ß-glucosidases from Saccharomyces cerevisiae: production, protein precipitation, characterization, and application in the enzymatic hydrolysis of delignified sugarcane bagasse.

ß-glucosidase is an essential enzyme for the enzymatic hydrolysis of lignocellulosic biomass, as it catalyzes the final stage of cellulose breakdown, releasing glucose. This paper aims to produce ß-glucosidase from Saccharomyces cerevisiae and evaluate the enzymatic degradation of delignified sugarcane bagasse. S. cerevisiae was grown in yeast peptone dextrose medium. Partial purification of the enzyme was achieved through precipitating proteins with ethanol, and the optimal activity was measured by optimizing pH and temperature. The effects of ions, glucose tolerance, and heat treatment were evaluated. Delignified sugarcane bagasse was hydrolyzed by the enzyme. ß-glucosidase showed a specific activity of 14.0712 ± 0.0207 U mg-1. Partial purification showed 1.22-fold purification. The optimum pH and temperature were 6.24 and 54 °C, respectively. ß-glucosidase showed tolerance to glucose, with a relative activity of 71.27 ± 0.16%. Thermostability showed a relative activity of 58.84 ± 0.91% at 90 °C. The hydrolysis of delignified sugarcane bagasse showed a conversion rate of 87.97 ± 0.10% in the presence of Zn2+, an ion that promoted the highest increase in enzymatic activity. S. cerevisiae produced an extracellular ß-glucosidase with good stability at pH and temperatures conventionally applied in the hydrolysis of lignocellulosic biomass, showing viability for industrial application.

Application of Immobilized ß-Glucosidase from Candida boidinii in the Hydrolysis of Delignified Sugarcane Bagasse.

Candida boidinii is a methylotrophic yeast with wide geographical distribution. In the present study, the microorganism was isolated from the Bahian semiarid and the enzymatic extract containing ß-glucosidase was obtained through submerged fermentation. Response surface methodology was employed to optimize of fermentation medium. The higher production of ß-glucosidase was obtained after 71 h of fermentation in an optimized medium composed of 3.35% glucose, 1.78% yeast extract and 1% peptone. The optimum pH and temperature of enzymatic activity were 6.8 (citrate-phosphate buffer) and 71.7 °C, respectively. Salts tested (10 mM) CaCl2, Na2SO4 and ZnSO4 promotes the increase of 91%, 45% and 80% of activity, respectively. The enzyme retained 64% ± 2.3 of its initial activity after 1 h heating at 90 °C. The production of reducing sugars was 95.94% after 24 h of hydrolysis and, with the addition of metal ions, this value increased more than 2 times. Among the supports analyzed for immobilization, chitosan showed higher residual activity during reuse. The immobilized enzyme showed higher activity at 60 °C with pH 6 and preserved almost 100% of the initial activity after 30 min at 70 °C.

Antinociceptive and anti-inflammatory properties of α-D-mannan from the yeast Kluyveromyces marxianus: evidence for a role in interleukin-6 inhibition.

The management of inflammatory states typically involves non-steroidal anti-inflammatory drugs (NSAIDs) and opiates. Understanding the mechanisms underlying the processing of nociceptive information from potential alternatives such as some polysaccharides may enable new and meaningful therapeutic approaches. In this study, α-D-mannan isolated from the Kluyveromyces marxianus cell wall produced antinociceptive effects in models of inflammatory pain (formalin and complete Freund's adjuvant tests). Furthermore, α-D-mannan reduced paw edema and interleukin-6 (IL-6) production after carrageenan-induced inflammation. The polysaccharide α-D-mannan was characterized by gas chromatography-mass spectrometry, methylation analysis, and spectroscopic techniques. Moreover, the Doehlert experimental design was applied to find the optimal conditions for biomass production, with the best conditions being 10.8 g/L and 117 h for the glucose concentration and the fermentation time, respectively. These results indicate that α-D-mannan from K. marxianus exerts anti-inflammatory and antinociceptive effects in mice, possibly via a mechanism dependent on the inhibition of IL-6 production.

