Adjuvant effect of a probiotic fermented milk in the protection against Salmonella enteritidis serovar typhimurium infection: mechanisms involved.

Probiotics may offer protection against Salmonella enteritidis serovar Typhimurium infection via different mechanisms. The aim of this study is to investigate, using mouse models, the effect of the administration of fermented milk containing the probiotic bacteria L. casei DN-114 001 in the protection against Salmonella enteritidis serovar Typhimurium when this product is administered continuously before and after infection or only post-infection. The adjuvant effect of this probiotic fermented milk (PFM) against S. Typhimurium was also evaluated in newborn mice, whose mothers received the PFM during the suckling period or their offspring after weaning. The results obtained showed that PFM administration after salmonella infection was useful to decrease the severity of the infection. The best effect was obtained with continuous PFM administration. In the newborn mice model, PFM administration to the newborn mice after weaning showed the best effect against the pathogen. PFM administration to the mother during the suckling period was beneficial against this enterophatogen when their offspring did not receive probiotics after weaning. Continuous PFM administration to adult mice (before and after infection) was important to maintain the intestinal barrier and the immune surveillance in optimal conditions to diminish the pathway of entrance of salmonella and the spread of this pathogen to deeper tissues. In the newborn mice model, it was observed that PFM administration to the offspring after weaning or their mother during the suckling period had a protective effect against salmonella infection, however, in the mice from mothers that received PFM during nursing which were fed with PFM after weaning, we found a down regulated immune maturity that was not protective against this infection.

Respiratory allergy control by probiotic fermented milk intake: a mouse model from weaning to maturity.

This study is based on our previous research showing that commercial probiotic fermented milk (PFM) intake mitigates respiratory allergy development to ovalbumin (OVA) in adult mice (6-weeks old) increasing specific immunoglobulin (Ig)G2a and interferon (IFN)-γ rather than IgE. The aim was to determine if PFM exerts a protective effect when an allergy model is induced 5 days after weaning and whether the mechanisms involved are similar to those previously reported. Before inducing allergy, a group of 21-day old BALB/c mice received PFM for 10 days to analyse the impact on intestinal epithelial cells (IECs) activation. Two more groups received PFM for 5 days and were sensitised with OVA; only one group continued taking PFM until the end of the experiment. Sensitisation scheme: 3 OVA injections 1% in phosphate buffered saline (PBS) plus 7 days OVA aerosol exposure and re-stimulus 15 days later. The contents of specific- IgE, IgG, total-secretory-IgA and Th1/Th2 balance in serum, bronchoalveolar lavage (BAL) and gut were measured at 7 and 15 days post-sensitisation (dPS) and 2 days post-re-stimulus (2dPR). Treg cells in lungs were also quantified. Results were compared with normal and sensitised controls. PFM induced mild activation of IECs increasing monocyte chemoattractant protein-1 (MCP-1 or CCL2) and interleukin (IL)-6 production. In sensitised mice, PFM controlled the response inducing IgG rather than IgE at 7 and 15-dPS and 2dPR (60 days old). Th1-balance (IFN-γ) was favoured by PFM in lungs at 7 dPS with low levels of IL-10 released to regulate the response. Total-S-IgA increased in lungs and gut; however, PFM intake did not affect Treg cells in lungs. PFM maintains controlled stimulation of the immune cells involved in Th1 response, favouring IgG at the respiratory mucosal site. Although the effect was not as strong as that reported previously, PFM promoted maturation and activation of gut immune cells preserving intestinal homeostasis and lung immune response.

Effect of long-term continuous consumption of fermented milk containing probiotic bacteria on mucosal immunity and the activity of peritoneal macrophages.

