Long-term safety of early consumption of Lactobacillus fermentum CECT5716: A 3-year follow-up of a randomized controlled trial.

Lactobacillus fermentum CECT5716 is a probiotic strain originally isolated from human breast milk. Previous clinical studies in infants showed that the early administration of a milk formula containing this probiotic strain was safe and may be useful for the prevention of community-acquired infections. This is a 3-year follow-up study aimed at evaluating the long-term effects produced by the early consumption of an infant formula supplemented with L. fermentum CECT5716 (experimental group, EG) compared with a control formula without the probiotic (control group, CG). The infants included in this follow-up study had previously completed a 5-month randomized double-blind controlled trial (from 1 to 6 months of age), where the safety and tolerance of the probiotic formula was evaluated. The main outcome of the follow-up study was the growth of the children. The secondary outcomes included the incidence of infectious and non-infectious diseases, parameters related with intestinal function and faecal microbiota. At 3 years, the mean values of weight, length and head circumference were similar in children of the EG compared with those of the CG. No differences were observed in the incidence of infectious and non-infectious diseases or disorders related with intestinal function. The pattern of faecal microbiota was also similar between both groups. In conclusion, this 3-year study shows that the early administration of the probiotic of L. fermentum CECT5716 in an infant formula is safe and it does not produce measurable differences in children compared with a control formula.

Safety assessment of the human milk-isolated probiotic Lactobacillus salivarius CECT5713.

The potential probiotic bacteria Lactobacillus salivarius CECT5713 has recently been isolated from human milk and characterized. The objective of the present study was to evaluate the oral toxicity of this potential probiotic bacteria in mice. With this aim, 50 Balb/C mice were divided in 5 groups (n = 10). Three of these groups were treated orally with different doses of L. salivarius CECT5713: 5 x 10(8), 2 x 10(9), or 10(10) cfu/mouse per d for 28 d. One additional group was administered the vehicle alone and was used as a control. The last group were injected intraperitoneally with 10(8) cfu/mouse in a single dose and killed 2 (n = 5) and 5 (n = 5) d after intraperitoneal injection. Food intake, body weight, bacterial translocation, serum alpha-amyloid protein, and different biochemical parameters were analyzed. Oral administration of L. salivarius CECT5713 to mice had no adverse effects on mouse body weight or food intake. No bacteremia was shown and there was no treatment-associated bacterial translocation to the liver or spleen. Intraperitoneal administration caused a significant bacterial translocation to the liver and spleen, but not to the blood. However, this translocation was not related to illness or death at either d 2 or d 5, although an increase in plasma serum alpha-amyloid protein was observed at d 2. These results suggest that the strain L. salivarius CECT5713 is nonpathogenic for mice, even in doses 10,000 times higher (expressed per kilograms of body weight) than those normally consumed by humans. Thus, this strain is likely to be safe for human consumption.

[Beneficial effects of consumption of a dairy product containing two probiotic strains, Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714 in healthy children]. / Efectos beneficiosos en niños sanos del consumo de un producto lácteo que contiene dos cepas probióticas. Lactobacillus coryniformis CECT5711 y Lactobacillus gasseri CECT5714.

OBJECTIVE: In the last decades there has been an increasing interest in the manipulation of intestinal microbiota with probiotics for the prevention and treatment of certain paediatric diseases. In addition, it has been suggested that probiotics could play a role in the development of immune system. Recent studies suggest that the administration of two probiotic strains, Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714 improves intestinal function of healthy adults and enhances the immune response. Since there are few studies reporting the use of probiotic in children, the main consumers of these products, the aim of the present study was to analyze the effects of the administration of the mentioned probiotic strains in healthy children. INTERVENTIONS: 30 children (age range 3-12) with no gastrointestinal pathology were included in the study. In addition to their usual diet, during the first 3 weeks they received 200 ml of a conventional yogurt containing Lactobacillus bulgaricus and Streptococcus thermophilus. During the following three weeks this yogurt was substi-tuted for 80 ml of a probiotic product (Max Defensas, Puleva Food S.L.) containing the same amounts of Streptococcus thermophilus and the L. bulgaricus was substituted by a mixture of the target probiotic strains: L. coryniformis CECT5711 and L. gasseri CECT5714. Samples of faeces and saliva were taken at the beginning of the protocol, at week 3 and at the end of the study. Intestinal microbiota, faecal citotoxicity and the inhibition of Salmonella cholerasusis ssp. cholerasuis adhesion to intestinal mucins by the faeces were analyzed. Finally, IgA concentration was determined in the faecal and saliva samples. RESULTS: Tolerance of the probiotic product was good in all the children included in the study. An increase in faecal lactobacilli counts was shown at the end of the experimental protocol (P < 0,05). In addition citotoxicity of faecal samples was significantly (p < 0.05) reduced after probiotic consumption. The inhibition of S. cholerasuis adhesion to intestinal mucins was significantly higher (P < 0.05) for faecal waters from children in week 6 compared to samples form week 0 and 3. Probiotic consumption was also shown to increase IgA concentration in faeces and saliva (P < 0.05). CONCLUSIONS: The consumption of a probiotic product containing L. coryniformis CECT5711 and L. gasseri CECT5714 improves intestinal flora of healthy children, enhancing the defence against gastrointestinal aggressions and infections both by inhibiting pathogen adhesion to intestinal mucins and enhancing the immune function.

