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Nutrición Hospitalaria

versión impresa ISSN 0212-1611

Nutr. Hosp. v.25 n.5 Madrid set.-oct. 2010

 

SPECIAL ARTICLE

 

 

Consensus statements from the Workshop "Probiotics and Health: Scientific Evidence"

Declaraciones consensuadas en el workshop "probióticos y salud: evidencia científica"

 

 

F. Guarner1, T. Requena2 and A. Marcos3

1Hospital Vall d'Hebrón. Barcelona.
2Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM). Madrid.
3Instituto de Ciencia y Tecnología de Alimentos y Nutrición, ICTAN (CSIC). Madrid. España

Correspondence

 

 


ABSTRACT

This report shows the level of scientific consensus on definition, characteristics and health benefits of probiotics. The content of the report has derived from the scientific meeting: Workshop on Probiotics and Health. Scientific evidence, that congregated several Spanish experts, including gastroenterologists, microbiologists, nutritionists, immunologists and food technologists, among others, who have agreed with the statements shown in this document. Each statement has been sustained with the most relevant scientific aspects that were discussed during the Workshop and the following evaluation of the report by all experts who approved and signed it.

Key words: Probiotic. Scientific evidence. Gastrointestinal health. Immunomodulation.


RESUMEN

En este documento se muestra una base de consenso en torno a la definición, características y propiedades beneficiosas de los probióticos. El contenido fue generado a partir de la reunión científica Workshop Probióticos y Salud. Evidencia Científica, que agrupó a una variedad de expertos españoles gastroenterólogos, microbiólogos, nutricionistas, inmunólogos y tecnólogos de alimentos, entre otros, que se han adherido en su mayoría a las sentencias que constituyen este documento. Para cada sentencia se establecen las aspectos científicos más relevantes que la respaldan y que son consecuencia del acuerdo al que se ha llegado tras el debate surgido en la reunión y la evaluación posterior del contenido por todos los expertos que han firmado este documento.

Palabras clave: Probiótico. Evidencia científica. Salud gastrointestinal. Inmunomodulación.


 

1. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host

This definition was proposed by a Joint FAO/WHO working group of experts,1 and has been widely accepted in the scientific literature, being the most referenced definition since it was first published. In general, aspects such as the viability of microorganisms, oral administration and demonstrated beneficial effects on health after consumption are permanent criteria in most definitions that have been proposed for probiotics. Although not explicitly reflected, the definition also implies that probiotics should survive passing through the gastrointestinal tract and preserve their beneficial capacity when they reach the sites where they interact with the host, which explains their mechanisms of action. The definition also implies the viability of probiotics throughout the shelf life of the product in which they are administered. Products should contain the quantity of microorganisms needed to provide the benefit.

 

2. Those substances that are constituents of microorganisms or are produced by them should not be considered probiotics even if they would produce a beneficial effect on health

Some mechanisms of action relative to beneficial effects of probiotics include, among others, the release of microbial enzymes in the intestine (e.g. β-galactosidase, which hydrolyses the lactose present in foods), the secretion of proteins and the extracellular proteins or other macromolecules associated to microbial envelopes that interact with pattern-recognising receptors in host cells2 or the production of metabolites or peptides with antimicrobial activity (e.g. bacteriocins). Although they are related to the mechanisms of action by which probiotic microorganisms exert their beneficial effect on the host, these substances should not be considered to be probiotics themselves because they are not live or viable organisms. Likewise, part of the resident or autochthonous intestinal microbiota can enable certain metabolic activities that human intestinal cells cannot, such as the degradation of non-digestible components in the diet, methanogenesis, gluconeogenesis, and detoxification of xenobiotics or the biosynthesis of essential amino acids, vitamins and isoprenoids. However, such activities do not necessarily confer to these microorganisms the qualification of probiotics.

 

3. For a microorganism to qualify as a probiotic, it must be scientifically demonstrated that it produces beneficial effects on the health of the host

The ability to produce a beneficial effect on health is an essential property that characterises a probiotic. In the 21st century, it is clear that an effect on health can and must be verified by scientific methods which guarantee the efficacy and safety of the agent which produces such effect. Therefore, health claims which are not based on scientific evidence3 are not acceptable. Thus, there must be scientific evidence showing that the consumption of a certain microorganism produces a specific health effect in order to support the probiotic status.

