Biological evaluation of recombinant human erythropoietin in pharmaceutical products

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Occurrence of Haemophilus influenzae strains in three Brazilian states since the introduction of a conjugate Haemophilus influenzae type b vaccine

Few vaccines in history have induced such a dramatic decline in incidence over such a short period of time as the Haemophilus influenzae type b (Hib) conjugate. This vaccine was introduced in 1988 in the United States, but only in 1999 was Hib immunization introduced by the Brazilian Ministry of Health as part of the routine infant National Immunization Program. The authors analyzed 229 H. influenzae (Hi) isolates from Public Health Laboratories in three Brazilian states: Pernambuco (Northeast, N = 54), Santa Catarina (South, N = 19), and Rio de Janeiro (Southeast, N = 156). The isolates were collected from Brazilian children 0-10 years of age with meningitis and other infections from 1990 to 2003 and were part of the research collection of the National Institute of Quality Control in Health, FIOCRUZ. Bacterial strains were characterized by serotyping and biotyping. During the pre-vaccination period the prevalence infection due to Hib was of 165 isolates and only 2 non-b Hi among all the notified meningitis infections caused by Hi. Our results showed a significant decrease in the prevalence of Hib meningitis from 165 to 33 isolates after 1999. However, during the post-vaccination period of 2001-2003 we observed an increase in the number of non-b Hi isolates: only 2 non-b strains isolated from 1990 to 1999 and 29 from 1999 to 2003. Based on the present data, the authors emphasize the need for more sensitive epidemiological and bacteriological studies aiming the improvement of the available Hib vaccine, in order to protect the susceptible population to infections due to other serological types of Hi and the reevaluation of immunization schedules used by the National Immunization Program.

Epidemiological studies in the information and genomics era: experience of the Clinical Genome of Cancer Project in São Paulo, Brazil

Genomics is expanding the horizons of epidemiology, providing a new dimension for classical epidemiological studies and inspiring the development of large-scale multicenter studies with the statistical power necessary for the assessment of gene-gene and gene-environment interactions in cancer etiology and prognosis. This paper describes the methodology of the Clinical Genome of Cancer Project in São Paulo, Brazil (CGCP), which includes patients with nine types of tumors and controls. Three major epidemiological designs were used to reach specific objectives: cross-sectional studies to examine gene expression, case-control studies to evaluate etiological factors, and follow-up studies to analyze genetic profiles in prognosis. The clinical groups included patients' data in the electronic database through the Internet. Two approaches were used for data quality control: continuous data evaluation and data entry consistency. A total of 1749 cases and 1509 controls were entered into the CGCP database from the first trimester of 2002 to the end of 2004. Continuous evaluation showed that, for all tumors taken together, only 0.5% of the general form fields still included potential inconsistencies by the end of 2004. Regarding data entry consistency, the highest percentage of errors (11.8%) was observed for the follow-up form, followed by 6.7% for the clinical form, 4.0% for the general form, and only 1.1% for the pathology form. Good data quality is required for their transformation into useful information for clinical application and for preventive measures. The use of the Internet for communication among researchers and for data entry is perhaps the most innovative feature of the CGCP. The monitoring of patients' data guaranteed their quality.

Shelf life of irradiated minimally processed (MP) watercress (Nasturtium officinale)

This study was carried out to evaluate the shelf life of minimally processed (MP) watercress exposed to gamma radiation with doses of 1.0; 3.0; 4.0 kGy. Packaged irradiated and non-irradiated MP watercress was subject to sensory analysis. A panel consisting of 25-30 non-trained members, aged 20-55 years, was used. Sensory evaluation was carried out on days 0, 2, 5, 7, 9 and 12 after treatment with packages maintained at 7 °C. The members of the panel rated each sample for overall liking on a hybrid 10 hedonic scale (0 = extremely dislike; 10 = extremely like). Microbiological analyses were also conducted. Compared to the non-irradiated sample, 1 kGy irradiated watercress increased its shelf life by one day (16 days). Shelf lives of samples exposed to higher doses were reduced to 9 days (3 kGy) and 6 days (4 kGy) due to changes in appearance. The microbiological quality was good throughout the experiment.