Cytokine distribution in mothers and breastfed children after omega-3 LCPUFAs supplementation during the last trimester of pregnancy and the lactation period: A randomized, controlled trial.

OBJECTIVE: To determine whether maternal diet supplementation with omega-3 long chain polyunsaturated fatty acids (omega-3 LC-PUFAs) during the last trimester of pregnancy and the breastfeeding period influences the levels of inflammatory cytokines in mother and infants. MATERIAL AND METHOD: This registered, double-blind randomized study included 46 pregnant women, who were randomly allocated to either an experimental group receiving 400mL/day of a fish oil-enriched dairy drink [320mg docosahexaenoic acid (DHA) + 72mg eicoapentaenoic acid] (FO group, n = 24) or to a control group receiving 400mL/day of a non-supplemented dairy drink (CT group, n = 22), from week 28 of pregnancy until the fourth month of lactation. During the study, maternal dietary patterns were monitored by a nutritionist, who encouraged compliance with current recommendations of fatty acids intake. DHA concentrations and cytokine levels (GM-CSF, IL-2, IL-4, IL-6, IL-10, INF-γ and TNF-α) were measured in maternal plasma at the moment of recruitment and in maternal (n = 46) and infant (n = 46) plasma at birth and 2.5 months after birth. RESULTS: Maternal plasmatic IL-4 levels were higher in FO than in CT subjects (p = 0.009). Additionally, a tendency was observed to higher IL-10 and IL-2 in the FO group. Plasmatic IL-6 however, was higher in CT mothers (p = 0.001). TNF-α was higher in CT infants at birth and 2.5 months after birth (p = 0.005). An analysis of possible relationships between DHA and the concentrations of different cytokines revealed negative correlation between maternal plasmatic IL-6 and DHA (higher plasmatic DHA corresponded to lower IL-6). CONCLUSIONS: Maternal dietary omega-3 LC-PUFAs supplementation during critical periods like pregnancy, lactation and early newborn development may influence the levels of certain inflammatory cytokines, reducing pro-inflammatory cytokines and promoting an anti-inflammatory "environment".

[Il-10 expression is involved in the regulation of the immune response by omega 3 fatty acids]. / La expresión de IL-10 interviene en la regulación de la respuesta inflamatoria por los acidos grasos omega 3.

INTRODUCTION: Polyunsaturated fatty acids play a key role in a huge number of biological functions. Western diets are highly rich in w-6 fatty acids. However the content of w-3 fatty acids is not suitable in those diets, despite of their importance in normal development of the human body and regulation of immune response. The aim of this work is to examine the effect of w-3 fatty acids enriched diet in the regulation of inflammatory response. MATERIAL AND METHODS: Balb/c mice were fed either w-6 fatty acids rich diet (100% sunflower oil) or w-3 fatty acids fortified diet (12% fish oil plus 88% sunflower oil) during 28 days. Twelve hours prior to sacrifice, the mice were treated with 2,4-ninitro-1-fluorobezene on the left ear to induce the inflammatory reaction. Afterwards the mice were sacrificed and the different samples collected were analized. RESULTS: Ear inflammation of mice fed the w-3 diet was significantly lower. Leukocyte infiltration and oxidative stress were also lower in those mice. To explain these results, cytokine expression and plasma eicosanoid concentration were measured. An increase in IL-10 levels and a down regulation of Th1 and Th2 responses were observed in mice fed the w-3 diet. CONCLUSION: Not only n-3 fatty acids exerts an antiinflammatory and an antialergical role but also they enhance some of the organism defenses. Our data suggest that w-3 fatty acids downregulate the inflammatory response by enhancing IL10 expression.

[Thickened infant formula, rheological study of the "in vitro" properties]. / Estudio del comportamiento reológico in vitro de las fórmulas antirregurgitación.