The effect of the long-term administration of commercial fermented milk containing probiotic bacteria in the mucosal immune response and peritoneal macrophages was analyzed. BALB/c mice were fed with fermented milk for 98 consecutive days. Small and large intestines were removed for histology; IgA, CD4, CD8 cells and cytokines-producing cells were counted. The influence on the immune cells associated with bronchus and mammary glands as well as on peritoneal macrophages was also analyzed. Continuous oral administration of fermented milk increased IgA+ cells in both parts of the intestine (small and large intestine). IL-10, a regulatory cytokine, increased in the intestinal cells in most samples. TNFalpha, IFNgamma and IL-2 producing cells were also enhanced. Values for CD4 and CD8(+) cell populations in lamina propria of the intestine were increased in relation to the control throughout the assay. No modifications in the histology of intestines were observed. Long-term consumption of fermented milk enhanced intestinal mucosa immunity, mediated by IgA+ cells and by cytokine production. This improvement of gut immunity was maintained and down-regulated by cytokines such as IL-10, preventing gut inflammatory immune response. The effect of this fermented milk on mucosal sites distant to the gut, such as bronchus and mammary glands, showed that in both tissues the increase in IgA+ cells was only observed at the beginning of the continuous consumption and no modifications in the number of cytokine positive cells were found. Similar observations were found when phagocytic activity of peritoneal macrophages was measured. It was demonstrated that the most evident effect of long-term consumption of fermented milk was observed in the intestine. Immunodulatory effects and the maintenance of intestinal homeostasis without secondary effects after long-term administration of fermented milk were also observed.

Iron bioavailability from fortified petit suisse cheese determined by the prophylactic-preventive method.

In this research, we measured the iron bioavailability of ferrous gluconate stabilized with glycine (SFG) when it is used to fortify petit suisse cheese using the prophylactic-preventive method in rats. Three groups of male, weaned rats received a basal diet (control diet; 5.2 ppm Fe), a reference standard diet (SO4Fe; 9.2 ppm Fe), and a basal diet using iron-fortified petit suisse cheese as the iron source (cheese diet; 8.8 ppm Fe) for 22 d. The iron bioavailability was calculated as the ratio between the mass of iron incorporated into hemoglobin and the total iron intake per animal during the treatment. These values (BioFe) were 68% and 72% for SFG and ferrous sulfate, respectively. The value of the Relative Biological Value (RBV) was 95% for SFG in petit suisse cheese. These results show that according to this method, the iron bioavailability from industrial fortified petit suisse cheese can be considered as a high bioavailability rate.

Dietary zinc effects on zinc, calcium, and magnesium content in bones of growing rats.

The aim of the present study was to assess dietary zinc effects on femur weight and mineral content in growing rats. For this purpose, 70 weanling Sprague-Dawley rats were divided into four groups. Each group was subject to a diet containing 2 (BZ), 5 (DZ), 10 (MZ), and 30 (CZ) ppm zinc. The calcium and magnesium content in all diets was 5 g/kg and 507 mg/kg, respectively. The animals were kept on this regime for 28 d and then sacrificed and their femurs were removed for analysis using atomic absorption spectrophotometry. The weights of the BZ and DZ groups were significantly different from the MZ and CZ groups (38.5+/-10.5, 89.9+/-13.7, 118.6+/-13.6, and 134+/-19.9 g, p<0.01) respectively. There were no differences between the MZ and CZ groups. Femur weight also varied with dietary zinc, as it was significantly different among all groups (BZ, 265+/-49 mg; DZ, 380+/-40 mg; MZ, 452+/-54 mg; CZ, 735+/-66 mg; p<0.01). The femur zinc content varied with diets, following a different pattern than the above parameters. Femur zinc from the BZ group (51.5+/-5.4 ppm) was significantly different from the MZ and CZ groups (115.9+/-14.2 and 175.0+/-13.5 ppm, respectively), whereas the DZ group (62.5+/-11.3 ppm) did not differ from the other three groups. The femur content of calcium (BZ, 83.2+/-9.8 mg/g; DZ, 88.0+/-9.2 mg/g; MZ, 90.2+/-13.6 mg/g; CZ, 83.1+/-14.7 mg/g) and magnesium (BZ, 1.82+/-0.13 mg/g; DZ, 1.98+/-0.09 mg/g; MZ, 1.93+/-14 mg/g; CZ, 1.83+/-0.19 mg/g) were not significantly different among the groups, nor was the calcium-magnesium ratio. These results suggest that although dietary zinc deficiency retards growth and causes bone fragility, bone deposition of calcium and magnesium and its ratio are not affected.

