WO2012107601A1 - Use of a strain of lactobacillus bulgaricus for treating immunosenescence - Google Patents

Use of a strain of lactobacillus bulgaricus for treating immunosenescence Download PDF

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WO2012107601A1
WO2012107601A1 PCT/ES2011/000329 ES2011000329W WO2012107601A1 WO 2012107601 A1 WO2012107601 A1 WO 2012107601A1 ES 2011000329 W ES2011000329 W ES 2011000329W WO 2012107601 A1 WO2012107601 A1 WO 2012107601A1
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strain
probiotic
increase
bulgaricus
lymphocytes
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PCT/ES2011/000329
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Spanish (es)
French (fr)
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WO2012107601A8 (en
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José Miguel DE LAS HERAS DEL RIO
Maria Baltadjieva
Carlos LOPEZ LARREA
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De Las Heras Del Rio Jose Miguel
Maria Baltadjieva
Lopez Larrea Carlos
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Publication of WO2012107601A1 publication Critical patent/WO2012107601A1/en
Publication of WO2012107601A8 publication Critical patent/WO2012107601A8/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/123—Bulgaricus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to the use of a strain of Lactobacillus bulgaricus for the preparation of a drug or food supplement intended for the treatment of immunosenecence or deterioration of the defensive capacity of the immune system as a consequence of chronological aging. Specifically, it refers to the use of the strain of Lactobacillus delbrueckii subsp. bulgaricus, isolated in the mountains of Bulgaria and deposited in the "National Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) under n ° 8481.
  • NBIMCC National Bank for Industrial Microorganisms and Cell Cultures
  • Probiotics are non-pathogenic microorganisms that exert a positive influence on the health or physiology of the host when ingested.
  • beneficial properties of these microorganisms is their immunomodulatory activity.
  • This process is mainly characterized by (1) the limitation of the repertoire of the T lymphocytes and the accumulation of oligoclonal effector memory cells directed against ubiquitous infectious agents; (2) thymic involution and disappearance of "na ' ⁇ ve"cells; and (3) a chronic inflammatory state.
  • Lactobacillus Among the probiotic organisms inherent to the digestive system of humans, the effect of Lactobacillus is known, presenting an effect on lactose intolerance, effect on the immune system, etc., due to its ability to control infections while maintaining an adequate balance in the microflora of the intestine.
  • Different strains of Lactobacillus have been used for the preparation of products with adequate organoleptic properties and with prophylactic and therapeutic characteristics.
  • the application of the known strains of Lactobacillus delbrueckii subsp. bulgaricus has been limited to the area of fermented milk products and its activity is limited to a short period of time.
  • the patents BG63961, BG63962 and BG63963 disclose that strains of Lactobacillus delbrueckii subsp. bulgaricus are used as crops for Bulgarian yogurt and other fermented dairy products.
  • the patent BG61476 discloses the possibility of combining a biologically active substance from the culture of Lactobacillus delbrueckii subsp. bulgaricus with natural products for the preparation of food additives.
  • Patents UA63201, UA63202 and UA64163 disclose that a strain of Lactobacillus delbrueckii subsp. bulgaricus 9702 grown under certain conditions produces a substance with hepatoprotective, gastroenterological and immune system modulating properties, but does not describe specific clinical experimental data that supports this claim.
  • Lactobacillus delbrueckii subsp. Strain is described in patent application WO2008 / 043161.
  • Bulgaricus deposited in the "National Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) under n ° 8481, and is indicated in a general way that can be used for the preparation of pharmaceutical or food products with prophylactic and therapeutic properties, showing effect at the local level of the digestive system.
  • NBIMCC National Bank for Industrial Microorganisms and Cell Cultures
  • the present invention relates to the use of the strain of Lactobacillus delbrueckii subsp. bulgaricus, deposited in the "National Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) under n ° 8481 for the preparation of drugs or food supplements intended for the treatment of immunosenescence or deterioration of the defensive capacity of the immune system due to chronological aging . As a consequence of age, a series of changes in the functioning of the immune system that imply a worse defensive capacity can be observed.
  • NBIMCC National Bank for Industrial Microorganisms and Cell Cultures
  • the pharmaceutical or food product prepared from this probiotic strain can be a tablet or capsule for oral administration to the patient.
  • each capsule contains 400 mg of the probiotic strain.
  • the pharmaceutical or food product can also be presented in the form of envelopes.
  • the administration of a drug containing this probiotic strain has been shown to have implications for a number of modifications in both the innate and adaptive immune responses.
  • the probiotic strain acts on different parameters such as:
  • SIRT1 protein sirtuin-1
  • the probiotic strain has an effect on the production of ⁇ -defensins, molecules that represent a defensive first line against bacteria and viruses.
  • - Figure 2 is a graphic representation of the distributions of CD8 + CD28 + CD8 + CD28 + CD28 + T lymphocytes in the probiotic group and placebo at zero time, three and six months after the ingestion of the probiotic.
  • FIG. 3 shows the distribution of different subpopulations in both TCD4 + and CD8 + lymphocytes in young and elderly individuals.
  • FIG. 4 is a graphic representation of the evolution of the frequency of populations of CD4 and CD8 T lymphocytes of the probiotic group
  • Figure 5 represents the population of CD45RA + cells in young and old.
  • FIG. 6 shows the evolution of CD4 + CD45RA + / CD31 + T lymphocyte populations in five individuals representative of the probiotic group and the placebo group.
  • FIG. 7 shows the population of ⁇ -defensins of the probiotic group and placebo group at zero, three and six months of the probiotic intake.
  • FIG. 8 shows levels of SIRT1 mRNA from the probiotic group and placebo at different times of probiotic intake.
  • FIG. 9 shows two 2D-PAGE gels (two-dimensional polyacrylamide electrophoresis), before (9a) and after (9b) of the probiotic intake.
  • the objective of the study on the immune system was to assess the efficacy of the intake of capsules of Lactobacillus delbrueckii subsp. lyophilized bulgaricus in the potentiation of the immune response in the elderly.
  • the main objective was to study the improvement that the exposure to the Lactobacillus delbrueckii subsp strain produces on different aspects of the immune response. bulgaricus in people older than 65 years.
  • Placebo group group of individuals given placebo capsules, without probiotic
  • IRP Immunological risk profile
  • T lymphocytes can be divided into: na ' ⁇ ve (NAIVE, CD45RA + CCR7 +), central memory (CM; CD54RA-CCR7 +), effector memory (E; CD45RA + CCR7) -) and terminally differentiated effector memory (EMRA, CD45RA + CCR7-).