 

4. Beneficial effects on health should be demonstrated through studies carried out on the human population with suitable scientific methodology

The usual methods to provide the scientific evidence of a certain effect were reviewed both by the group of experts from WHO and FAO,1 as well as by the working groups from PASSCLAIM;3 these two publications therefore offer an extensive view on the matter. There is unanimous consensus on the concept that there is no definitive proof of a given health effect if its efficacy has not been confirmed in individuals of the same species for which its use is intended. Therefore, the probiotics which are intended for human health must have proven efficacy in studies with human subjects.

 

5. Laboratory or animal model studies are a necessary requirement before performing studies in the human population, and they provide information regarding mechanisms of action; however, alone they should not be considered as sufficient proof of efficacy in human health.

The unavoidable need to demonstrate efficacy and safety in studies carried out in human subjects should not lead to the conclusion that laboratory or animal model studies are superfluous or even useless. In the case of probiotics, it is always necessary to start with an adequate characterisation of the microorganism that is intended to be selected for its use in humans. The adequate characterisation of the microorganism, according to the recommendations of the WHO/FAO group,1 must include its identification, including genus, species and strain using phenotypic methods (biological properties expressed in vitro) and genotypic methods (sequencing of its genes, especially of the 16SrRNA gene fragments, and a description of methods for its identification in biological media by specific techniques including fingerprinting techniques such as PFGE or similar techniques) and depositing the strain in internationally recognised collections. Adequate characterisation and safety studies in animal models are necessary before use in the human population. Moreover, laboratory and animal model studies are ideal for understanding and illustrating the mechanism of action of a given probiotic on human health.

 

6. Healthy effects demonstrated for a specific microbial strain cannot be extrapolated or attributable to other strains of the same species.

A microbial strain is defined by the different properties and characteristics that differentiate it from other microorganisms of its species, including phenotypic properties (morphological, physiological, biochemical, ecological, etc.), structural properties (cytoplasmic membrane, cell wall, capsule, etc.) and genetic properties (DNA, RNA). The strains that result from new microbial isolates should be evaluated to demonstrate whether a beneficial effect is present, regardless of whether they correspond to a species in which a probiotic strain has been reported. The development of molecular biology methodologies has led to the identification of different effector cell and molecule structures that are strain-dependent and which are related to the specific interactions between probiotic microorganisms and host cells.4 Some strain-dependent characteristics that have been described are, among others, the production of bacterial adhesins and exopolysaccharides, related to the probiotics which remain in the intestine, immunomodulating activity and the production of bacteriocins. Furthermore, the strain-specific characteristics of probiotics makes it necessary to develop DNA fingerprinting techniques in order to assess the authenticity of the microorganism claimed to be a probiotic, as well as its viability in the products in which it is administered.

 

7. A microbial strain properly qualified as probiotic due to its demonstrated efficacy for a specific indication (e.g. diarrhoea prevention) is not necessarily valid for other indications (e.g. allergy prevention).

Strains included in the probiotic category, depending on their characteristics, must have been proven effective for a specific condition. This is the case of Lactobacillus rhamnosus GG, the usefulness of which has been demonstrated in several studies on paediatric gastroenteritis and diarrhoea associated with treatment with antibiotics.5-7 Nevertheless, there are no recommendations for its use in other situations such as genitourinary infection prevention8. Randomised, placebocontrolled studies for intervention with L. rhamnosus GG, carried out to compare the preventative capacity of developing eczema and atopic sensitisation in at-risk children have given rise to contradictory results for eczema prevention in general and, in particular, negative results for the prevention of eczema with atopic sensitisation.9-12.