The improvement of guava (Psidium guajava) juice quality using crude multi-enzymatic extracts obtained from yeasts.

Guava juice is cloudy and viscous, which hinders filtration, decreases yield, and causes the loss of quality after its processing and during storage. This study aimed to evaluate enzymatic treatment effects using crude multi-enzymatic extracts (CME) obtained from Rhodotorula mucilaginosa, Rhodotorula orizycola, and Pseudozyma sp. produced by submerse fermentation in the extraction of juice guava. Mixtures of 100 ml of guava pulp and multi-enzymatic extracts proposed by Doehlert planning were incubated under constant agitation at 150 rpm and 50°C, and a Doehlert design was applied as a multivariate optimization strategy. The optimal conditions using the multi-enzymatic extract were: 0.4% (v/v) of CME for 131 min for the multi-enzymatic treatment using Pseudozyma sp.; 3.0% (v/v) of CME for 154 min using the R. mucilaginosa CME; and 5.0% (v/v) of CME for 90 min using R. oryzicola. The maximum viscosity reduction values for the juices treated with the CME of yeasts were 10.33%, 86.38%, and 13.33% for the juices treated with the CME of Pseudozyma sp., R. mucilaginosa, and R. orizycola, respectively. The physical-chemical properties were improved after treatment with CMEs, yielding a reduction of clarity, increase of total soluble solids and reducing sugars, and decreasing the acidity (pH) for all treatments with enzymatic extracts of all strains. The yeasts studied showed a potential for CME production to be applied to juice, improving the quality of the juice, and R. mucilaginosa was the most prominent yeast due to most significant reduction of viscosity in guava juice.

Production of ß-glucosidase by Rhodotorula oryzicola and use of enzyme for hydrolysis of sugarcane bagasse delignified.

Bioethanol is obtained by hydrolysis of sugarcane bagasse by cellulases. Commercial cellulases are expensive and have a low concentration of ß-glucosidase (EC 3.2.1.21), which decrease hydrolysis efficiency. The present work aims to produce supernatant rich in ß-glucosidase (BGL) using the yeast Rhodotorula oryzicola and apply it in the hydrolysis of delignified sugarcane bagasse. Yeast fermented in a modified YPD (Yeast Peptone Dextrose) medium with 0.5% (w/v) cellobiose and 1.0% (w/v) glucose produced BGL with a specific activity of 1.44 ± 0.013 U/mg. Partial purification of BGL by acetone showed a specific activity of 3.48 U/mg. The optimum pH and temperature were 6.02 and 65 °C, respectively. BGL partially purified (BGLppR.oryzicola) by acetone showed tolerance to glucose, with a relative activity of 82.89 ± 0.11%. The activity increased with the addition of iron sulfate and zinc sulfate and decreased with manganese sulfate. BGL partially purified was thermal stable, with a relative activity of 85.59% after 60 min at 90 °C. BGL partially purified applied in the hydrolysis of sugarcane bagasse delignified with 3% (w/w) NaOH + 6% (w/w) Na2SO3 showed a conversion rate of 72.46 ± 1.60%. The results showed that BGL partially purified is a glucose tolerant cellulase of low-cost, promising the application of bioethanol production.

ß-Glucosidase produced by Moniliophthora perniciosa: Characterization and application in the hydrolysis of sugarcane bagasse.

ß-Glucosidases (BGLs) belong to the group of enzymes of cellulases and act in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of BGL from Moniliophthora perniciosa in the hydrolysis of pretreated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein.

Production, characterization, and immobilization of protease from the yeast Rhodotorula oryzicola.