  • NAIVE CD45RA + CCR7 +
  • CM central memory
  • E effector memory
  • EMRA terminally differentiated effector memory
  • CD45RA + cells the expression of CD31 is related to a higher degree of immaturity, this phenotype corresponding to the cells of more recent thymic migration. As can be seen in the representative figure 5, young individuals have this population clearly increased compared to the older individuals.
  • Defensins are cysteine proteins rich in ions, they are found in vertebrates and invertebrates and function as natural antibiotics that are found on the surface of the skin. They are active against bacteria, fungi and viruses. Most defensins act by penetrating the microbial plasma membrane, once they have penetrated, they form a pore that produces cell death.
  • Defensins are peptides highly conserved in the evolutionary scale forming part of the innate immune response, they have a size of 2-5 kilodaltons (typically 30-45 amino acids) and have three disulfide bridges. In humans, they are synthesized mainly in epithelia and leukocytes. Its antimicrobial properties are broad spectrum (antibacterial, antifungal, antiviral) varying in microbicidal effectiveness according to its structure and net cationic charge. According to their molecular pattern of disulfide bridges and genomic distribution they are classified into the alpha (a), beta ( ⁇ ) and theta ( ⁇ ) families.
  • the a-defensins 1-4 have anti-HIV activity and it has been shown that although they have the property of binding to the C1q peptide and thus activating the classical pathway of the complement system. Additionally they have a chemotactic effect for T lymphocytes, monocytes and mature dendritic cells, in addition to inducing the synthesis of cytokines. In this study, no difference was found in the expression of a-defensin 1, neither in the people who took the placebo nor in those who took the probiotic. It is hypothesized that ⁇ -defensins block late events of the replication of viruses and other pathogens. They induce intracellular signals by interacting with chemokine receptors and Toll-like receptors (TLRs), whose pathways promote adaptive immune responses by recruiting dendritic cells and T to the site of invasion.
  • TLRs Toll-like receptors
  • SIRT1 Sirtuin-1
  • SIRT1 is the most studied member of the sirtuin family with respect to increased longevity and age-related diseases. It is believed that it plays a crucial role in the aging process for several reasons. SIRT1 is decreased in senescent cells and in aging. Caloric reduction, clearly related to longevity, induces the production of SIRT1, possibly increasing cell survival. The increase in its expression is also related to the increase in mitochondrial biogenesis, which is closely related to longevity and aging. One activator of SIRT1 is resveratrol whose beneficial effects on cell rejuvenation have been shown to be mediated by the increase in SIRT1.
  • proteomics is the large-scale study of proteins, particularly their structure and function.
  • the proteome is the complete complement of proteins, including the modifications made in a particular set of proteins, produced by an organism or system.
  • Figure 2 shows two 2D-PAGE gels (two-dimensional polyacrylamide electrophoresis) before and after the probiotic intake.

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Abstract

The invention relates to the use of a strain of Lactobacillus delbrueckii subsp. Bulgaricus for the preparation of a drug or food supplement for treating immunosenescence or deterioration of the immune system as a result of chronological ageing. Said strain exhibits systemic effects, both in the innate immune response and in the adaptive immune response.

Description

USO DE UNA CEPA DE LACTOBACILLUS BULGARICUS PARA EL TRATAMIENTO DE  USE OF A LACTOBACILLUS BULGARICUS SEED FOR THE TREATMENT OF
LA INMUNOSENESCENCIA  IMMUNOSENESCENCE
Objeto de la invención Object of the invention
La presente invención se refiere al uso de una cepa de Lactobacillus bulgaricus para la preparación de un fármaco o complemento alimenticio destinado al tratamiento de la inmunosenecencia o deterioro de la capacidad defensiva del sistema inmune como consecuencia del envejecimiento cronológico. Concretamente se refiere al uso de la cepa de Lactobacillus delbrueckii subsp. bulgaricus, aislada en la montañas de Bulgaria y depositada en el "Nacional Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) bajo el n° 8481. The present invention relates to the use of a strain of Lactobacillus bulgaricus for the preparation of a drug or food supplement intended for the treatment of immunosenecence or deterioration of the defensive capacity of the immune system as a consequence of chronological aging. Specifically, it refers to the use of the strain of Lactobacillus delbrueckii subsp. bulgaricus, isolated in the mountains of Bulgaria and deposited in the "National Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) under n ° 8481.
Antecedentes de la invención  BACKGROUND OF THE INVENTION
Los probióticos son microorganismos no patógenos que ejercen una influencia positiva sobre la salud o la fisiología del huésped al ser ingeridos. Entre las propiedades beneficiosas de estos microorganismos se encuentra su actividad inmunomoduladora. A lo largo de la vida se produce un envejecimiento del sistema inmune que ocasiona un deterioro de su capacidad defensiva que se conoce como inmunosenescencia y que implica una serie de modificaciones tanto en la respuesta innata como adaptativa. Esta proceso se caracteriza fundamentalmente por (1 ) la limitación del repertorio de los linfocitos T y la acumulación de células oligoclonales de memoria efectora dirigidas frente a agentes infecciosos ubicuos; (2) la involución tímica y desaparición de células "na'íve"; y (3) un estado inflamatorio crónico. El mecanismo basal de esta pérdida de eficacia inmunológica no se conoce pero tiene gran importancia desde un punto de vista clínico, particularmente la disfunción de la respuesta inmune humoral responsable de la eficacia de la vacunación. Existe una clara asociación entre la funcionalidad del sistema inmune y la longevidad de los individuos, encontrándose considerables evidencias de que la inmunosenescencia implica mayor morbilidad y mortalidad. En este sentido, evitar o retrasar el deterioro del sistema inmune desde edades tempranas se considera decisivo para conseguir un mejor estado general y una mayor longevidad. Probiotics are non-pathogenic microorganisms that exert a positive influence on the health or physiology of the host when ingested. Among the beneficial properties of these microorganisms is their immunomodulatory activity. Throughout life there is an aging of the immune system that causes a deterioration of its defensive capacity that is known as immunosenescence and that involves a series of modifications in both the innate and adaptive response. This process is mainly characterized by (1) the limitation of the repertoire of the T lymphocytes and the accumulation of oligoclonal effector memory cells directed against ubiquitous infectious agents; (2) thymic involution and disappearance of "na ' íve"cells; and (3) a chronic inflammatory state. The basal mechanism of this loss of immunological efficacy is not known but it is of great importance from a clinical point of view, particularly the dysfunction of the humoral immune response responsible for the effectiveness of the vaccination. There is a clear association between the functionality of the immune system and the longevity of individuals, with considerable evidence that immunosenescence implies greater morbidity and mortality. In this sense, avoiding or delaying the deterioration of the immune system from an early age is considered decisive to achieve a better general state and greater longevity.