 

8. The effectiveness of some probiotics strains is widely documented for specific gastrointestinal health conditions (e.g. some types of diarrhoea, constipation, irritable bowel and intestinal inflammation)

One main area in which probiotics have been applied is in the prevention and treatment of gastrointestinal disorders. There is extensive scientific evidence documenting the efficacy of certain probiotic strains in acute processes of the digestive tract. Some of these indications are already common in medical practice. In contrast, the use of probiotics or prebiotics in chronic gastrointestinal processes is still much more limited. Recently, the World Gastroenterology Organisation (WGO) published a practical guide on the use of probiotics and prebiotics in gastroenterology.13 The WGO guide was drafted by an international group of experts and translated into several languages; it is available on the organisation's website in English, French, Spanish, Portuguese, Russian and Mandarin Chinese. It is worth noting that the clinical guide includes a table of specific conditions that can be treated with certain probiotics, thereby avoiding generalised recommendations which are not based on scientific findings.

 

9. There are probiotic strains with demonstrated efficacy for specific indications of the immune system (e.g. prevention of infections)

Today, it is generally accepted and has been demonstrated by both in vitro and in vivo experiments that probiotic strains interact with the immune system of the host.14 For example, the formulation of multiple species contained in the probiotic preparation VSL#3 has shown a beneficial anti-inflammatory effect in mice by way of local stimulation of epithelial innate immune responses (increase in the synthesis of epithelial TNF-α and reestablishment of intestinal permeability).15.

Effects on the immune system that have been repeatedly observed in well-conducted intervention studies in human beings include:

- Modulation of phagocytic activaty. Lactobacillus johnsonii La1 may limit low-grade chronic inflammation due to overgrowth of intestinal bacteria in the elderly, decreasing phagocytosis.16 L. rhamnosus HN001 has demonstrated its ability to modulate innate cell immunity, increasing the phagocytic capacity of polymorphonuclear cells (PMN) and monocytes, as well as an increase in the phagocytic activity of natural killer cells (NK) in adults and the elderly.17,18 The same has been demonstrated for Bifidobacterium lactis HN019, although the two studies that have documented this were carried out in a small number of subjects.19,20.

- Coadjuvant effect in vaccination. In studies on the expression of activation markers and cytokine production in in vitro cultures of dendritic cells (derived from monocytes) and cocultivated Tcells with different species of lactobacillus (L. johnsonnii, L. reuteri, L. gasseri), it has been demonstrated that dendritic cells activated as such secrete interleukin (IL)-12 and IL-18 and polarise the response of lymphocytes toward a Th1-type response by inducing the secretion of interferon (IFN)-γ but not that of IL-4 or IL-13.20 The same has been observed with Lactobacillus fermentum CECT5716 as its administration together with anti-flu vaccination improves Th1 response and the production of antibodies.22 Likewise, an adjuvant effect in vaccination against the seasonal flu has also been shown in milk fermented with Lactobacillus casei DN 014 001 and live cultures in yoghurt.23.

- Mitigation of common winter infections. Although various studies carried out with probiotics have highlighted this positive effect on health, it cannot be attributed to one single species or strain because the evidence available comes either from studies that use combinations of bacteria species,24 or studies that use one single strain, such as L. johnsonii La125; it would be advisable to reproduce the evidence by subsequent independent studies.

 

10. Scientific evidence observed for one population group cannot be extrapolated to other populations of different age (children and the elderly) or physiological state (e.g. pregnancy and breastfeeding)

Several studies have attempted to demonstrate the efficacy and safety of probiotic in different population groups and physiological states. The studies which have demonstrated the scientific evidence of probiotics can only be attributed to the strain(s) analysed in each population group studied; they cannot be generalised for all populations and physiological states.26 The anatomical and physiological differences (not only in the intestinal microbiota) between children, adults and the elderly in the healthy population are significant, which is why every study must normally attempt to demonstrate the efficacy of probiotic bacteria in samples representative of specific population groups. The same can be said for special physiological states such as pregnancy and breastfeeding. In any case, the meta-analyses which have included different population groups have found different, non-extrapolative effects. In this respect, a meta-analysis carried out by Sazawal et al.27 found that the use of probiotics to prevent acute diarrhoea is more effective in children than in adults. Therefore, not all probiotics (or the combination thereof) act equally from one given population group to another, or in one physiological state or another, making it therefore necessary to demonstrate their effects in studies that are properly designed for each situation.