Chocolate rheology

Rheology is the science that studies the deformation and flow of solids and fluids under the influence of mechanical forces. The rheological measures of a product in the stage of manufacture can be useful in quality control. The microstructure of a product can also be correlated with its rheological behavior allowing for the development of new materials. Rheometry permits attainment of rheological equations applied in process engineering, particularly unit operations that involve heat and mass transfer. Consumer demands make it possible to obtain a product that complies with these requirements. Chocolate industries work with products in a liquid phase in conching, tempering, and also during pumping operations. A good design of each type of equipment is essential for optimum processing. In the design of every process, it is necessary to know the physical characteristics of the product. The rheological behavior of chocolate can help to know the characteristics of application of the product and its consumers. Foods are generally in a metastable state. Their texture depends on the structural changes that occur during processing. Molten chocolate is a suspension with properties that are strongly affected by particle characteristics including not only the dispersed particles but also the fat crystals formed during chocolate cooling and solidification. Chocolate rheology is extensively studied, and it is known that chocolate texture and stability is strongly affected by the presence of specific crystals

(E)-2-Nonenal determination in brazilian beers using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS)

(E)-2-nonenal is considered an important off-flavor of beer, related to the flavor of beer staling. In this study, a new method for determination of (E)-2-nonenal in beer using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS) was developed and applied in Brazilian beer samples. The extractions were carried out in CAR-PDMS (carboxen-polydimethylsiloxane) fiber and the best results were found with 15 minutes of equilibrium and 90 minutes of extraction at 50 °C. The method was linear in the range from 0.02 to 4.0 μg.L-1 with correlation coefficient of 0.9994. The limits of detection and quantification were 0.01 and 0.02 μg.L-1, respectively. 96.5% of recovery and 4% precision (RSD) were obtained in the fortification of beer samples with 2.0 μg.L-1 of (E)-2-nonenal. The developed method proved to be simple, efficient and highly sensitive to the determination of this analyte being easily applied in the quality control of the brewery. (E)-2-nonenal was found in all beer samples analyzed with levels between 0.17 and 0.42 μg.L-1.

Conversion of hydroxycinnamic acids into volatile phenols in a synthetic medium and in red wine by Dekkera bruxellensis

The conversion of p-coumaric acid, ferulic acid, and caffeic acid into 4-ethylphenol, 4-ethylguaiacol and 4-ethylcatechol was studied in Dekkera bruxellensis ISA 1791 under defined conditions in a synthetic medium and in a red wine. Liquid chromatography (HPLC-DAD) was used to quantify the phenolic acids, and gas chromatography (GC) coupled to a FID detector was used to quantify volatile phenols using a novel analytical methodology that does not require sample derivatization. Identification was achieved by gas chromatography-mass detection (GC-MS). The results show that phenolic acids concentration decreases while volatile phenols concentration increases. The proportion of caffeic acid taken up by Dekkera bruxellensis is lower than that for p-coumaric or ferulic acid; therefore less 4-ethylcatechol is formed. More important, 4-ethylcathecol synthesis by Dekkera bruxellensis in wine has never been demonstrated so far. These results contribute decisively to a better understanding of the origin of the volatile phenols in wines. The accumulation of these compounds in wine is nowadays regarded as one of the key factors of quality control.

Ocurrence of Vibrio spp., positive coagulase staphylococci and enteric bacteria in oysters (Crassostrea gigas) harvested in the south bay of Santa Catarina island, Brazil

The aim of this study was to assess the contamination of oysters (Crassostrea gigas), harvested in six different regions of the South Bay of Santa Catarina Island, with Coliforms at 45 ºC, Escherichia coli, Vibrio spp., positive coagulase staphylococci, and Salmonella sp. over a period of one year. One hundred eighty oyster samples were collected directly from their culture sites and analyzed. Each sample consisted of a pool of 12 oysters. All of the samples analyzed showed absence of Salmonella, 18 (10%) samples showed presence of Escherichia coli, 15 (8.3%) samples were positive for V. alginolyticus, and Vibriocholerae was detected in 4 samples (2.2%). The counts of positive-coagulase staphylococci varied from <10 to 1.9 x 102 CFU.g-1, whereas the counts of Coliforms at 45 ºC and E. coli ranged from <3 to 1.5 x 102 MPN.g-1 and <3 and 4.3 x 10 MPN.g-1, respectively. Counts of V. parahaemolyticus and V. vulnificus ranged between <3 and 7 MPN.g-1, for both microorganisms. This suggests the need for monitoring these Vibrios contamination in oysters. Based on the results of the microbiological assays, the samples analyzed showed acceptable bacteriological quality, i.e., they were within the parameters established by Brazilian Legislation.