Determination of relative bioavailability of zinc in a petit suisse cheese using weight gain and bone zinc content in rats as markers.

The aim of the study was to determine the relative bioavailability of zinc gluconate stabilized with glycine in a Petit Suisse cheese from an infant dessert. Weight gain and bone zinc content were the nutritional responses evaluated for the diets of different zinc content: 2 ppm (basal) and 5, 10, and 30 ppm from zinc gluconate stabilized with glycine and zinc sulfate. Nonlinear regression analysis of the fitted curves for weight gain determined a relative zinc bioavailability of 100% for the Ymax ratio and 96% for Ymax/t1/2 ratio for zinc gluconate stabilized with glycine (R2=0.7996 for zinc sulfate and 0.8665 for zinc gluconate stabilized with glycine). The slope ratio analysis from linear regression of femur zinc determined a relative zinc bioavailability of 93% for zinc gluconate stabilized with glycine (R2=0.8693 for zinc sulfate and 0.8307 for zinc gluconate stabilized with glycine). Zinc gluconate stabilized with glycine has similar bioavailability as zinc sulfate in a Petit Suisse cheese nutritional matrix, with the advantage that the stabilized compound does not modify the sensorial characteristics of the fortified cheese.

Normal growth rate in rats is recovered after a period of zinc deficiency by restoration of zinc supply by means of a zinc-fortified Petit Suisse cheese.

Fortification of a Petit Suisse cheese with zinc sulfate and zinc gluconate stabilized with glycine was used as a tool to overcome zinc-deficiency effects on total-body growth and skeletal growth. Animals were divided in 4 groups of 10 rats: basal (B), control (C), depletion-repletion 1 (DR1), and depletion-repletion 2 (DR2). These four groups were fed with four diets: basal (2 ppm Zn), control (30 ppm Zn), DR1, and DR2; they received a basal diet for 14 d and a control diet for the other 14 d of the experiment, using zinc sulfate for DR1 and zinc gluconate stabilized with glycine for DR2. After 28 d of the experiment, total-body weight and weight gain of the control and DR1 and DR2 animals were not statistically different (p<0.05), Femur weight and femur zinc content of DR1 and DR2 did not achieve the values of control animals (p<0.05), but they were higher than that of basal animals. Our results show that restoration of dietary zinc levels by means of food fortification normalized weight gain, as an indicator of total-body growth, and presented a trend to normalize bone weight, as a marker of skeletal growth, in young rats and independently of the zinc source used.

Collagenase in mineralized tissues of human teeth.

A collagenase cleaving native type I [14C]collagen but inactive against the synthetic substrate Pz-Pro-Leu-Gly-Pro-D-Arg was extracted from mineralized human dental tissue. The enzyme specifically degrades native collagen into characteristic products (3/4) and (1/4). Its apparent molecular mass of 68 kDa is relatively high in comparison with collagenases from other oral tissues. The enzyme is a metalloproteinase inhibited by low concentrations of the chelating agents EDTA, 1, 10-phenanthroline, alpha alpha'-dipyridyl, and not affected by diisopropylfluorophosphate, soybean trypsin inhibitor, and p-chloromercuribenzoate. It is stable to lyophilization and can be stored at-20 degrees C for at least 6 months.

A new procedure to fortify fluid milk and dairy products with high-bioavailable ferrous sulfate.

The behavior of SFE-171 used for food fortification was studied. The biological and nutritional properties of this new iron source are discussed in this work.

Ultrastructural study of the protein-polysaccharides distribution in enamel from rat incisors.

The distribution pattern of the protein-polysaccharides in the rat incisor enamel was similar whether stained for glycoproteins (phosphotungstic acid) or for proteoglycans (alcian blue, bismith nitrate). Except for the holes at the dentino-enamel junction, where these components seem to exist as amorphous gel, protein-polysaccharides are closely associated to the proteinic fibrous matrix in rods and interrods structures as in prism sheaths where they are particularly abundant.