 

Experts who have agreed to join the consensus:

1. Guillermo Álvarez Calatayud. Hospital General Universitario Gregorio Marañón. Madrid; 2. Rebeca Arroyo Rodríguez. Facultad de Veterinaria. Universidad Complutense de Madrid (UCM). Madrid; 3. Rosa Aznar Novella. Instituto de Agroquímica y Tecnología de Alimentos (CSIC, Valencia) y Universidad de Valencia (UVEG); 4. Fernando Azpiroz. Hospital Vall d'Hebron. Barcelona; 5. Begoña Bartolomé Sualdea. Instituto de Fermentaciones Industriales. CSIC. Madrid; 6. Miguel Bixquert. Hospital Arnau de Vilanova. Valencia; 7. Juan Borrero del Pino. UCM; 8. Mar Calvo Terrades. CAP Peralada. Girona; 9. Cristina Campanero Pintado. UCM; 10. Ma Cristina Castellote Bargallo. Universidad de Barcelona (UB). Barcelona; 11. August Corominas. Universitat Autònoma de Barcelona (UAB). Barcelona; 12. María Lourdes de Torres Aured. H. U. Miguel Servet. Zaragoza; 13. Lígia Esperanza Díaz. Instituto del Frío. CSIC. Madrid; 14. Lídia Domínguez Clavería. UAB. Barcelona; 15. Irene Espinosa. UCM. Madrid ; 16. Pilar Fernández de Palencia. Centro de Investigaciones Biológicas. CSIC. Madrid; 17. María Fernández. Instituto de Productos Lácteos. Asturias; 18. Julio Gálvez. Universidad de Granada (UG). Granada; 19. Ángel Gil. UG. Granada; 20. Sonia Gómez. Instituto del Frío. CSIC. Madrid; 21. Carolina Teresa Hernández Haro. UCM. Madrid; 22. Francisco Ibáñez Moya. Universidad Pública de Navarra. Pamplona; 23. Esther Jiménez Quintana. UCM. Madrid; 24. Pilar León Izard. Colegio de Farmacéuticos de Madrid. Madrid; 25. Maria Rosaura Leis Trabazo. Universidad de Santiago de Compostela. Santiago de Compostela; 26. Ma Elvira López Caballero Instituto del Frío. CSIC. Madrid; 27. Ana López de Lacey. Instituto del Frío. CSIC. Madrid; 28. María Marín Martínez. UCM. Madrid; 29. Abel Mariné. UB. Barcelona; 30. Abelardo Margolles. Instituto de Productos Lácteos. CSIC. Asturias; 31. Magdalena Martínez Cañamero. Universidad de Jaén (UJ). Jaén; 32. Carmen Martínez Rincón. UCM. Madrid; 33. Luis Alberto Menchén. Hospital Gregorio Marañón. Madrid; 34. Miguel Mínguez. Hospital Clínico Universitario. Valencia; 35. Ma Pilar Montero García. Instituto del Frío. CSIC. Madrid; 36. Ma Victoria Moreno Arribas. Instituto de Fermentaciones Industriales. CSIC. Madrid; 37. Esther Nova Rebato. Instituto del Frío-ICTAN. CSIC. Madrid; 38. Tamara Pozo. Instituto del Frío-ICTAN. CSIC. Madrid; 39. Ma Isabel Prieto Gómez. UJ. Jaén; 40. Daniel Ramón Vidal. Biopolis. Valencia; 41. Margarita Ribó. Instituto Nutrición Margarita Ribó. Barcelona; 42. María Rodríguez González. UAB. Barcelona; 43. Eva Rodríguez Mínguez. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Madrid; 44. Javier Romeo. Instituto del Frío. CSIC. Madrid; 45. Patricia Ruas Madiego. Instituto de Productos Lácteos. CSIC. Asturias; 46. Ma Pilar Rupérez Antón. Instituto del Frío. CSIC. Madrid; 47. Cristina Saro. Hospital de Cabueñes. Gijón ; 48. Germán Soriano Pastor. Hospital Sant Pau. Barcelona; 49. Paloma Torre Hernández. UN. Navarra; 50. Ana Isabel Vallejo. Directora Científica de NaturSanix. Madrid; 51. Soledad Vinuesa Vinuesa. Farmacéutica Oficial de Salud Pública; 52. Amelia Martí del Moral. Universidad de Navarra; 53. Ma Carme Vidal. UB. Barcelona. 54. Maite Dueñas. Universidad del País Vasco (EHU). San Sebastian. 55. Lluís Serra-Majem. Universidad de las Palmas de Gran Canaria. 56. Francisco Guarner. Hospital Vall d'Hebron; 57.Teresa Requena Rolanía. Instituto de Investigación en Ciencia de los Alimentos CIAL (CSIC-UAM). Madrid; 58.Ascensión Marcos Sánchez. Instituto de Ciencia y Tecnología de Alimentos y Nutrición, ICTAN (CSIC). Madrid.