Microbiota of sausages obtained by spontaneous fermentation produced in the South of Brazil

The study of the ecology of fermented sausage is fundamental to understand the physical and chemical changes that happen during fermentation and maturation. The aim of the present study was to determine the microbiological characteristics of sausages produced by spontaneous fermentation. Fifty samples of sausages produced in the South of Brazil by different small manufacturers were analyzed for the following microbiota: aerobic mesophilic bacteria; Micrococcaceae; mold and yeast; lactic acid bacteria; total and fecal coliforms; coagulase-positive Staphylococcus, and Salmonella. In most samples (72%), the count of lactic bacteria was higher than 6 log10 cfu.g-1, and the samples with the highest counts were above 8 log10 cfu.g-1. The counts of Micrococcaceae in most samples were between 5 log10 and 7 log10 cfu.g-1. With respect to the presence of molds and yeasts, there was a significant variation among the samples with counts ranging from 2 log10 cfu.g-1 and 6 log10 cfu.g-1. From the data obtained, it was possible to conclude that 24% of the analyzed samples did not comply with the current law in Brazil since the levels of fecal coliforms or coagulase-positive Staphylococcus exceeded the maximum limit allowed.

Reduction of carcass weight loss in swines

The carcass fast freezing is one of the aspects of great prominence to the final quality of pork. In order to reduce weight loss, two experiments were performed, in which the carcasses were monitored during 20 hours to evaluate the main variables involved during two different freezing processes (standard and proposed) as follows: microbiological quality, storage temperature, relative humidity (RH) and air velocity. In experiment I, the carcasses were submitted to a system using heat shock (2 hours in static tunnel at - 25 °C) and subsequently sent to the equalization chamber. In experiment II, the carcasses were submitted to the heat shock and stored in a chamber with RH between 80-85%. The chambers used in both experiments showed no change in the variables studied (internal temperature of 5 °C and air velocity of approximately 0.3 m/s). However, the relative humidity in the three chambers was evaluated and significant differences were found; as a consequence, high levels of weight loss were observed in both chambers In experiment II there was an increase of RH, which reduced the weight loss of the carcasses.

Stigmastadiene and specific extitntion (270 nm) to evaluate the presence of refined oils in virgin olive oil commercialized in Brazil

The increased marketing of olive oil in Brazil has intensified legal requirements to ensure regulation of this product. The measurement of the specific extinction at 270 nm (E 270) and content of stigmastadiene can be used to assess the presence of refined oils in virgin olive oil. During the vegetable oil refining process, compounds with conjugated double bonds are generated from unsaturated fatty acids that absorb at 270 nm and sterols, such as stigmasta-3,5-diene. To compare these parameters, seven samples of extra virgin olive oil and three samples of olive oil (blend of virgin and refined) were analyzed. Among the samples analyzed, four extra virgin samples had levels of stigmastadiene and E 270 higher than expected, among which two were adulterated with seed oil (rich in linoleic acid) and the other two with olive pomace oil. The results demonstrate the higher sensitivity of stigmastadiene to determine the presence of the refined oil in virgin olive oil and good agreement with determining E 270. The latter technique is a simple, quick, and low cost method of determination that can be easily implemented in laboratories to assist in the screening and regulation of olive oils sold in Brazil.

Biochemical characteristics of composite flours: influence of fermentation

The purpose of this study was to introduce yam in the development of two new composite flours containing soy and cassava. Two composite flours were obtained after fermentation of yam, soybean, and cassava in respectively 60, 30, and 10% proportions. Two varieties of yam were used: Dioscorea alata (variety "Bete bete") and Dioscorea cayenensis (variety "Lokpa"). Proximate composition, mineral content, some anti-nutritional factors (oxalates, phenols), microbiological quality, and α-amylase digestibility were determined for the fermented and unfermented composite flours. The results indicated that for the composite flours made of D. alata and D. cayenensis, fermentation increased ash and titrable acidity. Carbohydrates, pH, and energy decreased. Crude fat content was not affected by the fermentation process. Anti-nutritional factors such as oxalates and phenols were found to decrease significantly after the fermentation of the composite flours. Fermentation increased the mineral content (Mg, K, Fe, and Ca) of the composite flours. A decrease in P and Na was observed after fermentation. The microbiological study showed that safety flours contain no potential pathogenic germs. The in vitro α-amylase digestibility of the composite flours was significantly improved after fermentation. The biochemical characteristics and good hygienic quality of the obtained flours suggest that these flours can be considered as a feeding alternative for children in poor areas where yam is produced.