The protease was produced extracellularly in submerged fermentation by the yeast Rhodotorula oryzicola using different sources of nitrogen and maximum activity (6.54 × 10-3 U/mg) was obtained in medium containing 2% casein (w/v). Purification of the protease by gel filtration chromatography resulted in a 3.07-fold increase of specific protease activity. The optimal pH and temperature for enzyme activity were 6.51 and 63.04 °C, respectively. Incubation in the presence of some salts enhanced enzyme activity, which peaked under 0.01 M BaCl2 . The enzyme retained about 90% of enzymatic activity at temperatures 50-60 °C. The commercially available enzyme carriers evaluated, silica gel, Celite 545, and chitosan effectively immobilized the protease. The enzyme immobilized in Celite 545 retained 73.53% of the initial activity after 15 reuse cycles. These results are quite promising for large-scale production and immobilization of protease from R. oryzicola, as the high operational stability of the immobilized enzyme lowers production costs in biotechnological applications that require high enzymatic activity and stability under high temperatures.

Exploring "wear and tear" of joints and "muscle function" assumptions in skeletons with known occupation at death.

OBJECTIVES: Changes in the joints are believed to result from "wear and tear," a consequence of activity, as entheseal changes (EC) result from muscle use. However, clinical data showed that activity does not necessarily increase the likelihood of degenerative joint changes (DJC) and that exercise results in healthier joints. We tested whether individuals with continuous repetitive biomechanical efforts (Group 1) were more likely to exhibit EC and if occupations known to exert strenuous but discontinued efforts (Group 2) would more likely cause DJC. MATERIALS AND METHODS: Eighty-nine males with known occupations from Portuguese identified collections were used: shoemakers and carpenters (Group 1), workers (Group 2), and civil servants, and shop assistants as a control group. Major upper and lower limb joints and entheses sites were used. DJC and EC were tested between occupations - while controlling for age (overall approach) - and within occupation (occupation-specific approach). RESULTS: The overall approach showed that age - as a covariant - had a significant impact on DJC and EC development (p < 0.05), with occupation being non-significant (p > 0.05) despite the variability in the mean-values of lesions. The occupation-specific approach showed a significant variability of DJC and EC correlations, within and between occupations, with no clear trend of DJC and EC development according to occupation. DISCUSSION: The results showed that exploring overall patterns might conceal occupation-specific joint and muscle use, emphasizing age as a major contributor of changes; and that the occupation-specific approach highlighted particular aspects associated with occupations, allowing for a more informative assessment of strenuous repetitive or discontinuous activity-related technical gestures and their impact on skeletal biology.

Clarification of tangerine juice using cellulases from Pseudoyma sp.

Tangerine juice was treated with crude extract containing cellulase from Pseudozyma sp. obtained by liquid fermentation. The thermal stability of cellulase was investigated by incubating crude extract at different temperatures and times. The pulp, obtained from tangerine, was pasteurized at 85 °C for 5 min and then used in a clarification process with a Doehlert experimental design. The results showed that the cellulase obtained from Pseudozyma sp. is thermostable at temperatures of 60, 70 and 90 °C and retained 98%, 88% and 80% of activity, respectively, after a 1-h incubation time. The optimum conditions for clarification were verified by varying the enzyme extract concentration (%, v v-1) and the time (minutes) in a shaker at 150 rpm, at 50 °C. The optimum condition for clarification was obtained in the 80th min with a 1.25% enzymatic extract concentration (v v-1), resulting in a reduction of tangerine juice viscosity by 65%. The analysis of physical and chemical parameters of tangerine juice after clarification showed that the enzyme extract improved the process responsible for the clarification of tangerine juice. The results are promising since this is a methodology that can be used in the citrus juice industry.

The macroscopic and histomorphological properties of periosteal rib lesions and its relation with disease duration: evidence from the Luis Lopes Skeletal Collection (Lisbon, Portugal).

