A platform for anti-biofilm assays combining viability, biomass and matrix quantifications in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Bacteria can exist as planktonic, the lifestyle in which single cells exist in suspension, and as biofilms, which are surface-attached bacterial communities embedded in a selfproduced matrix. Most of the antibiotics and the methods for antimicrobial work have been developed for planktonic bacteria. However, the majority of the bacteria in natural habitats live as biofilms. Biofilms develop dauntingly fast high resistance towards conventional antibacterial treatments and thus, there is a great need to meet the demands of effective anti-biofilm therapy. In this thesis project it was attempted to fill the void of anti-biofilm screening methods by developing a platform of assays that evaluate the effect that screened compounds have on the total biomass, viability and the extracellular polysaccharide (EPS) layer of the biofilms. Additionally, a new method for studying biofilms and their interactions with compounds in a continuous flow system was developed using capillary electrochromatography (CEC). The screening platform was utilized with a screening campaign using a small library of cinchona alkaloids. The assays were optimized to be statistically robust enough for screening. The first assay, based on crystal violet staining, measures total biofilm biomass, and it was automated using a liquid handling workstation to decrease the manual workload and signal variation. The second assay, based on resazurin staining, measures viability of the biofilm, and it was thoroughly optimized for the strain used, but was then a very simple and fast method to be used for primary screening. The fluorescent resazurin probe is not toxic to the biofilms. In fact, it was also shown in this project that staining the biofilms with resazurin prior to staining with crystal violet had no effect on the latter and they can be used in sequence on the same screening plate. This sequential addition step was indeed a major improvement on the use of reagents and consumables and also shortened the work time. As a third assay in the platform a wheat germ agglutinin based assay was added to evaluate the effect a compound has on the EPS layer. Using this assay it was found that even if compounds might have clear effect on both biomass and viability, the EPS layer can be left untouched or even be increased. This is a clear implication of the importance of using several assays to be able to find “true hits” in a screening setting. In the pilot study of screening for antimicrobial and anti-biofilm effects using a cinchona alkaloid library, one compound was found to have antimicrobial effect against planktonic bacteria and prevent biofilm formation at low micromolar concentration. To eradicate biofilms, a higher concentration was needed. It was also shown that the chemical space occupied by the active compound was slightly different than the rest of the cinchona alkaloids as well as the rest of the compounds used for validatory screening during the optimization processes of the separate assays.

Nutritional status of hemodialysis patients with secondary hyperparathyroidism

The repercussions of secondary hyperparathyroidism on the nutritional status of chronic renal failure patients have not been well established. Therefore, the aim of this study was to compare the nutritional indices of hemodialysis patients with and without secondary hyperparathyroidism. Sixteen hemodialysis patients with serum parathyroid hormone (PTH) levels higher than 420 pg/ml (hyperparathyroidism group) were matched for gender, age and length of dialysis treatment to 16 patients with serum PTH between 64 and 290 pg/ml (control group). The following parameters were assessed: anthropometric indices (body mass index, skinfold thickness, midarm muscle circumference and body fat), 4-day food diaries, protein catabolic rate, biochemical indices (blood urea nitrogen, serum creatinine, albumin, ionized calcium, inorganic phosphorus, serum alkaline phosphatase, PTH, pH and HCO3) and dialysis efficiency. We did not observe differences in the anthropometric indices between the two groups. Only calcium intake was significantly different between groups (307.9 mg/day for the hyperparathyroidism group vs 475.8 mg/day for the control group). Protein catabolic rate tended to be higher in the hyperparathyroidism group compared to the control group (1.3 vs 0.9 g kg-1 day-1; P = 0.08). Except for blood urea nitrogen (86.4 vs 75.7 mg/dl), alkaline phosphatase (175 vs 65 U/l) and PTH (898 vs 155 pg/ml), no other differences were found between groups in the biochemical indices studied. PTH was directly correlated with protein catabolic rate (r = 0.61; P<0.05) and length of dialysis (r = 0.53; P<0.05) only in the hyperparathyroidism group. Considering the indices used, we could not demonstrate the deleterious effect of high PTH levels on the nutritional status of hemodialysis patients. Indirect evidence, however, suggests an action of PTH on protein metabolism.