Bioavailability, biodistribution, and toxicity of BioZn-AAS(1): a new zinc source. comparative studies in rats.

Food fortification with a proper zinc compound is an economic and effective strategy to prevent zinc deficiency. BioZn-AAS, a zinc gluconate stabilized with glycine, was compared with zinc sulfate (reference standard), zinc hydroxide, and zinc gluconate, all of them labeled with (65)Zn. This preclinical study was performed on Sprague-Dawley rats of both sexes, and the administered dose was 85 microg/kg of zinc. Bioavailability studies showed that absorption of BioZn-AAS was not statistically different than absorption from other sources in female rats (25.65% +/- 2.20% for BioZn-AAS, 28.24% +/- 4. 60% for ZnSO(4), 24.91% +/- 4.02% for Zn[OH](2), and 25.51% +/- 2. 70% for Zn-gluconate). In the case of the male rats, absorption of BioZn-AAS (27.97% +/- 4.20%) was higher (P<0.05) than that from the other compounds (23.15% +/- 2.90% for ZnSO(4), 22.62% +/- 3.90% for Zn[OH](2), and 22.30% +/- 3.90% for Zn-gluconate). Biodistribution studies demonstrated that the zinc from BioZn-AAS followed the same metabolic pathway as zinc from the other sources. Toxicity studies were performed with 50 female and 50 male rats. The value of oral lethal dose 50 (LD(50)) was 2000 mg/kg for female rats and 1900 mg/kg for male rats. Therefore, we conclude that BioZn-AAS has adequate properties to be considered a proper zinc compound for food fortification or dietary supplementation.

Bioavailability study of dried microencapsulated ferrous sulfate--SFE 171--by means of the prophylactic-preventive method.

Microencapsulated ferrous sulfate with soy lecithin (SFE-171) has been used as an iron source for the fortification of milk and dairy products. With the purpose to extend the use of this agent to other kind of foods or even to pharmaceutical preparations for oral administration, the SFE-171 was turned into a fluid powder (SFE-171-P) by means of vacuum drying. The iron bioavailability (BioFe) of SFE-171-P was evaluated in this work by means of the prophylactic-preventive method in rats, using ferrous sulfate as reference standard. Both iron sources were separately added to a basal diet of low iron content in a concentration of 10 mg iron/kg diet. Two groups of 10 weaned rats 25 days old received the fortified diets during 28 days, while a third group of the same size received the basal diet without iron additions. The weights and haemoglobin concentrations (HbC) of every animal were determined before and after the treatment, thus allowing the calculation of the mass of iron incorporated into haemoglobin (HbFe) during this period. The BioFe of the iron sources were obtained as the percentage ratio between the HbFe and the mass of iron consumed by each animal. The results were also given as Relative Biological Value (RBV), which relates the BioFe of the studied source with that of the reference standard. The liver iron concentration (LIC) of each animal was determined at the end of the experiment in order to evaLuate the influence of the studied iron sources on the liver iron stores. SFE-171-P presented BioFe, RBV and LIC values of (47 +/- 7)%, 109% and (46.6 +/- 3.4) mg/kg respectively, while the corresponding values for the reference standard were of (43 +/- 7)%, 100% and (45.0 +/- 4.7) mg/kg. These results show that the drying process used to produce the SFE-171-P does not affect its bioavailability, which is also adequate for the potential use of this product in food fortification or with pharmaceutical purposes.

Zinc status and immune system relationship: a review.

The essentiality of zinc for humans was first documented by Prasad in the 1960s. The main clinical manifestations associated with zinc deficiency are growth retardation, hypogonadism, diarrhea, and increased susceptibility to infectious diseases. Thus, in the past 25 yr, there was an increased interest of researchers in studying the role of zinc in human immunity. Although mechanistic research has been carried out using animal models, there are several studies in humans with similar results. This work is an attempt to review the information available in this field to understand the important role that zinc plays in the normal development and function of the immune system.

Zinc and diabetes mellitus: is there a need of zinc supplementation in diabetes mellitus patients?