Periosteal new bone formation (PNBF) is a common finding in a large spectrum of diseases. In clinical practice, the morphology and location of periosteal lesions are frequently used to assist in the differential diagnosis of distinct bone conditions. Less commonly reported is the presence of PNBF on the ribs. This contrasts with the data retrieved from the study of skeletonized human remains that shows a high frequency of cases and a strong, albeit not specific, association between periosteal rib lesions and pulmonary conditions (e.g. tuberculosis). Despite that, an overall disagreement regarding the specificity and non-specificity of periosteal reactions exists in the study of dry bone remains. The insufficient number of clinical models exploring the morphology and the pathophysiology of PNBF's and the lack of systematic studies of pathological samples with a known diagnosis are claimed as major reasons for the disagreements. This study aimed to describe and compare the macroscopic and the histomorphologic appearance of periosteal rib lesions and to discuss their usefulness as diagnostic indicators. To pursue this goal, an assemblage of 13 rib samples (males = 11, females = 2, mean age-at-death = 36.6 years old) was collected from the Luis Lopes Skeletal Collection (Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Portugal). The assemblage belongs to individuals who died from pulmonary-TB (group 1), non-TB pulmonary infections (group 2) and other conditions (group 3). Prior to sample preparation, the ribs were visually inspected and the PNBF described according to its thickness, the degree of cortical integration and the type of new bone formed (e.g. woven, lamellar or both). After sampling, each bone sample was prepared for histological analysis under plane and polarized light microscopy. Macroscopically, the results showed no differences in the new bone composition between cause-of-death groups. Only slight differences in the degree of cortical integration, which was most frequently classified as mild to high in the pulmonary-TB group, were observed. Histologically, no distinguishing features were identified by pathological group. However, new bone microarchitectures were observed compatible with (1) acute, fast-growing processes (e.g. spiculated reactions), (2) long-standing processes with a rapid bone formation (e.g. appositional layering of bone) and/or (3) chronic, slow-growing processes (e.g. layers of compact lamellae). To some extent, these distinct rates of disease progression resonate with the cause-of-death listed for some individuals. Despite the small sample size, the results of this investigation are in agreement with previous studies, according to which the macroscopic and histological appearance of periosteal formations are not specific for a particular pathological conditions. Nevertheless, the results support the conclusion that the morphology of periosteal lesions is a good biological indicator for inferring the rate of progression and duration of pathological processes. This study provides important reference data regarding the histomorphology of periosteal lesions that can be used for comparative purposes, as well as to narrow down the differential diagnosis in unidentified skeletal remains.

Production and partial characterization of ß-1,3-glucanase obtained from Rhodotorula oryzicola.

The current study aims to assess the kinetics of population growth of Rhodotorula oryzicola and the production of ß-1,3-glucanase (EC 3.2.1.39) enzyme by this yeast. It also aims to obtain the optimum conditions of ß-1,3-glucanase enzymatic activity by varying the pH as well as to study the enzyme thermostability. R. oryzicola population doubled within 12 hr. During this period, 9.26 generations were obtained, with 1 hr and 29 min of interval from one generation to the other, with specific growth rate (µ) of 0.15 (hr-1). The entire microorganism growth process was monitored during ß-1,3-glucanases production, and the maximum value was obtained in the stationary phase in the 48-hr fermentation period. pH and temperature optimum values were 4.7 and 96°C, respectively. The enzyme maintained 88% of its activity when submitted to the temperature of 90°C for an incubation period of 1 hr. The results show that the enzyme can be used in industrial processes that require high temperatures and acidic pH.

The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells.

The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Application of aqueous biphasic systems as strategy to purify tannase from Aspergillus tamarii URM 7115.

The aims of the current study are to assess the influence of polyethylene glycol (PEG) concentration, molar mass, pH, and citrate concentrations on aqueous biphasic systems based on 24 factorial designs, as well as to check their capacity to purify tannase secreted by Aspergillus tamarii URM 7115. Tannase was produced through submerged fermentation at 26°C for 67 h in Czapeck-Dox modified broth and added with yeast extract and tannic acid. The factorial design was followed to assess the influence of PEG molar mass (MPEG 600; 4,000 and 8,000 g/ mol), and PEG (CPEG 20.0; 22.0 and 24.0% w/w) and citrate concentrations (CCIT 15.0, 17.5, and 20.0%, w/w), as well as of pH (6.0, 7.0, and 8.0) on the response variables; moreover, partition coefficient (K), yield (Y), and purification factor (PF) were analyzed. The most suitable parameters to purify tannase secreted by A. tamarii URM 7115 through a biphasic system were 600 (g/mol) MPEG, 24% (w/w) CPEG, 15% (w/w) CCIT at pH 6.0 and they resulted in 6.33 enzyme partition, 131.25% yield, 19.80 purification factor and 195.08 selectivity. Tannase secreted by A. tamarii URM 7115 purified through aqueous biphasic systems composed of PEG/citrate can be used for industrial purposes, since it presents suitable purification factor and yield.

