1.58; RMSEC = 0.75) and tyramine (R2 = 0.87; RMSEP = 1.87; RMSEC = 0.68); as well as phenolic compounds and antioxidant activity by Rancimat® (R2 = 0.98; RMSEP = 0.32; RMSEC = 0.21) and DPPH (R2 = 0.97; RMSEP = 4.05; RMSEC = 1.66). The wavenumbers of amines vibrations are among those that most affected antioxidant prediction models, confirming the contribution of amines to the antioxidant activity of chocolates.

In vitro bioaccessibility of amino acids and bioactive amines in 70% cocoa dark chocolate: What you eat and what you get.

Chocolate is an important source of free bioactive amines and amino acids which play important roles in human health. Considering the limited information on the bioaccessibility of these compounds from chocolate, the objective of this study was to characterize their profiles and bioaccessibility in 70% cocoa dark chocolate through in vitro simulation of oral, gastric and intestinal digestions. Seven amines were detected; polyamines were predominant before in vitro digestion, whereas tyramine, cadaverine and spermidine after digestion. All amines showed high bioaccessibility with slight influence of digestive enzymes. Amines increased after gastrointestinal digestion: tyramine (13-fold), tryptamine (9-fold), others (2.4-4.2-fold) and histamine appeared. All amino acids, GABA and ammonia were detected in chocolate, and their contents increased after in vitro digestion due to digestive enzymes (4.6, 2.8 and 2.1, respectively). Dark chocolate protein is a good source of tryptophan, phenylalanine + tyrosine, isoleucine, histidine, but limiting for lysine, leucine, and threonine.

Chemical implications and time reduction of on-farm cocoa fermentation by Saccharomyces cerevisiae and Pichia kudriavzevii.

The use of starters during fermentation has been gaining momentum as it can warrant high-quality chocolate. The objective of this study was to investigate the influence of Saccharomyces cerevisiae (Sc) and Pichia kudriavzevii (Pk) during on-farm fermentation on physico-chemical and microbiological characteristics and levels of methylxanthines and bioactive amines of cocoa. Four treatments were used: ScPk (1:1), only Sc, only Pk, and no starter (control). The starters lead to changes throughout fermentation, but provided fermented cocoa with similar pH, titratable acidity, reducing sugars and phenolic compounds. ScPk shortened fermentation time by 24 h. The ScPk fermented and dried cocoa had higher levels of monomeric phenols, methylxanthines, phenylethylamine and lower levels of the putrefactive amines - putrescine and cadaverine (p < 0.05). The results were confirmed by multivariate analysis. Based on these results, the mixture of both yeasts species is a promising starter for cocoa fermentation decreasing duration time and modulating high-quality components.

In vitro digestion of spermidine and amino acids in fresh and processed Agaricus bisporus mushroom.

Mushrooms are significant sources of amino acids and bioactive amines; however, their bioaccessibility can be affected by processing and during in vitro digestion. Fresh Agaricus bisporus mushroom was submitted to cooking and canning and samples were submitted to in vitro gastric and gastric-intestinal digestions. An UHPLC method was used for the simultaneous determination of 18 free amino acids, 10 biogenic amines and ammonia in the samples. Fresh mushroom contained 14 free amino acids, with predominance of alanine and glutamic acid; spermidine was the only amine detected; and ammonia was also detected. Spermidine levels were not affected by cooking, but there was a significant loss (14%) after canning. Spermidine levels were not affected by the in vitro gastric and intestinal digestion, suggesting full bioaccessibility. There was a significant decrease on total amino acids levels after cooking and canning, with higher losses for aspartic and glutamic acids in cooked and for aspartic acid and valine in canned mushrooms. After the in vitro gastric and intestinal digestions, the total levels of amino acids increased and two additional amino acids (arginine and methionine) were detected. During in vitro digestion many of the amino acids were released mainly in the intestinal phase. After in vitro digestion, amino acids per gram of protein of mushrooms are adequate for most FAO amino acid pattern for adults. Multivariate analysis confirmed that protein hydrolysis in processed mushrooms is higher in intestinal phase. Bioaccessibility data for spermidine in A. bisporus is a novelty and increase the value of this food.

Total mercury in commercial fishes and estimation of Brazilian dietary exposure to methylmercury.