Diabetes mellitus is a group of metabolic disorders, the incidence of which varies widely throughout the world. The treatment of diabetes mellitus includes insulin, oral antidiabetic agents, and dietary regimens. Although the emphasis is on macronutrients intakes, there is strong evidence that there is an abnormal metabolism of several micronutrients in diabetic individuals. Zinc is one of the essential micronutrients of which status and metabolism is altered in this condition. This work is a short review about the close relation among zinc, glucose metabolism, and insulin physiology, as well as about the few experimental data about zinc absorption and zinc supplementation in diabetes mellitus patients.

Bioavailability and stability of microencapsulated ferrous sulfate in fluid milk: studies in mice.

Iron deficiency is the most important nutritional problem all over the world. Fluid milk is an attractive vehicle for iron fortification, since it is a food with a high nutritional value, accessible to the whole population and easy to be given to children. Fortification of this food with iron has the disadvantage of the interaction of the iron with the constitutive elements of milk, diminishing its bioavailability and changing its sensorial properties, making it unacceptable. Nowadays, this problem can be overcome by the implementation of a new technological procedure, which consists in the microencapsulation of the ferrous sulfate with lecithin, thus avoiding the interaction of iron with the food. The absorption obtained in mice for milk-ferrous sulfate was 7.9 +/- 3.2%, while for microencapsulated ferrous sulfate-milk the result was 11.6 +/- 4.5%. Comparing these data with those obtained with the ferrous ascorbate in water 13.1 +/- 4.9% and ferrous sulfate in water 13.2 +/- 4.3%, both of them considered as reference standards, no statistically significant difference between them and the microencapsulated ferrous sulfate in milk can be observed. However, this difference becomes significant (p < 0.01) when these products are compared to the non-encapsulated ferrous sulfate in milk. On the other hand, we demonstrated that this product is stable to heat-processing (100 degrees C, 30 min) and storage at a room temperature up to 6 months that lacteous products are usually submitted to.

New procedure to fortify fluid milk and derivatives with iron: a comparative study in mice.

It is known that the fortification of milk and its derivatives with iron has been recognized as a worldwide necessity, since the deficiency of this element produces different metabolic disorders. With this purpose, we have studied the iron absorption in mice after the administration of fluid milk and yogurt with FeSO4, stabilized by microencapsulation with soy lecithin (SFE-171) or with FeSO4, both labeled with 59Fe, which was used in the same experimental conditions for comparative purposes. The absorption of iron is influenced by the presence of some additives which usually are ingested together with milk, such as cacao, coffee, tea, "Argentine green herbs tea or maté" or cereals. The experimental results demonstrate that the iron absorption from SFE-171 is (12.3 +/- 2.9)%, whereas that from FeSO4 is (7.7 +/- 2.7)%, this difference is highly significant at p < 0.01. In all the cases, groups of 25 mice each were used to increase the statistical robustness of the experimental results. On the other hand, it could be demonstrated that the presence of 10% w/v cacao (Nesquik) increases the iron absorption in both cases, whereas 1% w/v cacao (Nesquik) and maté has no influence on the iron absorption if SFE-171 is used. Cereals (Nestum 3 cereals), yogurt and other additives like tea and coffee, for different reasons, decrease the absorption of this element.

Microencapsulated ferrous sulfate to fortify cow milk: absorption and distribution in mice.

To determine the absorption and biodistribution of iron from microencapsulated ferrous sulfate (SFE-171), used to fortify dairy products with iron, a comparative study in four groups of 30 mice each was carried out. In two of the groups, the absorption of iron from ferrous ascorbate in water (13.3 +/- 4.3%) and from ferrous sulfate in water (12.7 +/- 3.9%) was determined and taken as reference standards. In the third group the iron absorption from SFE-171 in milk was determined, giving a value of 12.1 +/- 4.2%, which statistically does not differ from the data obtained with either reference standard. In the fourth group, the absorption of iron from ferrous sulfate in milk showed a value of 7.7 +/- 3.4%, which statistically differs with a p < 0.01 from the data corresponding to the other three groups. The biodistribution studies showed that the iron from the SFE-171 follows the same metabolic pathway as the iron from the reference standards thus, giving a higher radioactivity percentage and radioactivity concentration in organs or systems, principally blood, that are closely related to iron metabolism. Our studies allow us to conclude that the iron from SFE-171 in milk follows the same behavior as the nonhemic iron, with a higher absorption than that of ferrous sulfate in milk.