 

References

1. FAO/WHO. Probiotics in food. Health and nutritional properties and guidelines for evaluation. En "FAO Food and Nutrition Paper 85", 2006, ISBN 92-5-105513-0. Available at: ftp://ftp.fao.org/docrep/fao/009/a0512e/a0512e00.pdf.        [ Links ]

2. Lebeer S, Vanderleyden J, De Keersmaecker SCJ. Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat Rev Microbiol 2010; 8 (3): 171-184.        [ Links ]

3. Aggett PJ, Antoine JM, Asp NG, Bellisle F, Contor L, Cummings JH, Howlett J, Müller DJ, Persin C, Pijls LT, Rechkem-mer G, Tuijtelaars S, Verhagen H. PASSCLAIM: consensus on criteria. Eur J Nutr 2005 Jun; 44 Suppl 1: i5-30.        [ Links ]

4. Kleerebezem M, Vaughan EE. Probiotic and gut lactobacilli and bifidobacteria: Molecular approaches to study diversity and activity. Ann Rev Microbiol 2009; 63: 269-290.        [ Links ]

5. Vanderhoof JA, Whitney DB, Antonson DL, Hanner TL, Lupo JV, Young RJ. Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children. The Journal of Pediatrics 1999, 135(5); pp 564-568.        [ Links ]

6. NASPGHAN Nutrition Report Committee. Clinical efficacy of probiotics: review of the evidence with focus on children. J Pediatr Gastroenterol Nutr 2006; 43: 550-7. Disponible en http://www.naspghan.org/userassets/Documents/pdf/PositionPapers/probiotics.pdf        [ Links ]

7. Hojsak I, Snovak N, Abdovi S, Szajewska H, Misak Z, Kola ek S. Lactobacillus GG in the prevention of gastrointestinal and respiratory tract infections in children who attend day care centers: A randomized, double-blind, placebo-controlled trial Clinical Nutrition, In Press, Corrected Proof, Available online 5 November 2009.        [ Links ]

8. Falagas ME, Betsi GI, Tokas T, Athanasiou S. Probiotics for prevention of recurrent urinary tract infections in women: a review of the evidence from microbiological and clinical studies. Drugs 2006; 66(9): 1253-61.        [ Links ]

9. Kalliomäki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001; 357(9262): 1076-9.        [ Links ]

10. Kalliomäki M, Salminen S, Poussa T, Isolauri E. Probiotics during the first 7 years of life: a cumulative risk reduction of eczema in a randomized, placebo-controlled trial. J Allergy Clin Immunol 2007 Apr; 119(4): 1019-21.        [ Links ]

11. Kopp MV, Hennemuth I, Heinzmann A, Urbanek R. Randomized, double-blind, placebo-controlled trial of probiotics for primary prevention: no clinical effects of Lactobacillus GG supplementation. Pediatrics 2008 Apr; 121(4): e850-6.        [ Links ]

12. Salfeld P, Kopp MV. Probiotics cannot be generally recommended for primary prevention of atopic dermatitis. J Allergy Clin Immunol 2009 Jul; 124(1): 170; author reply 170-1.        [ Links ]

13. World Gastroenterology Organization. Global Guideline 'Probiotics and Prebiotics in Gastroenterology, http://www.worldgastroenterology.org        [ Links ]

