Ursula M. Lanfer Marquez

Distribution of intracellular nitrogen in marine microalgae: Calculation of new nitrogen-to-protein conversion factors

Nitrogen budgets in microalgae are strongly affected by growth conditions and physiological state of the cultures. As a consequence, protein N (PN) to total N (TN) ratio may be variable in microalgae grown in batch cultures, and this may limit the usefulness of the nitrogen-to-protein conversion factors (N-Prot factors), the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of specific N-Prot factors, and experimental data are needed to fill this gap. Complementing a previous study, the present work was designed to quantify the fluctuations of the main nitrogenous compounds during the growth of 12 species of marine microalgae, as well as to determine N-Prot factors for them. The microalgae were cultured in two experimental conditions: (a) using a N-replete culture medium (initial N concentration, 1.18 mM) and aeration, and (b) with a N-depleted culture medium (initial N concentration, 235 μM) and no aeration. The distribution of intracellular nitrogen was studied by constructing budgets of different nitrogen pools in different growth phases of the cultures. In all species, large variations occurred in the distribution of PN and non-protein N (NPN) in the treatments tested and in different growth phases. Intracellular inorganic nitrogen (NO3  − , NO2  −  and NH3 + NH4  + ) was the most important NPN component (0.4 – 30.4% of TN) in all species, followed by nucleic acids (0.3 – 12.2% of TN), and chlorophylls (0.1 – 1.8% of TN). The relative importance of NPN was greater in the exponential phase, decreasing during growth. PN ranged from 59.3 to 96.8% of TN. N-Prot factors are proposed for each of the species studied, based on the ratio of amino acid residues to TN, with values ranging from 2.53 to 5.77. Based on current results and on the previous study, we establish an overall average N-Prot factor for all species, treatments and growth phases of 4.78 ± 0.62 (n = 354). This study confirms that the use of the traditional factor 6.25 is unsuitable for marine microalgae, and the use of the N-Prot factors proposed here is recommended.

Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds

The use of nitrogen‐to‐protein conversion factors (N‐Prot factors) is the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of N‐Prot factors specific to individual species. Experimental data are needed to allow the use of this methodology with seaweeds. The present study was designed to characterize the amino acid composition and to establish specific N‐Prot factors for six green, four brown and nine red marine algae. Mean values for individual amino acids tended to be similar among the three groups, but some differences were found. Green algae tended to show lower percentages of both aspartic acid and glutamic acid than the other two groups of algae. The percentages of both lysine and arginine were higher in red algae, while brown algae tended to show more methionine than green and red algae. The actual protein content of the species, based on the sum of amino acid residues, varied from 10.8% (Chnoospora minima, brown algae) to 23.1% (Aglaothamnion uru‐guayense, red algae) of the dry weight. Nitrogen‐to‐protein conversion factors were established for the species studied, based on the ratio of amino acid residues to total nitrogen, with values ranging from 3.75 (Cryptonemia seminervis, red algae) to 5.72 (Padina gymnospora, brown algae). The relative importance of non‐protein nitrogen is greater in red algae, and consequently lower N‐Prot factors were calculated for these species (average value 4.59). Conversely, protein nitrogen content in both green and brown algae tends to be higher, and average N‐Prot factors were 5.13 and 5.38, respectively. An overall average N‐Prot factor for all species studied of 4.92 ± 0.59 (n = 57) was established. This study confirms that the use of the traditional factor 6.25 is unsuitable for seaweeds, and the use of the N‐Prot factors proposed here is recommended.