INTRODUCTION: Thickened infant formulas, specially formulated to increase the viscosity, are commonly used in the treatment of regurgitation in the non-complicated gastroesophageal reflux. OBJECTIVE: To analyse viscosity and the rheological behaviour of different thickened standard formulas on the Spanish market compared to a standard formula with or without the addition of 10 g/100mL of gluten-free cereals. METHODS: Viscosity of the samples was evaluated in a Bohlim CS-1o controlled-stress rheometer and was performed at basal conditions (25 degrees C, pH 7) and at simulated gastric conditions (37 degrees C, pH 4 and 10 g/100mL of pepsin) at time 0, 30 and after 60 min of incubation. Values were expressed as centipoises (cp) (1 cp=1/100 p). RESULTS: All formulas show a viscosity increase both in basal conditions and in gastric simulated conditions but the behaviour is very heterogeneous. Formulas containing bean gum (carob seed flour) with 2.9 g/100g and a protein ratio similar to cow's milk (80 casein/20 whey) showed the highest and consistent viscosity (70 cp and 90 cp), with significant differences compared to the standard formula in all the measurements. When this thickener is in formulas with a protein ratio similar to breast milk (40 casein/60 whey) the viscosity was lower and reached 50 cp only with the thickener at a concentration of 4.7 gr/100g, achieving significant differences versus standard formula. The formulas with starch thickeners (rice, potatoes and corn) achieved a lower and less consistent viscosity, with no significant difference. The viscosity reached after the addition of cereals both in basal conditions and in gastric simulated conditions was similar to that achieved with more effective thickeners. Lipid concentration is not involved in viscosity and rheological behaviour. CONCLUSION: The viscosity of the thickened infant formula depends on the agent used, concentration and protein ratio. Not all reach a viscosity of 50 cp, hypothetical value to reach, since it doubles the viscosity of a standard formula. The ideal viscosity to be reached and the role of other components of the formula in the viscosity and rheological behaviour is still unclear.

Safety assessment of two probiotic strains, Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714.

AIMS: The object of the present study was to evaluate the oral toxicity of the recently isolated probiotic bacteria Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714. METHODS AND RESULTS: Enzymatic activity and antibiotic resistance profile were evaluated in vitro. Then, the oral toxicity was analysed by an in vivo experiment using 20 Balb/C mice, which were orally treated with CECT5711 or CECT5714 (10(10) CFU mouse(-1) day(-1)) during 30 days. Results showed that CECT5711 and CECT5714 have no deleterious enzymatic activities and present intrinsic antibiotic resistance profile. Administration of both strains to mice had no adverse effects on body weight or food intake. No bacteraemia was present in liver or spleen and there was no treatment-associated bacterial translocation to these tissues. Liver glutathione content as well as plasma malondialdehide concentration were not statistically different in probiotic-treated mice when compared with control mice. Probiotic treatment did not cause changes in the biochemical and haematological parameters analysed. CONCLUSIONS: These results suggest that strains CECT5711 and CECT5714 are nonpathogenic and likely to be safe for human consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals the oral safety of two new lactobacilli strains that are aimed to be used as probiotics in food and pharmaceutical applications.

Two Lactobacillus strains, isolated from breast milk, differently modulate the immune response.

AIMS: The ability of two different Lactobacillus strains (Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716), isolated from human breast milk, to modulate the immune response was examined. METHODS AND RESULTS: In rodent bone-marrow-derived macrophages (BMDM), the presence of Lact. fermentum CECT5716 induced pro-inflammatory cytokines, in contrast to the activation of IL-10 induced by Lact. salivarius CECT5713. Although both strains reduced the lipopolysaccharide (LPS)-induced inflammatory response in BMDM, the effect of Lact. salivarius CECT5713 was more efficient, probably because of the production of higher amounts of IL-10 cytokine. In vivo assays in mice showed similar results; the consumption of Lact. fermentum CECT5716 enhanced the production of Th1 cytokines by spleen cells and increased the IgA concentration in faeces. However, the consumption of Lact. salivarius CECT5713 induced IL-10 production by spleen cells. CONCLUSION: Therefore, in general, the effect of Lact. fermentum CECT5716 is immunostimulatory in contrast to the anti-inflammatory effect of Lact. salivarius CECT5713. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that two Lactobacillus strains isolated from breast milk can exert different and even opposing effects on immune response demonstrating the specificity of each strain.

The balance between caseins and whey proteins in cow's milk determines its allergenicity.

Cow's milk allergy is quite common in the first years of human life. Protein composition plays an important role in this pathology, particularly the casein/whey protein ratio. It is known that milks from different species have different sensitization capacities although their protein sources are quite similar. Thus, the objective of this work was to compare the allergenicity of native cow's milk and milk with a modified ratio of casein and whey proteins in a murine model of atopy. Twenty-four Balb/c mice were orally sensitized to native cow's milk or modified cow's milk with a casein/whey protein ratio of 40:60. During the sensitization period, the number of mice suffering from diarrhea was significantly higher in the native cow's milk-sensitized group than in the modified milk-sensitized group. Once mice were killed, plasma histamine levels were shown to be significantly higher in native cow's milk-sensitized mice. In addition, cow's milk proteins induced a higher lymphocyte sensitization in the native milk-sensitized mice, with a significant increase in the specific proliferation ratio of these cells. These results suggest that the balance between caseins and whey proteins plays an important role in the sensitization capacity of cow's milk, and its modification might be a way to reduce the allergenicity of cow's milk.