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

Nutritional status and body composition after 6 months of patients switching from continuous ambulatorial peritoneal dialysis to automated peritoneal dialysis

Our objective was to determine if automated peritoneal dialysis (APD) leads to changes in nutritional parameters of patients treated by continuous ambulatory peritoneal dialysis (CAPD). Twenty-six patients (15 males; 50.5 ± 14.3 years) were evaluated during CAPD while training for APD and after 3 and 6 months of APD. Body fat was assessed by the sum of skinfold thickness and the other body compartments were assessed by bioelectrical impedance. During the 6-month follow-up, 12 patients gained more than 1 kg (GW group), 8 patients lost more than 1 kg (LW group), and 6 patients maintained body weight (MW group). Except for length on dialysis that was longer for the LW group compared with the GW group, no other differences were found between the groups at baseline. After 6 months on APD, the LW group had a reduction in body fat (24.5 ± 7.7 vs 22.1 ± 7.3 kg; P = 0.01), body cell mass (22.6 ± 6.2 vs 21.6 ± 5.8 kg, P = 0.02) and phase angle (5.4 ± 0.9 vs 5.1 ± 0.8 degrees, P = 0.004). In the GW group, body fat (25 ± 7.6 vs 27.2 ± 7.6 kg, P = 0.001) and body cell mass (20.1 ± 3.9 vs 20.8 ± 4.0 kg, P = 0.05) were increased. In the present study, different patterns of change in body composition were found. The length of previous dialysis treatment seems to be the most important factor in determining these nutritional modifications.

Sensory evaluation and nutritional value of cakes prepared with whole flaxseed flour

The objective of this study was to evaluate the nutritional value, the oxidative stabilitiy, and consumer acceptance of cakes containing four different concentrations of flaxseed flour (5, 15, 30 and 45%) as partial replacement for wheat flour. The oxidative stability of polyunsaturated fatty acids was evaluated through the lipid peroxidation test (TBARS) in the flour and cakes. Linolenic acid was determined by gas chromatography as well as contents of protein, lipid, ash, and dietary fiber. Consumer acceptance was assessed using a structured hedonic scale of nine points. The oxidative stability of lipid flaxseeds was not affected by the heat treatment during flour processing and cake baking. Cakes made with 5, 15, and 30% of flaxseed flour, the most accepted by consumers, had dietary fiber levels ranging from 3.5 to 6.2 g and linolenic acid ranging from 445 to 2,500 mg.100 g-1 of the product. The cakes received claims of good and excellent source of dietary fiber and linolenic acid, respectively, both are bioactive compounds. The use of up to 30% of flaxseed flour in the preparation of cakes is a useful strategy to optimize the consumption of food rich in functional ingredients.

Partial chemical and functional characterization of milk whey products obtained by different processes

Whey protein samples (S-1 to S-5) were tested in vivo and in vitro for nutritional properties and selected bioactivities. Weanling male Wistar rats fed modified AIN-93G (12 g protein.100 g-1) diets for 21 days were used the in vivo studies. The nutritional parameters did not differ among the protein diets tested. Erythrocyte glutathione content was considered high and was higher for S-3, but liver glutathione was the same for all dietary groups. For S-3, cytokine secretion (IL-10 and TNF-α) by human peripheral blood mononuclear cells (in RPMI-1640 medium) was higher in the absence of antigen than in the presence of BCG antigen. Interleukin-4 secretion was repressed in all treatments. The IC50, whey protein concentration required to inhibit 50% of the melanoma cell proliferation, was 2.68 mg.mL-1 of culture medium for the S-3 sample and 3.66 mg.mL-1 for the S-2 sample. Based on these results, it was concluded that S-3 (whey protein concentrate enriched with TGF-β and lactoferrin) produced better nutritional and immunological responses than the other products tested.

Physical and chemical characterization of the pulp of different varieties of avocado targeting oil extraction potential

The aim of this study was to evaluate the physicochemical properties of avocado pulp of four different varieties (Avocado, Guatemala, Dickinson, and Butter pear) and to identify which has the greatest potential for oil extraction. Fresh avocado pulp was characterized by moisture, protein, fat, ash, carbohydrates and energy contents were determined. The carotenoids and chlorophyll contents were determined by the organic solvent extraction method. The results showed significant differences in the composition of the fruit when varieties are compared. However, the striking feature in all varieties is high lipid content; Avocado and Dickinson are the most suitable varieties for oil extraction, taking into account moisture content and the levels of lipids in the pulp. Moreover, it could be said that the variety Dickinson is the most affected by the parameters evaluated in terms of overall quality. Chlorophyll and carotenoids, fat-soluble pigments, showed a negative correlation with respect to lipids since it could be related to its function in the fruit. The varieties Avocado and Dickinson are an alternative to oil extraction having great commercial potential to be exploited thus avoiding waste and increasing farmers’ income.