BACKGROUND: Mercury, in particular its most toxic form methylmercury, poses a risk to public health. Dietary methylmercury exposure is mainly by fish, and it can vary with fish contamination and by dietary habits of the population. This study aimed to quantify total mercury levels in different fish from Brazil and to estimate Brazilian exposure to methylmercury by fish consumption. METHODS: Total mercury occurrence was investigated in 18 different fish species by atomic absorption spectrometry with thermal decomposition and gold amalgamation. Dietary exposure to methylmercury was estimated by a deterministic method for different groups considering consumption by sex, different Brazilian geographical regions and habitat (rural or urban). RESULTS: Carnivorous fish showed higher levels of mercury (0.01 to 0.93 mg/kg) compared to non-strictly carnivorous fish (<0.01 to 0.30 mg/kg). Farmed fishes showed significantly lower levels compared to wild fish. Mean Brazilian fish consumption achieves FAO/WHO health recommendation of about two portions of fish per week. However, there is a large difference between fish consumption at urban and rural homes and among Brazilian geographic regions. These differences in consumption impacted estimated methylmercury intake that was higher in the Northern (1.85 µg/kg bw week) and in the Northeastern (0.72 µg/kg bw week) regions and also by rural population (1.08 µg/kg bw week). These values were compared with the toxicological reference dose for neurotoxicity of 1.6 µg/kg bw week. CONCLUSION: Even though total levels of mercury in fish were lower than Brazilian and international legislations, in the Northern Brazilian region methylmercury intake overpassed the toxicological reference dose for neurotoxicity and in rural areas it achieved 68% of this reference dose.

A simple and sensitive HPLC-FL method for simultaneous determination of angiotensin II receptor antagonists in human plasma.

Angiotensin II receptor antagonists are one of the most widely used classes of antihypertensive drugs. In this study, an HPLC fluorescence method after protein precipitation (PPT) extraction was developed and validated for determination of olmesartan, losartan, irbesartan, and valsartan in human plasma. The separation was carried out on a Luna cyano (250 × 4.6 mm i.d.; 5 µm particle size) column and the mobile phase was composed of acetonitrile and 0.1 % phosphoric acid in gradient elution, at a flow rate of 1.2 mL min-1. A PPT method was optimized by a two-level factorial design with triplicate at the central point. The parameters that could affect the extraction (sample volume and acetonitrile/plasma volume ratio) were evaluated and the method was compared to microextraction by packed sorbent (MEPS) and liquid-liquid extraction (LLE). The developed method allowed the simultaneous quantification of the analytes employing a simple and cheap sample preparation method and a short chromatographic run (13 min). This method was fully validated showing selectivity, precision, accuracy, and linearity over the range of 25.0-1500.0 ng mL-1 for olmesartan and valsartan, 25.0-2500.0 ng mL-1 for irbesartan, and 35.0-2500.0 ng mL-1 for losartan. Finally, the method was successfully applied in the analysis of human plasma from volunteers.

Teores de mercúrio em mexilhões do mercado consumidor e influência do tratamento térmico / Mercury contents in mussels from the retail market and influence of thermal processing

A contaminação por mercúrio é um desafio para a saúde humana e animal. Os moluscos bivalves, especialmente os mexilhões, absorvem, retém e concentram o mercúrio. Este trabalho teve como objetivo determinar os teores de mercúrio em mexilhões do mercado varejista de Belo Horizonte, MG e investigar a influência do tratamento térmico. Foram analisadas 42 amostras de mexilhão marrom procedentes de cultivo marinho. Os teores de mercúrio foram determinados por espectrometria de absorção atômica de combustão após amalgamação em ouro. Todas amostras tinham mercúrio em teores que variaram de 0,020 a 0,076 mg/kg. Nenhuma amostra apresentou teor de mercúrio superior ao limite estabelecido pela legislação brasileira (0,05 mg/kg para crustáceos, moluscos e cefalópodes). O cozimento do mexilhão por 3 e 6 min não foi eficaz na eliminação do Hg.

Cadmium, copper and lead levels in different cultivars of lettuce and soil from urban agriculture.

Urban agriculture plays an important role in sustainable food supply. However, because of the atmospheric pollution and soil contamination associated with urban areas, this activity may be of concern. In fact, contamination of soil with metals and the transference of contaminants to vegetables can represent health and safety risks associated with urban agriculture. The objective of this study was to evaluate the concentrations of selected trace metals (cadmium, copper and lead) in three lettuce cultivars produced in three different urban gardens in the metropolitan region of Belo Horizonte, Brazil and their respective soils. Samples of lettuce and soil were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrometry (AAS-GF), respectively, and their transfer coefficients were calculated. The methods were optimized and were fit for the purpose. Copper was the prevalent metal found in soils and lettuce, with an average of 27.9 ±â€¯13.9 and 0.608 ±â€¯0.157 mg kg-1 respectively, followed by lead (19.4 ±â€¯7.7 and 0.037 ±â€¯0.039 mg kg-1), and cadmium (0.16 ±â€¯0.03 and 0.009 ±â€¯0.005 mg kg-1). Cadmium presented the largest transfer coefficients, ranging from 0.34 to 1.84 with an average of 0.92 ±â€¯0.45, which may indicate a potential risk of accumulation in vegetables in the case of high soil contamination. A significant positive correlation was observed (p < 0.01) between cadmium in lettuce and in soil. Even though lead concentrations varied in the soils from the different urban areas, ranging from 11.88 to 30.01 mg kg-1, no significant difference (p < 0.05) was found among the lettuce, probably due to its low mobility (transfer coefficient = 0.02). The copper and cadmium levels found in lettuce indicate safe lettuce production in the three urban gardens.