Entre los organismos probióticos inherentes al aparato digestivo de los humanos, es conocido el efecto de los Lactobacillus, presentando un efecto en la intolerancia a la lactosa, efecto sobre el sistema inmune, etc, debido a su capacidad para controlar las infecciones manteniendo un equilibrio adecuado en la microflora del intestino. Diferentes cepas de Lactobacillus se han utilizado para la preparación de productos con adecuadas propiedades organolépticas y con características profilácticas y terapeúticas. Dentro de los Lactobacillus, la aplicación de las cepas conocidas de Lactobacillus delbrueckii subsp. bulgaricus ha estado limitada al área de productos lácticos fermentados y su actividad está limitada a un corto plazo de tiempo. Among the probiotic organisms inherent to the digestive system of humans, the effect of Lactobacillus is known, presenting an effect on lactose intolerance, effect on the immune system, etc., due to its ability to control infections while maintaining an adequate balance in the microflora of the intestine. Different strains of Lactobacillus have been used for the preparation of products with adequate organoleptic properties and with prophylactic and therapeutic characteristics. Within the Lactobacillus, the application of the known strains of Lactobacillus delbrueckii subsp. bulgaricus has been limited to the area of fermented milk products and its activity is limited to a short period of time.
Como ejemplos, las patentes BG63961 , BG63962 y BG63963 divulgan que cepas de Lactobacillus delbrueckii subsp. bulgaricus son usadas como cultivos para yogur búlgaro y otros productos lácteos fermentados. La patente BG61476 divulga la posibilidad de combinación de una sustancia biológicamente activa procedente del cultivo de Lactobacillus delbrueckii subsp. bulgaricus con productos naturales para la preparación de aditivos alimenticios.  As examples, the patents BG63961, BG63962 and BG63963 disclose that strains of Lactobacillus delbrueckii subsp. bulgaricus are used as crops for Bulgarian yogurt and other fermented dairy products. The patent BG61476 discloses the possibility of combining a biologically active substance from the culture of Lactobacillus delbrueckii subsp. bulgaricus with natural products for the preparation of food additives.
Las patentes UA63201 , UA63202 y UA64163 divulgan que una cepa de Lactobacillus delbrueckii subsp. bulgaricus 9702 cultivada bajo determinadas condiciones produce una sustancia con propiedades hepatoprotectoras, gastroenterológicas y moduladores del sistema inmune, pero no describen datos experimentales clínicos concretos que soporten esta afirmación. Patents UA63201, UA63202 and UA64163 disclose that a strain of Lactobacillus delbrueckii subsp. bulgaricus 9702 grown under certain conditions produces a substance with hepatoprotective, gastroenterological and immune system modulating properties, but does not describe specific clinical experimental data that supports this claim.
En la solicitud de patente WO2008/043161 se describe la cepa de Lactobacillus delbrueckii subsp. Bulgaricus, depositada en el "Nacional Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) bajo el n° 8481 , y se indica de forma general que puede ser usada para la preparación de productos farmacéuticos o alimenticios con propiedades profilácticas y terapéuticas, mostrando efecto a nivel local del aparato digestivo. The Lactobacillus delbrueckii subsp. Strain is described in patent application WO2008 / 043161. Bulgaricus, deposited in the "National Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) under n ° 8481, and is indicated in a general way that can be used for the preparation of pharmaceutical or food products with prophylactic and therapeutic properties, showing effect at the local level of the digestive system.
En la presente invención, se describe el uso, hasta ahora no descrito de la cepa de Lactobacillus delbrueckii subsp. Bulgaricus, NBIMCC n° 8481 , evaluando parámetros a nivel sistémico. La mayoría de los estudios inmunológicos realizados hasta ahora con probióticos sólo han demostrado efectos a nivel local de mucosa intestinal y no a nivel de sangre periférica. In the present invention, the use, hitherto not described, of the strain of Lactobacillus delbrueckii subsp. Bulgaricus, NBIMCC n ° 8481, evaluating parameters at a systemic level. Most immunological studies done so far with probiotics have only shown local effects of intestinal mucosa and not peripheral blood level.
Descripción de la invención Description of the invention
La presente invención se refiere al uso de la cepa de Lactobacillus delbrueckii subsp. bulgaricus, depositada en el "Nacional Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) bajo el n° 8481 para la preparación de fármacos o complementos alimenticios destinados al tratamiento de la inmunosenescencia o deterioro de la capacidad defensiva del sistema inmunitario debido al envejecimiento cronológico. Como consecuencia de la edad, se pueden observar una serie de cambios en el funcionamiento del sistema inmune que implican una peor capacidad defensiva.  The present invention relates to the use of the strain of Lactobacillus delbrueckii subsp. bulgaricus, deposited in the "National Bank for Industrial Microorganisms and Cell Cultures" (NBIMCC) under n ° 8481 for the preparation of drugs or food supplements intended for the treatment of immunosenescence or deterioration of the defensive capacity of the immune system due to chronological aging . As a consequence of age, a series of changes in the functioning of the immune system that imply a worse defensive capacity can be observed.
El producto farmacéutico o alimenticio preparado a partir de esta cepa de probiótico puede ser un comprimido o cápsula para su administración oral al paciente. Preferiblemente, cada cápsula contiene 400mg de la cepa de probiótico.  The pharmaceutical or food product prepared from this probiotic strain can be a tablet or capsule for oral administration to the patient. Preferably, each capsule contains 400 mg of the probiotic strain.
El producto farmacéutico o alimenticio también puede presentarse en forma de sobres. La administración de un fármaco conteniendo esta cepa de probiótico ha demostrado tener implicaciones en una serie de modificaciones tanto en la respuesta inmunitaria innata como en la adaptativa. The pharmaceutical or food product can also be presented in the form of envelopes. The administration of a drug containing this probiotic strain has been shown to have implications for a number of modifications in both the innate and adaptive immune responses.