Comparison between the univariate and multivariate analysis on the partial characterization of the endoglucanase produced in the solid state fermentation by Aspergillus oryzae ATCC 10124.

Endoglucanase production by Aspergillus oryzae ATCC 10124 cultivated in rice husks or peanut shells was optimized by experimental design as a function of humidity, time, and temperature. The optimum temperature for the endoglucanase activity was estimated by a univariate analysis (one factor at the time) as 50°C (rice husks) and 60°C (peanut shells), however, by a multivariate analysis (synergism of factors), it was determined a different temperature (56°C) for endoglucanase from peanut shells. For the optimum pH, values determined by univariate and multivariate analysis were 5 and 5.2 (rice husk) and 5 and 7.6 (peanut shells). In addition, the best half-lives were observed at 50°C as 22.8 hr (rice husks) and 7.3 hr (peanut shells), also, 80% of residual activities was obtained between 30 and 50°C for both substrates, and the pH stability was improved at 5-7 (rice hulls) and 6-9 (peanut shells). Both endoglucanases obtained presented different characteristics as a result of the versatility of fungi in different substrates.

Below the Callus Surface: Applying Paleohistological Techniques to Understand the Biology of Bone Healing in Skeletonized Human Remains.

OBJECTIVES: Bone trauma is a common occurrence in human skeletal remains. Macroscopic and imaging scrutiny is the approach most currently used to analyze and describe trauma. Nevertheless, this line of inquiry may not be sufficient to accurately identify the type of traumatic lesion and the associated degree of bone healing. To test the usefulness of histology in the examination of bone healing biology, we used an integrative approach that combines gross inspection and microscopy. MATERIALS AND METHODS: Six bone samples belonging to 5 adult individuals with signs of bone trauma were collected from the Human Identified Skeletal Collection from the Museu Bocage (Lisbon, Portugal). Previous to sampling, the lesions were described according to their location, morphology, and healing status. After sampling, the bone specimens were prepared for plane light and polarized light analysis. RESULTS: The histological analysis was pivotal: (1) to differentiate between types of traumatic lesions; (2) to ascertain the posttraumatic interval, and (3) to diagnose other associated pathological conditions. CONCLUSION: The outer surface of a bone lesion may not give a complete picture of the biology of the tissue's response. Accordingly, microscopic analysis is essential to differentiate, characterize, and classify trauma signs.

Exploring poverty: skeletal biology and documentary evidence in 19th-20th century Portugal.

BACKGROUND: The inference of the state of wealth or poverty from human skeletal remains is a difficult task, as the limited number of skeletal changes are mediated by numerous other physiological, biomechanical and pathological events. In recent years, identified skeletal collections have become valuable resources in enabling aetiologies of these changes to be understood while controlling for some known causative factors, e.g. age, sex and occupation. This has favoured more rigorous data analysis and interpretation. AIM: This study compares the presence of osteological makers of occupation - specifically degenerative joint changes (DJC) - between socio-economically framed occupational groups whilst controlling for age-at-death. MATERIALS AND METHODS: A total of 603 individuals were distributed into seven occupational groups used as a proxy for their socio-economic status. RESULTS: The results demonstrated that age was a contributing factor for DJC. Differences between occupational groups were only found for the hips, right shoulder and ankle. CONCLUSIONS: Differences found were not necessarily representative of low vs high socio-economic status. Furthermore, there are limitations associated with the use of occupation-at-death, based on documentary evidence, which does not necessarily reflect wealth-status.

Exploring the relationship between entheseal changes and physical activity: a multivariate study.