DISTRIBUTION OF INTRACELLULAR NITROGEN IN MARINE MICROALGAE : BASIS FOR THE CALCULATION OF SPECIFIC NITROGEN-TO-PROTEIN CONVERSION FACTORS

The utilization of nitrogen‐to‐protein conversion factors (N‐Prot factors) is a widely accepted and practical way to determine total protein content. The accuracy of protein determination depends on the establishment of specific N‐Prot factors, since the conventional factor of 6.25 may be unsuitable for all species. This study was designed to determine the concentrations of the main nitrogenous compounds and to establish N‐Prot factors specific for the following marine microalgae: Chlorella minutissima, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana, Nannochloropsis oculata, Phaeodactylum tricornutum, Prorocentrum minimum, Skeletonema costatum, Synechococcus subsalsus, and Tetraselmis gracilis. Cultures were maintained under a 12‐h photoperiod (300 μmol photons·m−2·s−1) at temperatures of 20.0°± 1.0° C (dark) to 23.0°± 2.0° C (light) in Walne’s culture medium without additional external carbon sources. The distribution of intracellular nitrogen was studied by determining total nitrogen (TN, by CHN [carbon, hydrogen, and nitrogen] analysis), protein N (PN, by analysis of total amino acids), and nonprotein N (NPN, determined by analysis of DNA, RNA, chlorophylls (chl) a,b, and c, and intracellular inorganic nitrogen—NO3−, NO2−, and NH3+ NH4+) in logarithmic and stationary growth phases of cultures. Variations occurred in both accumulation and distribution of PN and NPN among the species, as well as in each species during the different growth phases. Inorganic nitrogen compounds were observed to be the most important NPN source (from 6.4 ± 0.1% to 41.8 ± 4.2% of total N) in all species (except D. tertiolecta), followed by nucleic acids (from 0.8 ± 0.1% to 26.1 ± 2.4% of TN) and chlorophylls (from 0.2 ± 0.0% to 3.1 ± 0.3% of TN). Total amino acid residues ranged from 63.1 ± 4.6% up to 88.1 ± 11.2% of TN, which is in agreement with the presence of high NPN concentrations. N‐Prot factors are proposed for each growth phase in the studied species, based on the ratio of amino acid residues to TN, establishing specific N‐prot factors ranging from 3.60 ± 0.27 to 4.99 ± 0.64. The mean N‐Prot factor for all species/growth phases was 4.58 ± 0.11. The present study shows that the use of the traditional factor 6.25 is not suitable for these marine microalgae, and possibly for other species, because it overestimates their actual protein content.

Importância do diagnóstico e tratamento da fenilcetonúria

Phenylketonuria is the most common inborn error of amino acid metabolism. It is due to a deficiency of phenylalanine hydroxylase, which normally converts phenylalanine to tyrosine. A diet low in phenylalanine starting in the first month of life can significantly reduce mental retardation, the most important feature of the disease. The aim of the review is to discuss the difficulties found in the diagnosis of PKU and its variants, ranging from classic phenylketonuria to mild hyperphenylalaninaemia, and the effects of dietary restriction of phenylalanine on the growth and development of children. Also, we present the current controversies about the age of discontinuing the dietary treatment. This review summarizes the benefits and problems emerging from a prolonged therapy taking into account dietary compliance in different age groups, and discusses dietary alternatives to the synthetic amino acid mixtures free of phenylalanine, based on low phenylalanine protein hydrolysates. In addition, we show some information about the effects of maternal phenylketonuria on pregnancy outcome and infant development, if exposed to high phenylalanine levels intra uterineo.A fenilcetonuria (PKU) e o mais comum dos erros congenitos do metabolismo de aminoacidos. Resulta da deficiencia da fenilalanina hidroxilase, enzima que catalisa a conversao de fenilalanina em tirosina. A introducao de uma dieta com baixo teor de fenilalanina deve ter inicio nos primeiros meses de vida, de preferencia no primeiro mes, para evitar o retardo mental, manifestacao clinica mais severa da doenca. Foi elaborada revisao sobre essa tematica, que aborda desde a PKU classica ate a hiperfenilalaninemia branda, incluindo relato sobre a PKU maternal e os efeitos da exposicao do utero a altos niveis de fenilalanina sobre o feto.

Chemical and biochemical characterization of soybean produced under drought stress.