En cuanto a la respuesta adaptativa, la cepa de probiótico actúa sobre distintos parámetros como son:  Regarding the adaptive response, the probiotic strain acts on different parameters such as:
- la frecuencia de la subpoblación de linfocitos T CD8+CD28. Esta población es el principal factor que define el perfil de riesgo inmunológico (IRP), asociado a la longevidad de los individuos, su disminución representa un efecto claramente beneficioso ralentizando la senescencia del sistema inmune  - the frequency of the CD8 + CD28 T lymphocyte subpopulation. This population is the main factor that defines the profile of immunological risk (IRP), associated with the longevity of individuals, its decrease represents a clearly beneficial effect slowing the senescence of the immune system
- la frecuencia de las subpoblaciones de células na'íve CD45RA+CCR7+La capacidad de un individuo de reconocer y responder frente a nuevo antígenos depende en gran medida de la frecuencia de estas subpoblaciones. El aumento de las mismas implicaría una mayor y mejor capacidad de respuesta frente a patógenos, tumores,.. - the frequency of cell subpopulations na ' íve CD45RA + CCR7 + The ability of an individual to recognize and respond to new antigens depends to a large extent on the frequency of these subpopulations. The increase of them would imply a greater and better capacity of response against pathogens, tumors, ..
- los linfocitos T CD4+ de reciente migración tímica, que representan un estado muy inmaduro, cuyo aumento podría estar reflejando cierto grado de "rejuvenecimiento" del sistema inmunológico.  - the CD4 + T lymphocytes of recent thymic migration, which represent a very immature state, whose increase could be reflecting a certain degree of "rejuvenation" of the immune system.
- la proteína sirtuína-1 (SIRT1), que tiene un papel demostrado en evitar la senescencia celular y aumentar la longevidad.  - the protein sirtuin-1 (SIRT1), which has a proven role in preventing cellular senescence and increasing longevity.
Además de los efectos de la cepa de probiótico sobre la inmunidad adaptativa también se han encontrado efectos en determinados parámetros de la inmunidad innata, como son los niveles de células NKs, implicadas fundamentalmente en la defensa anti-viral y anti-tumoral. Esta población presente una clara disminución en el proceso de envejecimiento, con lo que su aumento resultaría claramente beneficioso. Dentro de la respuesta innata, la cepa de probiótico tiene efecto en la producción de β-defensinas, moléculas que suponen una primera línea defensiva frente a bacterias y virus.  In addition to the effects of the probiotic strain on adaptive immunity, effects have also been found in certain parameters of innate immunity, such as the levels of NK cells, mainly involved in anti-viral and anti-tumor defense. This population presents a clear decrease in the aging process, with which its increase would clearly be beneficial. Within the innate response, the probiotic strain has an effect on the production of β-defensins, molecules that represent a defensive first line against bacteria and viruses.
El efecto de la cepa Lactobacillus delbrueckii subsp. Bulgaricus, NBIMCC n° 8481 en los parámetros antes mencionados ha sido demostrada en un estudio que a continuación se describirá y que ilustra los efectos de la misma sobre el sistema inmune de los individuos a los que se administra un fármaco que contiene dicha cepa (grupo probiótico) frente a los individuos que reciben un fármaco con placebo (grupo placebo).  The effect of the strain Lactobacillus delbrueckii subsp. Bulgaricus, NBIMCC n ° 8481 in the aforementioned parameters has been demonstrated in a study that will be described below and that illustrates the effects thereof on the immune system of individuals administered a drug containing said strain (group probiotic) versus individuals receiving a drug with placebo (placebo group).
Breve descripción de ios dibujos Brief description of the drawings
- La figura 1 es una representación gráfica de las distribuciones de poblaciones de linfocitos B, T y células NK en el grupo placebo y en el grupo probiótico, a tiempo cero (antes de la ingesta del probiótico, t=0), a los tres meses (t=3) y a los seis meses (t=6) de la ingesta de probiótico. - La figura 2 es una representación gráfica de las distribuciones de linfocitos T CD8+CD28 y linfocitos T CD8+CD28+CD27+ en el grupo probiótico y placebo a tiempo cero, tres y seis meses de la ingesta del probiótico. - Figure 1 is a graphic representation of the population distributions of B lymphocytes, T cells and NK cells in the placebo group and in the probiotic group, at time zero (before the ingestion of the probiotic, t = 0), at three months (t = 3) and at six months (t = 6) of the probiotic intake. - Figure 2 is a graphic representation of the distributions of CD8 + CD28 + CD8 + CD28 + CD28 + T lymphocytes in the probiotic group and placebo at zero time, three and six months after the ingestion of the probiotic.
- La figura 3 muestra la distribución de distintas subpoblaciones tanto en linfocitos TCD4+ como CD8+ en individuos jóvenes y ancianos.  - Figure 3 shows the distribution of different subpopulations in both TCD4 + and CD8 + lymphocytes in young and elderly individuals.
- La figura 4 es una representación gráfica de la evolución de la frecuencia de poblaciones de linfocitos T CD4 y CD8 del grupo probiótico  - Figure 4 is a graphic representation of the evolution of the frequency of populations of CD4 and CD8 T lymphocytes of the probiotic group
- La figura 5 representa la población de células CD45RA+ en jóvenes y ancianos.  - Figure 5 represents the population of CD45RA + cells in young and old.
- La figura 6 muestra la evolución de poblaciones de linfocitos T CD4+CD45RA+/CD31 + en cinco individuos representativos del grupo probiótico y del grupo placebo.  - Figure 6 shows the evolution of CD4 + CD45RA + / CD31 + T lymphocyte populations in five individuals representative of the probiotic group and the placebo group.
- La figura 7 muestra la población de β-defensinas del grupo probiótico y grupo placebo a cero, tres y seis meses de la ingesta del probiótico.  - Figure 7 shows the population of β-defensins of the probiotic group and placebo group at zero, three and six months of the probiotic intake.
- La figura 8 muestra niveles de mRNA SIRT1 del grupo probiótico y placebo a diferentes tiempos de la ingesta del probiótico.  - Figure 8 shows levels of SIRT1 mRNA from the probiotic group and placebo at different times of probiotic intake.
- La figura 9 muestra dos geles 2D-PAGE (electroforesis de poliacrilamida de dos dimensiones), antes (9a) y después (9b) de la toma del probiótico. - Figure 9 shows two 2D-PAGE gels (two-dimensional polyacrylamide electrophoresis), before (9a) and after (9b) of the probiotic intake.