Effect of incorporation of amaranth on the physical properties and nutritional value of cheese bread

At the present celiac disease has no known cure, and its only treatment is a strict lifelong adherence to a gluten-free diet. Cheese bread is a traditional Brazilian product and a safe option for celiacs. However, like other gluten-free breads, it has inherent low levels of fibers and minerals. The objective of this study was to evaluate the effect of incorporation of whole amaranth flour on the physical properties and nutritional value of cheese bread. Amaranth flour was incorporated at 10, 15, and 20% proportions in different formulations. The increasing amaranth levels darkened the product, reduced specific volume, and increased compression force. Ten percent amaranth-content cheese breads exhibited slight differences in physical properties compared with the controls. These results demonstrated the possibility of incorporating 10% of whole amaranth flour in the formulation of cheese bread resulting in a product with higher dietary fiber and iron contents and the same level of acceptance as that of the conventional formulation. The aim of this approach is to increase the availability of gluten-free bakery products with added nutritional value contributing to increase the variety of the diet of celiac patients.

Stability and microbiological quality of rice bran subjected to different heat treatments

Rice bran is a byproduct commonly used for animal feeding; however its nutritional value and potential application in human diet have attracted market interest. Its preservation for safe use is still a challenge, so the objective of this study was to determine the quality of commercially available rice bran samples subjected to different heat treatments (extruding, parboiling, toasting, and microwave oven heating) in order to promote stabilization during storage under room temperature. Rice bran samples were collected from two industries, and each treatment was divided in three parts, each corresponding to three repetitions. All samples were evaluated for moisture content, total microorganisms, mold and yeast counting, hydrolytic rancidity, and lipase activity during 90 days of storage. Most of the heat treatments, including domestic and thermoplastic extrusion, generated products which may be used for human consumption under the tested conditions in terms of physicochemical and microbiological quality. The domestic treatments were more efficient in eliminating microorganisms or keeping them within acceptable limits. The toasted rice bran showed satisfactory results in terms of moisture, hydrolytic rancidity control, and lipase activity.

Genetic diversity and comparison of physicochemical and nutritional characteristics of six quinoa (Chenopodium quinoa willd.) genotypes cultivated in Chile

The present study was focused on the analysis of agronomical, nutritional, physicochemical, and antioxidant properties of six genetically different quinoa (Chenopodium quinoa Willd) genotypes cultivated in three distinctive geographical zones of Chile. Ancovinto and Cancosa genotypes from the northern Altiplano (19 ºS), Cáhuil and Faro from the central region (34 ºS), and Regalona and Villarica from the southern region (39 ºS) are representative of high genetic differentiation among the pooled samples, in particular between Altiplano and the central-southern groups. A Common-Garden Assay at 30 ºS showed significant differences among seed origins in all morphometric parameters and also in yields. Altiplano genotypes had larger panicule length but no seed production. A significant influence of the different quinoa genotypes on chemical composition and functional properties was also observed. Protein concentration ranged from 11.13 to 16.18 g.100 g-1 d.m., while total dietary fiber content ranged from 8.07-12.08 g.100 g-1 d.m., and both were the highest in Villarrica ecotype. An adequate balance of essential amino acids was also observed. Sucrose was the predominant sugar in all genotypes. Antioxidant activity was high in all genotypes, and it was highest in Faro genotype (79.58% inhibition).

Nutritional value and fatty acid composition of some traditional Argentinean meat sausages