Brazil is the second soybean (Glycine max L. Merrill) producer and exporter in the world. In 2005, soybean cultivated in the southeastern region of the country suffered drought stress imposed by adverse high temperatures and low humidity during its reproductive stage. Little information is available regarding the effect of drought stress on the quality of grains. In this study chemical and biochemical characteristics of five soybean samples belonging to three different cultivars grown under drought stress were evaluated. The samples did not meet standards for marketing and contained high amounts of green seeds. Grains were analyzed for appearance, 100 seed weight, humidity, water activity, proteins, lipids, lipoxygenase 1 activity, peroxides, and pigment contents after harvest and after 20 months of storage at room temperature. Acidity was measured also after 30 months of storage. The values of water activity and humidity were 0.6-0.7 and 8.7-11.9%, respectively, and they did not change during storage time, but there was an increase in acidity, which alludes to lipase activity. The activity of lipoxygenase 1 was greatly affected. Immediately after harvest, the green pigments were represented mainly by pheophytin a, followed by pheophytin b, small quantities of chlorophyll b and chlorophyll a, and traces of other chlorophyll derivatives. After 20 months of storage almost all green pigments had disappeared. Drought stress probably enhanced membrane permeability, which led to a lower pH and promoted transformation of chlorophylls to pheophytins.

Composição química de tabletes de caldo de carne: nitrogênio protéico, não-protéico e fenilalanina

The dietary intervention to lower the phenylalanine (Phe) serum levels of patients with Phenylketonuria (PKU) requires a restricted supply and the precise knowledge of Phe contents in foods, in order to control the intake of this amino acid. This work aimed to get more accurate data for protein and Phe contents in soup cubes of two commercial products to give dieticians data they need for planning diets. The amino acid contents were estimated by ion-exchange chromatography and moisture, lipids, crude protein (Nx6.25) contents were determined according to AOAC (1995) methods. Fiber was determined enzymatically. Protein nitrogen was determined after acid precipitation. Our results showed in both products, similar moisture and lipid contents with mean values of 3.7 and 8.4%, respectively. Dietary fiber contents were lower than 2% but it is worthy to mention the high mineral contents 61% in product A and 54% in product B. The comparison of total nitrogen (TN) and protein nitrogen (PN), shows that approximately 95% of TN corresponds to N of non protein origin. The real protein contents were only 0.71g/100g and 0.84g/100g in samples A and B, respectively and we were not able to detect phenylalanine. Theoretically, based solely on calculations and considering that proteins contain about 4% Phe in their constitution, the samples contain a maximum of 34mg Phe /100g, meaning 3.6mg Phe per each cube of 10.5g. These small amounts of Phe could explain the enormous difficulty in detecting this amino acid. The high amount of non-protein N is due to the presence of monosodium glutamate, which does not allow a reliable protein estimation by Nx6.25, resulting in protein and Phe overestimation.

Biological importance of γ-glutamyl-S-methylcysteine of kidney bean (Phaseolus vulgaris L.)

The effects of S-methyl-L-cysteine (SMC) on growth and food intake by rats were investigated and in vivo absorption studies of gamma-glutamyl-S-methyl-L-cysteine and free SMC were performed. The results showed that the peptide is slowly absorbed and only free SMC could be detected in the blood. The diets (10% casein), containing SMC, depressed food intake and animals’ growth. Both effects were proportional to the SMC contents in the diets. The results obtained with pair-fed groups led to the conclusion that the depression in growth was caused by the refusal of the SMC diets. The addition of cystine to the SMC diet only partially restored normal growth. Histological examination of livers, kidneys and spleens of rats fed with SMC showed no tissue alterations, except for a significant increase in the kidney weights. The hematological features did not show any significant differences, but there was a slight increase in erythroid precursors and young forms in the bone marrow, which might indicate an increased erythrocyte turnover. These results suggest that the reduction in food intake could be associated with some defence mechanism against toxic substances.