Descripción detallada de la invención Detailed description of the invention
Para completar la descripción se incluye un estudio que ilustra los efectos de la cepa de probiótico sobre el sistema inmune de los individuos a los que se administra un fármaco que contiene dicha cepa  To complete the description, a study is included that illustrates the effects of the probiotic strain on the immune system of the individuals to whom a drug containing said strain is administered.
Estudio realizado Study carried out
En el estudio realizado se han evaluado las posibles propiedades beneficiosas sobre la respuesta inmune de una cepa seleccionada in vitro de Lactobacillus delbrueckii subsp. bulgaricus NBI CC n°8481 como probiótico.  In the study carried out, the possible beneficial properties on the immune response of a strain selected in vitro from Lactobacillus delbrueckii subsp. bulgaricus NBI CC n ° 8481 as probiotic.
El objetivo del estudio sobre el sistema inmune fue valorar la eficacia de la ingesta de cápsulas de Lactobacillus delbrueckii subsp. bulgaricus liofilizado en la potenciación de la respuesta inmune en ancianos. El objetivo principal fue estudiar la mejoría que produce sobre distintos aspectos de la respuesta inmune la exposición a la cepa de Lactobacillus delbrueckii subsp. bulgaricus en personas mayores de 65 años. The objective of the study on the immune system was to assess the efficacy of the intake of capsules of Lactobacillus delbrueckii subsp. lyophilized bulgaricus in the potentiation of the immune response in the elderly. The main objective was to study the improvement that the exposure to the Lactobacillus delbrueckii subsp strain produces on different aspects of the immune response. bulgaricus in people older than 65 years.
Para este análisis se seleccionó un grupo de individuos mayores de 65 años en los que, como consecuencia de la edad, se puede observar una serie de cambios en el funcionamiento de su sistema inmune que implican una peor capacidad defensiva. Los efectos beneficiosos del probiótico resultan más evidentes en los individuos de edad avanzada debido a este estado de inmunodeficiencia asociada a la edad. For this analysis, a group of individuals older than 65 years was selected in which, as a consequence of age, a series of changes in the functioning of their immune system can be observed that imply a worse defensive capacity. The beneficial effects of probiotic are more evident in elderly individuals due to this age-related immunodeficiency state.
Los pasos seguidos en el desarrollo del proyecto fueron los siguientes: -Captación de voluntarios para el desarrollo del estudio T ES2011/000329 The steps followed in the development of the project were the following: -Captation of volunteers for the development of the study T ES2011 / 000329
-Extracción y procesamiento de las muestras de sangre periférica a tiempo cero (nivel basal), a los tres y a los seis meses del inicio del estudio -Seguimiento del cumplimiento del protocolo del estudio por parte de los voluntarios y recogida de datos clínicos por parte de los investigadores -Extraction and processing of peripheral blood samples at zero time (basal level), at three and six months after the start of the study -Follow-up of compliance with the study protocol by the volunteers and collection of clinical data by the researchers
Materiales y Métodos Materials and methods
• Caracterización de distintas subpoblaciones celulares por citometria de flujo • Characterization of different cellular subpopulations by flow cytometry
• Cuantificación de citocinas en suero de los voluntarios mediante tecnología xMAP de Luminex • Quantification of serum cytokines from volunteers using Luminex xMAP technology
• Cuantificación en suero de inmunoglobulinas (Ig A, Ig G, Ig M) y factores del complemento por turbidimetría y nefelometría.  • Serum quantification of immunoglobulins (Ig A, IgG, IgM) and complement factors by turbidimetry and nephelometry.
• Estimulación in-vitro (anti-CD3 y LPS) de células mononucleares de células de sangre periférica para determinar posibles cambios en la producción de distintas citocnas/factores solubles en el sobrenadante de los mismos.  • In-vitro stimulation (anti-CD3 and LPS) of peripheral blood cell mononuclear cells to determine possible changes in the production of different cytokines / soluble factors in their supernatant.
• Capacidad oxidativa/fagocítica de la población polimorfonuclear  • Oxidative / phagocytic capacity of the polymorphonuclear population
· Valoración de la función tímica (cuantificación de TRECS por RT-PCR), expresión de CD31 por citometria de flujo. · Evaluation of the thymic function (quantification of TRECS by RT-PCR), expression of CD31 by flow cytometry.
• Recogida y almacenamiento de suero, DNA y RNA de los individuos en estudio  • Collection and storage of serum, DNA and RNA of the individuals under study
• Cuantificación por ELISA de α y β-defensinas  • ELISA quantification of α and β-defensins
• Cuantificación por RT-PCR de la sirtuina-1  • Quantification by RT-PCR of sirtuin-1
· Comparación de los proteinogramas de voluntarios antes y después de la ingesta de cápsulas de probiótico mediante técnicas proteómicas. · Comparison of the proteinograms of volunteers before and after the ingestion of probiotic capsules using proteomic techniques.
• Analíticas de control (hematología, bioquímica y análisis de orina)  • Control analytics (hematology, biochemistry and urinalysis)
• Los resultados se analizaron con el programa estadístico SPSS 15.0. En aquellos casos en que los datos mostraron una distribución normal (test de Koimogorov-Smirnov) se realizaron las comparaciones entre los distintos tiempos utilizando la prueba T para datos pareados. En los casos en los que la distribución de los datos no fue normal se compararon mediante el test de Wilcoxon.  • The results were analyzed with the statistical program SPSS 15.0. In those cases in which the data showed a normal distribution (Koimogorov-Smirnov test) comparisons were made between the different times using the T test for paired data. In the cases in which the distribution of the data was not normal, they were compared using the Wilcoxon test.
Desarrollo del estudio Development of the study
En la visita cero se comprobaron los criterios de inclusión necesarios para poder participar y se realizaron las pruebas necesarias para el estudio.  At the zero visit, the necessary inclusion criteria were verified in order to participate and the necessary tests for the study were carried out.