Bioavailability, absorption mechanism, and toxicity of microencapsulated iron (I) sulfate: studies in mice.

The iron compounds used for food fortification have to meet certain requisites related to their bioavailability, absorption mechanism, and toxicity, since they will be consumed by a massive population group. With these purposes, we evaluated a new product used for the iron fortification of milk and lacteous derivatives, called SFE-171, which is a ferrous sulfate, microencapsulated with phospholipids. The bioavailability studies were carried out using four groups of 30 female mice each. In two groups, we studied the absorption of ferrous ascorbate and ferrous sulfate, both in water as reference standards, which show absorptions of 13.1+/-4.9% and 13.2+/-4.3%, respectively. With the third group, we studied the absorption of ferrous sulfate in milk; its value, 7.9+/-3.2%, is significantly lower than that of the remaining groups, with a p < 0.01. The studies with SFE-171 in milk, were performed on the fourth group, with a result of 11.6+/-4.5%, demonstrating that its absorption does not differ significantly from that of the reference standards. The absorption mechanism was determined by means of in vivo self-displacement studies of the ferrous ion and the SFE-171, taking ferrous sulfate as the reference compound. For this study, 210 female mice were used, and no significant difference between the absorption mechanism of both products could be observed. Toxicity studies of the new product with regard to ferrous sulfate were carried out with two groups of 70 female mice each and two groups of 70 male mice each. The lethal dose 50% LD50 for SFE-171 and for ferrous sulfate was 1200 and 680 mg/kg for female mice and 1230 and 670 mg/kg for male mice, respectively, demonstrating that the toxicity of the first product is substantially lower than that of the reference standard. We conclude that the iron product under study has a high bioavailability, an absorption mechanism equal to that of nonhemic iron, and lower toxicity than ferrous sulfate.

Study of industrial microencapsulated ferrous sulfate by means of the prophylactic-preventive method to determine its bioavailability.

Radio-iron tests are frequently used to measure the bioavailability of different iron sources for food fortification. As the labeling procedures must be done under laboratory conditions, complementary studies should be carried out to evaluate the bioavailability of iron sources produced on an industrial scale. The iron bioavailability of SFE-171 (ferrous sulfate microencapsulated with phospholipids) was studied in previous reports using the compounds labeled with 59Fe and 55Fe; the results showed an iron bioavailability similar to that of ferrous sulfate. In the present work, the iron bioavailability of industrial SFE-171 was studied by the prophylactic-preventive method in rats using ferrous sulfate as the reference standard. Elemental iron powder was also studied by the same method for comparative purposes. The liver iron concentration of each animal was determined at the end of the experiment in order to evaluate the influence of each iron source on the liver iron stores. Relative biological values of 98 and 34% were found for SFE-171 and elemental iron powder, respectively, while the corresponding relative liver iron concentrations were 104 and 45%. The results provided by the prophylactic-preventive method show that the iron bioavailability of industrial SFE-171 is similar to that of ferrous sulfate; these results are also in agreement to those obtained with the radioactive compounds. We can conclude that the SFE-171 obtained by industrial procedures for massive use in iron food fortification has the same bioavailability as that of the SFE-171 produced and labeled under laboratory conditions.

Zinc deficiency and growth: current concepts in relationship to two important points: intellectual and sexual development.

Zinc deficiency remains a serious health problem worldwide affecting developed as well as developing countries. Despite the evidence proving that zinc deprivation during the periods of rapid growth negatively affects the cognitive brain as well as sexual development, there are few complete studies carried out in children. The present article proposes a revision of the evidence gathered until now on the relationship existing between zinc deficiency and intellectual and sexual development during the stages of childhood, preadolescence, and adolescence.