A simple and rapid LC-MS/MS method for the determination of amphenicols in Nile tilapia.

A quantitative method for analysis of amphenicols (chloramphenicol - CAP, thiamphenicol - TAP and florfenicol - FF) in Nile tilapia using LC-MS/MS is described. A simple sample preparation procedure was optimized using a Plackett-Burman design. The method was validated in accordance with Decision 2002/657/EC. Repeatability and reproducibility were less than 10.7% and 16%, respectively, for all compounds. Recoveries varied from 79.8% to 92.0%. CCα was 0.019, 54.81 and 54.93 µg.kg-1 for CAP, FF and TAP, respectively. CCß was 0.068, 64.88 and 58.91 µg.kg-1 for CAP, FF and TAP, respectively. Limits of quantification (LOQ) were 12.5 µg.kg-1 for FF and TAP and 0.15 µg.kg-1 for CAP. Nile tilapia fillets (n = 32) analyzed did not contain chloramphenicol. Thiamphenicol was detected in one sample (3.1%) and florfenicol was detected in every sample, all of them at concentrations below the maximum residue limit.

Assessment of the quality of refrigerated and frozen pork by multivariate exploratory techniques.

Pork loin and leg were evaluated 24h after slaughter and during refrigerated (5°C) and frozen storage for microbial counts, pH, total volatile base nitrogen (TVB-N), thiobarbituric acid reactive substances (TBARS) and bioactive amines. Spermine was the prevalent amine in fresh pork loin and leg, followed by spermidine and agmatine. During refrigerated storage, pH, TVB-N, mesophilic and psychrotrophic counts increased and no changes (p<0.05) were observed on polyamines; however putrescine, cadaverine and histamine were produced and accumulated throughout storage. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) for all parameters indicated a shelf life of 8days for both cuts, which was also coherent with safety regarding histamine levels. During frozen storage, there was no change on amines and pH, TVB-N decreased, and TBARS increased. None biogenic amine was produced. PCA and HCA were not able to classify frozen pork based on the analyzed parameters; however, a shelf life of 90days was suggested for the frozen cuts based on lipid oxidation.

Quinolones and tetracyclines in aquaculture fish by a simple and rapid LC-MS/MS method.

The determination of antimicrobials in aquaculture fish is important to ensure food safety. Therefore, simple and fast multiresidue methods are needed. A liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of 14 antimicrobials (quinolones and tetracyclines) in fish. Antimicrobials were extracted with trichloroacetic acid and chromatographic separation was achieved with a C18 column and gradient elution (water and acetonitrile). The method was validated (Decision 2002/657/EC) and it was fit for the purpose. Linearities were established in the matrix and the coefficients of determination were ≥0.98. The method was applied to Nile tilapia and rainbow trout (n = 29) and 14% of them contained enrofloxacin at levels above the limit of quantification (12.53-19.01 µg.kg-1) but below the maximum residue limit (100 µg.kg-1). Even though prohibited in Brazil and other countries, this antimicrobial reached fish. Measures are needed to ascertain the source of this compound to warrant human safety.

Effect of Huanglongbing or Greening Disease on Orange Juice Quality, a Review.