Frog meat microbiota (Lithobates catesbeianus) used in infant food

Captive breeding of bullfrog (Lithobates catesbeianus) is of great economic potential, mainly for its thighs and leather. The nutritional quality of frog meat includes properly balanced amino acids with a protein profile of high biological value, low fat and low cholesterol, and high digestibility due to its short chain molecule structure. It is recommended by doctors and nutritionists, especially for protein restricted children or malnourished children. Aiming to aggregate value to the segment and offer a product with nutritional properties that meet the need of children aged six months and above, a meat product based on the composition of frog meat was developed experimentally. To ensure raw material quality after bleaching and deboning, the microbiota present in the frog meat was determined. The analyses were performed according to Brazilian laws. It was observed that the resident and transient microbiota met the standards set by regulations. The results found were: mesophyll 4.5 x 10(4) CFU/g; Staphylococcus coagulase positive 2.0 x 10² CFU/g; negative for Salmonella sp. and Aeromonas spp. The findings indicate that the raw material showed satisfactory sanitation even in terms of family industry.

Chemical composition and bioactive compounds of grape pomace (Vitis vinifera L.), Benitaka variety, grown in the semiarid region of Northeast Brazil

Grape pomace (Vitis vinifera L.), Benitaka variety, grown in the semiarid region of Northeast Brazil was evaluated in relation to chemical composition, and content of minerals and functional properties. Its microbiological quality and toxic potential, using Artemia salina sp, were also investigated. The results showed that the flour obtained from these residues had below neutral pH (3.82), moisture (3.33g/100g), acidity of (0.64g of citric acid/100g), and ash (4.65 g/100g). The amount of total dietary fiber (46.17g/100g) stood out quantitatively compared to the content of carbohydrate (29.2g/100 g), protein (8.49g/100g), and lipids (8.16g/100g). The total energy was 224Kcal/100g. With regard to the compounds with functional properties, higher values of insoluble fiber 79% (36.4 g/100 g); vitamin C (26.25 mg of acid ascorbic/100g), and anthocyanins (131mg/100g) were found. The minerals iron, potassium, zinc, manganese, and calcium were present in higher concentrations. There were no significant copper values. The results showed that the grape residues are an important source of nutrients and compounds with functional properties suggesting that they can be incorporated as an ingredient in the diet and/or used as a dietary supplement aiming at health benefits. The residues did not show microbiological contamination and were considered nontoxic.

Chemical composition, fatty acid profile and bioactive compounds of guava seeds (Psidium guajava L.)

This study aimed to characterize the chemical composition, determine the fatty acid profile, and quantify the bioactive compounds present in guava seed powder (Psidium guajava L.). The powder resulted from seeds obtained from guava pulp processing. The agro-industrial seeds from red guava cv. paluma were used, and they were donated by a frozen pulp fruit manufacturer. They contain varying amounts of macronutrients and micronutrients, with a high content of total dietary fiber (63.94 g/100g), protein (11.19 g/100g), iron (13.8 mg/100g), zinc (3.31 mg/100g), and reduced calorie content (182 kcal/100g). Their lipid profile showed a predominance of unsaturated fatty acids (87.06%), especially linoleic acid (n6) and oleic acid (n9). The powder obtained contained significant amounts of bioactive compounds such as ascorbic acid (87.44 mg/100g), total carotenoids (1.25 mg/100 g) and insoluble dietary fiber (63.55 g/100g). With regard to their microbiological quality, the samples were found suitable for consumption. Based on these results, it can be concluded that the powder produced has favorable attributes for industrial use, and that use of these seeds would be a viable alternative to prevent various diseases and malnutrition in our country and to reduce the environmental impact of agricultural waste.