The aim of this study was to determine the nutritional composition (moisture, protein, carbohydrates, and total fat) of some meat products produced in the northeastern Argentina, analyzing fatty acids composition, polyunsaturated/saturated fatty acid ratio PUFA/SFA ratio (polyunsaturated/ saturated fatty acids), n-6/n-3 ratio, and CLA (conjugated linoleic acid) content. Thirty traditional meat products produced by different processes were used. The samples were classified into 4 different categories as follows: salamín (dry cured and fermented sausage), chorizos (raw sausage), chorizo ahumado (cooked and smoked sausage), and morcilla (cooked sausage). From the results obtained it can be said that the total carbohydrate contents of the salamín studied were slightly lower; fat content of raw chorizo was significantly lower, and protein content of chorizo ahumado was significantly higher than those comparison from databases from other regions of Argentina, USA, and Spain. Except for chorizo, which has a value lower than 0.4, the PUFA/SFA-stearic ratio of the other products were a little higher than those reported by other researchers. CLA (Conjugated linoleic acid) contents between 0.03% and 0.19% were detected. The results obtained indicate that salamín produced in northeastern Argentina, Chaco state, shows high protein and PUFA (Polyunsaturated fatty acids) contents, and low atherogenic and thrombogenic indexes, which makes it a more healthful product than those of similar composition produced in other countries.

Sesame and flaxseed oil: nutritional quality and effects on serum lipids and glucose in rats

This study evaluated the nutritional value of sesame and flaxseed oils and their effects on the lipid and glucose profile of rats fed diets containing different fat combinations. Fatty acid composition, refractive index, and iodine and saponification values were analyzed to characterize the oils. In the biological assay, Wistar rats were fed different diets, whose fat composition consisted of varying combinations of flaxseed oil, sesame oil, and animal fat. The primary constituents of the sesame oil were oleic (28.6%), linoleic (28.4%), and lauric acid (14.6%); for the flaxseed oil they were alpha-linolenic (39.90%), oleic (17.97%) and linoleic acid (12.25%). The iodine and saponification values of the oils were within the reference range. Rats fed flaxseed oil-based diets had the lowest serum cholesterol values, whereas rats fed diets with flaxseed oil + sesame oil + animal fat had the highest glucose levels. HDL levels decreased significantly with flaxseed oil. Sesame and flaxseed oils are sources of polyunsaturated fatty acids (PUFA), and the flaxseed oil-based diet had a hypocholesterolemic effect, whereas sesame oil showed oxidative stability since it contains high levels of monounsaturated and saturated fatty acids.

Prevalent fatty acids in cashew nuts obtained from conventional and organic cultivation in different stages of processing

Brazil is one of the three largest producers of fruits in the world, and among those fruit trees, the cashew tree stands out due to the high nutritional and commercial value of its products. During its fruit processing, there are losses in some compounds and few studies address this issue. Over the last decade the conventional system of food production has been substituted for the organic cultivation system, which is a promising alternative source of income given the global demand for healthy food. Therefore, this research aimed to characterize and quantify the prevalent fatty acids found in cashew nuts obtained from conventional and organic cultivation during various stages of processing. The prevalent fatty acids found were palmitic, linoleic, oleic, and stearic acid. The average of these fatty acids were 6.93 ± 0.55; 16.99 ± 0.61; 67.62 ± 1.00 and 8.42 ± 0.55 g/100 g, respectively. There was no reduction in the palmitic, oleic and stearic fatty acid contents during processing. Very little difference was observed between the nuts obtained from conventional and organic cultivation, indicating that the method of cultivation used has little or no influence on the content of cashew nut fatty acids.

Nutritional composition of Annona crassiflora pulp and acceptability of bakery products prepared with its flour

Annona crassiflora offers an edible fruit native to the Savanna. This study aimed to develop a flour meal from Annona crassiflora pulp; analyze the chemical composition of the fresh pulp and its flour; develop and verify the acceptance of formulations with different concentrations of the flour of Annona crassiflora pulp. Fruit used were selected and processed. The pulp was dried in an oven at 60-65 ºC/48h. We analyzed the chemical composition, and two formulations of breads were prepared with 10 and 20% Annona crassiflora pulp. The results showed that the drying of Annona crassiflora pulp enriched its nutritional value. The Annona crassiflora pulp showed important chemical components, as insoluble fibers (pulp and flour), minerals (potassium, calcium, manganese and others) and antioxidant compounds. The formulations were well-accepted in a sensory point of view and proved to be a good alternative to the exploitation of the fruit.

Quality characteristics of Moroccan sweet paprika (Capsicum annuum L.) at different sampling times

"La Niora" is a red pepper variety cultivated in Tadla Region (Morocco) which is used for manufacturing paprika after sun drying. The paprika quality (nutritional, chemical and microbiological) was evaluated immediately after milling, from September to December. Sampling time mainly affected paprika color and the total capsaicinoid and vitamin C contents. The commercial quality was acceptable and no aflatoxins were found, but the microbial load sometimes exceeded permitted levels.