Se clasificaron de forma aleatoria a los participantes en uno de los dos grupos:  The participants were randomly classified into one of the two groups:
- Grupo probiótico (grupo de individuos a los que se administraron cápsulas de probiótico) - Probiotic group (group of individuals given probiotic capsules)
- Grupo placebo (grupo de individuos a los que se administraron cápsulas de placebo, sin probiótico) - Placebo group (group of individuals given placebo capsules, without probiotic)
Se realizaron todas las pruebas del estudio, recogida de datos y analíticas y se les proporcionaron las cápsulas con Lactobacillus delbrueckii subsp. bulgaricus NBIMCC n° 8481 liofilizado o con placebo. Cada dos meses los voluntarios recibieron el lote de cápsulas con placebo o Lactobacillus delbrueckii subsp. bulgaricus NBIMCC n° 8481 liofilizado y se les evaluó tras 3 y 6 meses (se les realizó recogida de datos y analítica) desde el inicio del tratamiento, comparando los resultados con los de la situación basal al inicio del estudio (visita cero). All the study, data collection and analytical tests were performed and the capsules were provided with Lactobacillus delbrueckii subsp. bulgaricus NBIMCC n ° 8481 lyophilized or with placebo. Every two months the volunteers received the batch of capsules with placebo or Lactobacillus delbrueckii subsp. bulgaricus NBIMCC n ° 8481 lyophilized and were evaluated after 3 and 6 months (data collection and analysis were performed) from the beginning of treatment, comparing the results with those of the baseline situation at the beginning of the study (zero visit).
En el estudio se incluyeron 52 sujetos con edad superior a 65 años. Los voluntarios se dividieron en dos grupos (probiotico, n=28; placebo, n =24), El diseño fue idéntico a un ensayo clínico: prospectivo, doble ciego, controlado con placebo. A los individuos en estudio se les administraron 3 cápsulas al día, tanto de probiotico como de placebo, durante seis meses. The study included 52 subjects older than 65 years. The volunteers were divided into two groups (probiotic, n = 28, placebo, n = 24), The design was identical to a clinical trial: prospective, double-blind, placebo-controlled. The individuals under study were given 3 capsules a day, both probiotic and placebo, for six months.
Resultados Results
• Características hematológicas de los ancianos en estudio:  • Hematological characteristics of the elderly in the study:
Se analizaron por citometría de flujo las subpoblaciones leucocitarias de los individuos en estudio. No se encontraron diferencias en los porcentajes de células polimorfonucleares, monocitos ni linfocitos. Tampoco se encontraron diferencias en la distribución de las poblaciones de linfocitos B y T en ninguno de los dos grupos. En los individuos que tomaron el probiotico se produjo un aumento significativo en el porcentaje de células NK tanto a los tres como a los seis meses (figura 1).  The leukocyte subpopulations of the individuals under study were analyzed by flow cytometry. No differences were found in the percentages of polymorphonuclear cells, monocytes or lymphocytes. No differences were found in the distribution of B and T lymphocyte populations in either group. In the individuals who took the probiotic there was a significant increase in the percentage of NK cells both at three and six months (Figure 1).
• Perfil de riesgo inmunológico (IRP):  • Immunological risk profile (IRP):
En el estudio inmnunológico OCTO (Pawelec G., Akbar A., Caruso C, Effros R., Grubeck- Loebenstein B., Wikby A.,"ls inmunosenescence infections?" Trends inmunol. August 2004. Ferguson FG, Wikby A, Maxon P, Olsson J, Johansson B " Immune parameters in a longitudinal study of a very oíd population of Swedish people: a comparison of survivors and nonsurvivors" J Gerontol A Biol Se/ Med Sci. 1995" realizado en Suecia entre personas de más de ochenta años se identificó un perfil de riesgo inmunológico (IRP) que relaciona ciertos parámetros inmunológicos con una mortalidad aumentada. La principal variable asociada con una mayor mortalidad fue la subpoblación celular de linfocitos T CD8+CD28nu". Estas células representan un estadio altamente diferenciado dentro del conjunto de linfocitos T CD8+, con una capacidad reducida para enfrentarse a nuevas patologías. In the OCTO inmnunological study (Pawelec G., Akbar A., Caruso C, Effros R., Grubeck-Loebenstein B., Wikby A., "Immunosenescence infections ls" Trends immunol August 2004. Ferguson FG, Wikby A, Maxon P, Olsson J, Johansson B "Immune parameters in a longitudinal study of a very old population of Swedish people: a comparison of survivors and nonsurvivors" J Gerontol A Biol Se / Med Sci. 1995 "conducted in Sweden among people over eighty years profile immunological risk (IRP) relating certain immunological parameters with increased mortality was identified. the main variable associated with increased mortality was cell subpopulation of CD8 + CD28 nu "cells. These cells represent a highly differentiated stage within of the set of CD8 + T lymphocytes, with a reduced capacity to face new pathologies.
Los individuos que tomaron el probiotico presentaron una disminución significativa en el porcentaje de esta población envejecida a los tres y a los seis meses del inicio de la ingesta. Los niveles se mantuvieron estables a lo largo del tiempo en los individuos del grupo placebo. The individuals who took the probiotic presented a significant decrease in the percentage of this aged population at three and six months after the beginning of the intake. The levels remained stable over time in the individuals of the placebo group.
Dentro de la población de linfocitos T CD8+, en los individuos del grupo probiotico, también encontramos diferencias significativas en los niveles de la subpoblación CD27+CD28+, que representa un grupo de células poco diferenciadas, capaces de reconocer nuevos antígenos (figura 2). 329 Within the population of CD8 + T lymphocytes, in the individuals of the probiotic group, we also found significant differences in the levels of the CD27 + CD28 + subpopulation, which represents a group of poorly differentiated cells, capable of recognizing new antigens (figure 2). 329
• Estado de diferenciación en las distintas subpoblaciones de células T en individuos de edad avanzada: • State of differentiation in the different T cell subpopulations in elderly individuals:
Existen diversos marcadores cuya expresión permite distinguir el estado de diferenciación de los linfocitos T, fundamentalmente en el caso de las células T CD8+, más afectadas por el proceso de envejecimiento ya desde edades tempranas. Dependiendo de la expresión de CD45RA y del receptor de quimiocinas CCR7, los linfocitos T se pueden dividir en: na'íve (NAIVE; CD45RA+CCR7+), memoria central (CM; CD54RA-CCR7+), memoria efectora (E ; CD45RA+CCR7-) y memoria efectora diferenciada de forma terminal (EMRA; CD45RA+CCR7-). Aquellos individuos que presenten un sistema inmunológico "joven" y más funcional presentarán mayor frecuencia de la subpoblación CD45RA+CCR7+. La población CD45RA+CCR7- representa aquellas células más envejecidas y con menor capacidad de respuesta y será más frecuente en individuos con sistemas inmunológicos más senescentes (figura 3). There are several markers whose expression allows us to distinguish the state of differentiation of T lymphocytes, fundamentally in the case of CD8 + T cells, which are more affected by the aging process and from an early age. Depending on the expression of CD45RA and the CCR7 chemokine receptor, the T lymphocytes can be divided into: na ' íve (NAIVE, CD45RA + CCR7 +), central memory (CM; CD54RA-CCR7 +), effector memory (E; CD45RA + CCR7) -) and terminally differentiated effector memory (EMRA, CD45RA + CCR7-). Those individuals with a "younger" and more functional immune system will have a higher frequency of the CD45RA + CCR7 + subpopulation. The population CD45RA + CCR7- represents those cells that are more aged and less responsive and will be more frequent in individuals with more senescent immune systems (figure 3).