Estudo da composição química de flocos de cereais com ênfase nos teores de fenilalanina

The chemical composition and the amount of phenylalanine in 13 types of breakfast cereals produced in Brazil and marketed in Sao Paulo were studied. All products showed high amounts of carbohydrates, higher than 77%, supplying a minimum of 345 kcal/100g. The protein contents (Nx5.7) ranged from 3.8 to 7.3%. The phenylalanine levels varied between 224 and 451 mg/100g of the products and the lowest levels were detected in cereals with a reduced protein content, corresponding to cereal flakes with high sugar contents. The amino acid analysis revealed that phenylalanine accounted for 5.96±0.12 g/100g recovered amino acids, with no significant statistical differences among the 13 products studied and showed to be independent of the cereal type. It was possible to establish a linear correlation between the micro-Kjeldahl nitrogen and the phenylalanine level of the samples (correl. coeff. 0.9887) and consequently estimate the phenylalanine contents based only on a simple nitrogen analysis and multiplying the protein content by 5.96. The calculated phenylalanine contents in the 13 products did not differ statistically from the data obtained by amino acid analysis.

Chemically determined total and available methionine in beans (Phaseolus vulgaris L.) and isolated protein fractions

Abstract The total and non-oxidized methionine contents in Phaseolus vulgaris L. whole beans and in isolated protein fractions from seven bean varieties were studied. The total methionine contents determined by classical ion-exchange chromatography (IEC) after performic acid oxidation and HCl hydrolysis, ranged in bean meals from 1.30 to 1.53% with an average of 1.42%. Total methionine in bean protein extracts, which accounted for both water- and salt-soluble proteins was similar (1.45%); water-soluble albumins showed an average methionine content of 1.20%. The non-oxidized (considered available) methionine contents in whole beans measured by reaction with BrCN and gas chromatography of methylthiocyanate formed, were unexpectedly higher than total methionine. These results might indicate that interfering compounds, probably S-methylcysteine or its dipeptide with glutamic acid, present in variable amounts in leguminous seeds, over-estimated the potentially available methionine in whole beans. The protein extracts and isolated albumin fractions were devoid of these interfering substances and, as expected, significantly lower available methionine contents were observed. The protein extracts showed values of methionine equivalents, determined as methylthiocyanate, between 0.70 and 0.90% in protein, 37% lower than total methionine determined by IEC, corresponding to an available methionine content of 63%. The albumins showed methylthiocyanate values varying between 0.65 and 0.85% in protein, on average 32% lower than values obtained by IEC, corresponding to 68% available methionine.

Nutritional reserves of Vochysiaceae seeds: chemical diversity and potential economic uses

Contents of proteins, carbohydrates and oil of seeds of 57 individuals of Vochysiaceae, involving one species of Callisthene, six of Qualea, one of Salvertia and eight of Vochysia were determined. The main nutritional reserves of Vochysiaceae seeds are proteins (20% in average) and oils (21. 6%). Mean of carbohydrate contents was 5. 8%. Callisthene showed the lowest protein content (16. 9%), while Q. cordata was the species with the highest content (30% in average). The contents of ethanol soluble carbohydrates were much higher than those of water soluble carbohydrates. Oil contents lay above 20% for most species (30. 4% in V. pygmaea and V. pyramidalis seeds). The predominant fatty acids are lauric (Q. grandiflora), oleic (Qualea and Salvertia) or acids with longer carbon chains (Salvertia and a group of Vochysia species). The distribution of Vochysiaceae fatty acids suggests for seeds of some species an exploitation as food sources (predominance of oleic acid), for other species an alternative to cocoa butter (high contents or predominance of stearic acid) or the production of lubricants, surfactants, detergents, cosmetics and plastic (predominance of acids with C(20) or C(22) chains) or biodiesel (predominance of monounsaturated acids). The possibility of exploitation of Vochysiaceae products in a cultivation regimen and in extractive reserves is discussed.