14. Corthesy B, Gaskins HR, Mercenier A. Cross-talk between probiotic host immune system. J Nutr 2007; 137: 781S-90S.        [ Links ]

15. Pagnini C, Saeed R, Bamias G, Arseneau KO, Pizarro TT, Cominelli F. Probiotics promote gut health through stimulation of epithelial innate immunity. Proc Natl Acad Sci U S A. 2010 Jan 5; 107(1): 454-9.        [ Links ]

16. Schiffrin EJ, Parlesak A, Bode C, Bode JC, van't Hof MA, Grathwohl D, Guigoz Y Probiotic yogurt in the elderly with intestinal bacterial overgrowth: endotoxaemia and innate immune functions. Br J Nutr 2009 Apr; 101(7): 961-6.        [ Links ]

17. Sheih YH, Chiang BL, Wang LH, Liao CK, Gill HS. Systemic immunity enhancing effects in healthy subjects following dietary consumption of the lactic acid bacterium Lactobacillus rhamnosus HN001. J Am Coll Nutr 2001; 20: 149-56.        [ Links ]

18. Gill H S; Cross M L; Rutherfurd K J; Gopal P K. Dietary probiotic supplementation to enhance cellular immunity in the elderly. British journal of biomedical science 2001; 58(2): 94-6.        [ Links ]

19. Arunachalam K, Gill HS, Chandra RK. Enhancement of natural immune function by dietary consumption of Bifidobacterium lactis (HN019). Eur J Clin Nut 2000 Mar; 54(3): 263-7.        [ Links ]

20. Gill HS, Rutherfurd KJ, Cross ML, Gopal PK. Enhancement of immunity in the elderly by dietary supplementation with the probiotic Bifidobacterium lactis HN019. Am J Clin Nu 2001 Dec; 74(6): 833-9        [ Links ]

21. Mohamadzadeh M, Olson S, Kalina WK, Ruthel G., Demmin G.L. , Warfield K.L, Bavari S.and Klaenhammer TR. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization, Proc Natl Acad Sci USA 2005; 102: 2880-2885.        [ Links ]

22. Olivares M, Diaz-Ropero MP, Sierra S, Lara-Villoslada F, Fonolla J, Navas M, Rodriguez JM, Xaus J. Oral intake of Lactobacillus fermentum CECT5716 enhances the effects of influenza vaccination. Nutrition 2007; 23: 254-60.        [ Links ]

23. Boge T, Rémigy M, Vaudaine S, Tanguy J, Bourdet-Sicard R, van der Werf S. A probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised controlled trials. Vaccine 2009 Sep 18; 27(41): 5677-84.        [ Links ]

24. De Vrese M, Winkler P, Rautenberg P, Harder T, Noah C, Laue C, Ott S, Hampe J, Schreiber S, Heller K, Schrezenmeir J. Effect of Lactobacillus gasseri PA 16/8, Bifidobacterium longum SP 07/3, B-bifidum MF 20/5 on common cold episodes: A double blind, randomized, controlled trial. Clin Ntr 2005. Aug; 24(4): 481-91        [ Links ]

25. Fukushima Y MS, Yamano T, Kaburagi T, Iino H, Ushida K, Sato K. Improvement of nutritional status and incidence of infection in hospitalized enterally fed elderly by feeding of fermented milk containing probiotic Lactobacillus johnsonii La1(NCC533). Br J Nutr 2007; 98: 969-77.        [ Links ]

26. Olveira Fuster G, González-Molero I. Probiotics and prebiotics in clinical practice. Nutr Hosp 2007; 22(2): 26-34. Review.        [ Links ]

27. Sazawal S, Hiremath G, Dhingra U, Malik P, Deb S, Black RE. Efficacy of probiotics in prevention of acute diarrhoea: a metaanalysis of masked, randomised, placebo-controlled trials. Lancet Infect Dis 2006; 6: 374-82.        [ Links ]

 

 

Correspondence:
Ascensión Marcos.
Departamento Metabolismo y Nutrición.
ICTAN. Instituto del Frío. CSIC.
José Antonio Novais, 10
28040 Madrid.

Recibido: 20-V-2010.
Aceptado: 11-VI-2010.

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