En los individuos del grupo probiótico se observó un aumento de la población CD45RA+CCR7+ (células na'íve) tanto en linfocitos T CD4+ como en linfocitos T CD8+. El aumento en esta población, esencial para responder y desarrollar respuestas inmunológicas frente a nuevos antígenos, es mucho más rápido en los linfocitos T CD8+, produciéndose un aumento importante de esta población a los tres meses y manteniéndose los niveles hasta los seis meses. Sin embargo en los linfocitos T CD4+ este aumento es mucho más gradual y constante. En los voluntarios que tomaron el placebo no se observó ninguna diferencia dentro de estas poblaciones (figura 4). In the individuals of the probiotic group, an increase in the CD45RA + CCR7 + population (na ' íve cells) was observed both in CD4 + T lymphocytes and in CD8 + T lymphocytes. The increase in this population, essential to respond and develop immune responses against new antigens, is much faster in CD8 + T lymphocytes, producing a significant increase in this population at three months and maintaining levels until six months. However, in CD4 + T lymphocytes this increase is much more gradual and constant. In the volunteers who took the placebo, no difference was observed within these populations (figure 4).
• Proximidad tímica de linfocitos T CD4+ tras la ingesta del probiótico.  • Thymic proximity of CD4 + T lymphocytes after the ingestion of the probiotic.
En las células CD45RA+ la expresión de CD31 se relaciona con mayor grado de inmadurez, correspondiendo este fenotipo a las células de más reciente migración tímica. Como se puede observar en la figura 5 representativa los individuos jóvenes presentan esta población claramente incrementada respecto a los individuos de mayor edad.  In CD45RA + cells the expression of CD31 is related to a higher degree of immaturity, this phenotype corresponding to the cells of more recent thymic migration. As can be seen in the representative figure 5, young individuals have this population clearly increased compared to the older individuals.
Al analizar la expresión de CD31 en las células CD45RA+ se observó un aumento significativo (p<0,05) de ésta tanto a los tres como a los seis meses en aquellos individuos que tornaron el probiótico (figura 6).  When analyzing the expression of CD31 in the CD45RA + cells, a significant increase (p <0.05) was observed both at three and at six months in those individuals who returned the probiotic (Figure 6).
· Cuantificación de α y β-defensinas: · Quantification of α and β-defensins:
Las defensinas son proteínas de cisteína ricas en iones, se encuentran en vertebrados e invertebrados y funcionan como antibióticos naturales que se hallan en la superficie de la piel. Son activas contra bacterias, hongos y virus. La mayoría de las defensinas actúan al penetrar en la membrana plasmática microbiana, una vez que han penetrado, forman un poro que produce la muerte celular.  Defensins are cysteine proteins rich in ions, they are found in vertebrates and invertebrates and function as natural antibiotics that are found on the surface of the skin. They are active against bacteria, fungi and viruses. Most defensins act by penetrating the microbial plasma membrane, once they have penetrated, they form a pore that produces cell death.
Las defensinas son péptidos altamente conservados en la escala evolutiva formando parte de la respuesta inmune innata, tienen un tamaño de 2-5 kilodaltons (típicamente de 30-45 aminoácidos) y presentan tres puentes disulfuro. En el humano se sintetizan principalmente en epitelios y leucocitos. Sus propiedades antimícrobianas son de amplio espectro (antibacterianas, antifúngicas, antivirales) variando en efectividad microbicida según su estructura y carga catiónica neta. De acuerdo con su patrón molecular de puentes disulfuro y distribución genómica se clasifican en las familias alfa (a), beta (β) y teta (Θ). Defensins are peptides highly conserved in the evolutionary scale forming part of the innate immune response, they have a size of 2-5 kilodaltons (typically 30-45 amino acids) and have three disulfide bridges. In humans, they are synthesized mainly in epithelia and leukocytes. Its antimicrobial properties are broad spectrum (antibacterial, antifungal, antiviral) varying in microbicidal effectiveness according to its structure and net cationic charge. According to their molecular pattern of disulfide bridges and genomic distribution they are classified into the alpha (a), beta (β) and theta (Θ) families.
Las a-defensinas 1-4 tienen actividad anti— VIH y se ha demostrado que aunque tienen la propiedad de unirse al péptido C1q y activar de esta forma la vía clásica del sistema del complemento. Adicionalmente tienen un efecto quimiotáctico para linfocitos T, monocitos y células dendríticas maduras, además de inducir la síntesis de cítocinas. En este estudio no se ha encontrado ninguna diferencia en la expresión de a-defensina 1 , ni en las personas que tomaron el placebo ni en las que tomaron el probiótico. Se hipotetiza que las β-defensinas bloquean eventos tardíos de la replicación de virus y otros patógenos. Inducen señales intracelulares al interactuar con receptores de quimiocinas y receptores similares a Toll (TLRs), cuyas vías promueven respuestas inmunes adaptativas reclutando células dendríticas y T al sitio de invasión.  The a-defensins 1-4 have anti-HIV activity and it has been shown that although they have the property of binding to the C1q peptide and thus activating the classical pathway of the complement system. Additionally they have a chemotactic effect for T lymphocytes, monocytes and mature dendritic cells, in addition to inducing the synthesis of cytokines. In this study, no difference was found in the expression of a-defensin 1, neither in the people who took the placebo nor in those who took the probiotic. It is hypothesized that β-defensins block late events of the replication of viruses and other pathogens. They induce intracellular signals by interacting with chemokine receptors and Toll-like receptors (TLRs), whose pathways promote adaptive immune responses by recruiting dendritic cells and T to the site of invasion.