Analyses of entheseal changes (EC) in identified skeletal samples employ a common research strategy based on the comparison between occupations grouped on the basis of shared biomechanical and/or social characteristics. Results from this approach are often ambiguous, with some studies that point to differences in EC between occupational samples and others failing to provide evidence of behavioral effects on EC. Here we investigate patterns of EC among documented occupations by means of a multivariate analysis of robusticity scores in nine postcranial entheses from a large (N = 372) contemporary skeletal sample including specimens from one Italian and two Portuguese identified collections. Data on entheseal robusticity, analyzed by pooled sides as well by separated sides and levels of asymmetry, are converted in binary scores and then analyzed through nonlinear principal component analysis and hierarchical cluster analysis. Results of these analyses are then used for the classification of occupations. Differences between occupational classes are tested by MANOVA and pairwise Hotelling's test. Results evidence three classes which separate occupations related to farming, physically demanding but generalized occupation, and physically undemanding occupations, with the more consistent differences between the first and the last classes. Our results are consistent with differences in biomechanical behavior between the occupations included in each class, and point to the physical and social specificity of farming activities. On the other hand, our study exemplifies the usefulness of alternative analytical protocols for the investigation of EC, and the value of research designs devoid of a priori assumptions for the test of biocultural hypotheses.

Bone Diagenesis and its Implication for Disease Diagnosis: The Relevance of Bone Microstructure Analysis for the Study of Past Human Remains.

When bone is exposed to the burial environment it may experience structural changes induced by multiple agents. The study of postmortem alterations is important to differentiate decomposition phenomena from normal physiological processes or pathological lesions, as well as to assess bone tissue quality. Microscopy is of great utility to evaluate the integrity of bone microstructure and it provides significant data on long-term bone decomposition. A total of 18 human bone sections (eight archeological and ten retrieved from an identified skeletal collection) were selected for analysis under plane light and polarized light. The aim of this exploratory study was to analyze the impact of diagenesis and taphonomy on the bone microstructure, as well as on the differential diagnosis of pathological conditions. The results showed that the microscopy approach to bone tissues contributed materially as an aid in the detailed description of the main diagenetic changes observed. It showed that gross inspection does not provide a realistic assessment of bone tissue preservation, which can impact in the characterization of lesions present and subsequent disease diagnosis. Therefore, researchers should continue to consider the application of histological techniques if the aim is to comprehend tissue integrity and its association with decomposition or disease.

Production, Characterization and Application of a Thermostable Tannase from Pestalotiopsis guepinii URM 7114.

Tannase (EC 3.1.1.20) is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun (Syzygium cumini (L.) Skeels) leaves, and identified as Pestalotiopsis guepinii, in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL). Response surface methodology was employed to evaluate the effect of variables (pH and temperature), and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. Km was found to be 7.18·10-4 mol/L and vmax =250.00 U/mL. The tannase activity was the highest in the presence of Ca2+ at a concentration of 5·10-3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3% of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology.

Reference intervals for serum immunoglobulins G, M and E levels in Brazilian children aged 1 to 10 years: a population-based study.

OBJECTIVE: To determine the reference intervals (RI) for serum levels of IgG, IgM, and IgE in healthy children aged 1-10 years living in central Brazil. METHOD: A sample of 1743 healthy children was randomly selected from kindergartens and public schools. Reference intervals were defined by non-parametric rank (Clinical Laboratory Standards Institute, USA), bootstrapping, and Horn's robust methods. RESULTS: By the rank method, the IgG RI was 792-1771 mg/dL for children of both sexes aged 1-10 years. IgM RI were different for gender and age groups, being 45-196 mg/dL and 34-190 mg/dL for boys aged 1-2 years and 3-10 years, respectively. For girls, the IgM RI were 50-212 mg/dL and 39-212 mg/dL, for ages 1-4 and 5-10 years, respectively. The IgE RI for both sexes and ages 1-10 years was 6-1005 mg/dL. The bootstrap method showed RI similar to the rank method but with slightly different confidence intervals. Horn's robust method determined RI different from those obtained by previous methods. CONCLUSION: RI for serum concentrations of IgG, IgM, and IgE were established for Brazilian children aged 1-10 years. This definition will be useful for Brazilian physicians, who will have more adequate parameters for their clinical decision-making.