Huanglongbing (HLB) or citrus greening is the most severe citrus disease, currently devastating the citrus industry worldwide. The presumed causal bacterial agent Candidatus Liberibacter spp. affects tree health as well as fruit development, ripening and quality of citrus fruits and juice. Fruit from infected orange trees can be either symptomatic or asymptomatic. Symptomatic oranges are small, asymmetrical and greener than healthy fruit. Furthermore, symptomatic oranges show higher titratable acidity and lower soluble solids, solids/acids ratio, total sugars, and malic acid levels. Among flavor volatiles, ethyl butanoate, valencene, decanal and other ethyl esters are lower, but many monoterpenes are higher in symptomatic fruit compared to healthy and asymptomatic fruit. The disease also causes an increase in secondary metabolites in the orange peel and pulp, including hydroxycinnamic acids, limonin, nomilin, narirutin, and hesperidin. Resulting from these chemical changes, juice made from symptomatic fruit is described as distinctly bitter, sour, salty/umami, metallic, musty, and lacking in sweetness and fruity/orange flavor. Those effects are reported in both Valencia and Hamlin oranges, two cultivars that are commercially processed for juice in Florida. The changes in the juice are reflective of a decrease in quality of the fresh fruit, although not all fresh fruit varieties have been tested. Earlier research showed that HLB-induced off-flavor was not detectable in juice made with up to 25% symptomatic fruit in healthy juice, by chemical or sensory analysis. However, a blend with a higher proportion of symptomatic juice would present a detectable and recognizable off flavor. In some production regions, such as Florida in the United States, it is increasingly difficult to find fruit not showing HLB symptoms. This review analyzes and discusses the effects of HLB on orange juice quality in order to help the citrus industry manage the quality of orange juice, and guide future research needs.

Synephrine - A potential biomarker for orange honey authenticity.

A LC-MS/MS method for synephrine as a biomarker for orange honey authenticity was developed and validated. The sample was extracted with 5% TCA and cleaned up with Florisil providing 83.7% recoveries. Ions transitions for quantification and identification were 168→135.0 and 168→107.0, respectively. The limits of detection and quantification were 0.66 and 1.0ng/g, respectively. Synephrine was detected in orange honey at levels from 79.2 to 432.2ng/g, but not in other monofloral honeys. It was also present in some wildflower honeys (9.4-236.5ng/g), showing contribution of citrus to this polyfloral honey. Results were confirmed by qualitative pollen analysis. No citrus pollen was detected in honey containing synephrine levels ≤43.8ng/g, suggesting that synephrine in honey is more sensitive compared to pollen analysis. Synephrine was found in citrus but not in other apiculture flowers. Therefore, synephrine is a botanical marker to differentiate and attest authenticity of orange honey.

Advances on the chromatographic determination of amphenicols in food.

Antibiotics are widely used in veterinary medicine to treat and prevent diseases and their residues can remain in food of animal origin causing adverse effects to human health. Amphenicols (chloramphenicol, thiamphenicol, and florfenicol) may be found in foodstuffs, although the use of chloramphenicol has been prohibited in many countries due to its high toxicity. Since these antibiotics are usually present at trace levels in food, sensitive and selective techniques are required to detect them. This paper reviews analytical methods used since 2002 for the quantitative analysis of amphenicols in food. Sample preparation and separation/detection techniques are described and compared. The advantages and disadvantages of these procedures are discussed. Furthermore, the worldwide legislation and occurrence of these antibiotics in food matrices as well as future trends are also presented.

A simple, fast and sensitive screening LC-ESI-MS/MS method for antibiotics in fish.

The objective of this study was to develop and validate a fast, sensitive and simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the screening of six classes of antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, sulfonamides and tetracyclines) in fish. Samples were extracted with trichloroacetic acid. LC separation was achieved on a Zorbax Eclipse XDB C18 column and gradient elution using 0.1% heptafluorobutyric acid in water and acetonitrile as mobile phase. Analysis was carried out in multiple reaction monitoring mode via electrospray interface operated in the positive ionization mode, with sulfaphenazole as internal standard. The method was suitable for routine screening purposes of 40 antibiotics, according to EC Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines, taking into consideration threshold value, cut-off factor, detection capability, limit of detection, sensitivity and specificity. Real fish samples (n=193) from aquaculture were analyzed and 15% were positive for enrofloxacin (quinolone), one of them at a higher concentration than the level of interest (50µgkg-1), suggesting possible contamination or illegal use of that antibiotic.

Quality assurance of histamine analysis in fresh and canned fish.

Histamine determination is relevant for fish safety, quality and trade. Recently a study by the European Union (EU) compared the Codex and the EU mandated methods for the analysis of histamine and observed that they underestimated and overestimated the results, respectively. To solve this problem, a simple and efficient procedure for the extraction and quantification of histamine by ion-pair HPLC method with post-column derivatization and fluorimetric detection is proposed. It was optimized and validated for the analysis of histamine in fish. The method attended the performance criteria established by Commission Decision 2002/657/CE. The method was also submitted to proficiency testing; uncertainty was calculated; and the stability of solutions and standards was investigated. There was no matrix effect. The LOD, LOQ, CCα and CCß were fit for the purpose. The method was successfully used in the analyses of freshwater fish and fresh and canned tuna.