En este estudio se ha encontrado un aumento significativo de estos péptidos antimicrobianos tanto a los tres como a los seis meses de comenzada la ingesta de las cápsulas de probiótico. No se encontró ninguna diferencia en los voluntarios que tomaron placebo (figura 7).  In this study, a significant increase in these antimicrobial peptides has been found both at three and six months after the ingestion of the probiotic capsules. No difference was found in the volunteers who took placebo (figure 7).
· Estudio de lo niveles de sirtuina-1 mediante RT-PCR: · Study of sirtuin-1 levels by RT-PCR:
La sirtuina-1 (SIRT1), es el miembro de la familia de las sirtuinas mejor estudiado con respecto al aumento de longevidad y a enfermedades relacionadas con la edad. Se cree que juega un papel crucial en el proceso de envejecimiento por varias razones. La SIRT1 está disminuida en células senescentes y en el envejecimiento. La reducción calórica, claramente relacionada con la longevidad, induce la producción de SIRT1 , posiblemente aumentando la supervivencia celular. El aumento de su expresión también está relacionado con el incremento en la biogénesis mitocondrial, muy relacionado este proceso con la longevidad y el envejecimiento. Un activador de la SIRT1 es el resveratrol cuyos efectos beneficiosos sobre el rejuvenecimiento celular se ha demostrado que están mediados por el aumento de SIRT1.  Sirtuin-1 (SIRT1) is the most studied member of the sirtuin family with respect to increased longevity and age-related diseases. It is believed that it plays a crucial role in the aging process for several reasons. SIRT1 is decreased in senescent cells and in aging. Caloric reduction, clearly related to longevity, induces the production of SIRT1, possibly increasing cell survival. The increase in its expression is also related to the increase in mitochondrial biogenesis, which is closely related to longevity and aging. One activator of SIRT1 is resveratrol whose beneficial effects on cell rejuvenation have been shown to be mediated by the increase in SIRT1.
Se cuantificaron por RT-PCR los niveles de expresión de mRNA de SIRT1 en células mononucleares de sangre periférica en los individuos en estudio. En los voluntarios que tomaron el probiótico se observó un aumento en los niveles de SIRT1 a los tres meses. A los seis meses los niveles de SIRT1 siguen aumentados respecto al tiempo cero, pero han disminuido respecto a los 3 meses. No se observaron cambios en el grupo placebo (figura 8). 9 000329 The expression levels of SIRT1 mRNA in peripheral blood mononuclear cells in the individuals under study were quantified by RT-PCR. In the volunteers who took the probiotic an increase in the levels of SIRT1 was observed at three months. At six months the levels of SIRT1 continue to increase with respect to time zero, but have decreased with respect to 3 months. No changes were observed in the placebo group (figure 8). 9 000329
• Estudios de fagocitosis y oxidación • Studies of phagocytosis and oxidation
Se realizaron varios experimentos para evaluar a capacidad fagocítica y oxidativa de la población de polimorfonucleares de los voluntarios. No se encontró ninguna diferencia significativa en los parámetros de fagocitosis y oxidación después de la ingesta del probiótico. Los valores en el grupo de individuos que tomaron el probiótico y en el grupo placebo fueron similares.  Several experiments were carried out to evaluate the phagocytic and oxidative capacity of the polymorphonuclear population of the volunteers. No significant difference was found in the parameters of phagocytosis and oxidation after ingestion of the probiotic. The values in the group of individuals who took the probiotic and in the placebo group were similar.
• Estudios de proteómica  • Proteomics studies
La proteómica es el estudio a gran escala de las proteínas, en particular de su estructura y función. El proteoma es la dotación completa de proteínas, incluyendo las modificaciones hechas en un conjunto particular de proteínas, producidas por un organismo o sistema.  Proteomics is the large-scale study of proteins, particularly their structure and function. The proteome is the complete complement of proteins, including the modifications made in a particular set of proteins, produced by an organism or system.
Se realizó el estudio proteómico de cuatro muestras de voluntarios antes y después de la ingesta del probiótico y no se encontraron diferencias en su proteograma. En la siguiente figura 9 se muestran dos geles 2D-PAGE (electroforesis de poliacrilamida de dos dimensiones), antes y después de la toma del probiótico.  The proteomic study of four samples of volunteers before and after the ingestion of the probiotic was performed and no differences were found in its proteogram. Figure 2 shows two 2D-PAGE gels (two-dimensional polyacrylamide electrophoresis) before and after the probiotic intake.
Tras el análisis estadístico de los resultados obtenidos no se observó ninguna diferencia significativa en el proteinograrna de los individuos en estudio.  After the statistical analysis of the results obtained, no significant difference was observed in the proteinogram of the individuals under study.
Conclusiones  Conclusions
Tras el análisis de tos resultados se ha llegado a las siguientes conclusiones sobre los efectos que la cepa probiótica ejerce sobre el organismo que la ingiere: After analyzing the results, the following conclusions have been reached about the effects that the probiotic strain exerts on the body that ingests it:
• Disminución de la frecuencia de la subpoblación de linfocitos T CD8+CD28nu", lo que representa un efecto claramente beneficioso ralentizando la senescencia del sistema inmune. En contrapartida se observó un incremento de la subpoblación menos diferenciada CD8+CD27+CD28+. • Decrease in the frequency of the subpopulation of CD8 + CD28 lymphocytes nu ", which represents a clearly beneficial effect slowing down the senescence of the immune system In contrast there was an increase in the less differentiated subpopulation CD8 + CD27 + CD28 +.
· Aumento tanto en linfocitos T CD4+ como CD8+ de la subpobtación na'íve CD45RA+CCR7 tanto a los tres como a los seis meses, lo que implica una mayor y mejor capacidad de respuesta frente a patógenos, tumores... · Increase in both CD4 + and CD8 + T lymphocytes of the subpopulation na ' íve CD45RA + CCR7 both at three and six months, which implies a greater and better response capacity against pathogens, tumors ...
• Aumento en la proporción de linfocitos T CD4+ de reciente migración tímica, lo que refleja, junto con la disminución de poblaciones muy diferenciadas, cierto grado de rejuvenecimiento del sistema inmunológico.  • Increase in the proportion of CD4 + T lymphocytes of recent thymic migration, which reflects, along with the decrease in highly differentiated populations, a certain degree of rejuvenation of the immune system.
• Incremento en los nieles de mRNA de SIRT1 tras la ingesta del probiótico, lo que podría traducirse en un aumento de longevidad y evitaría la senescencia celular.  • Increase in the levels of mRNA of SIRT1 after ingestion of the probiotic, which could result in an increase in longevity and avoid cellular senescence.
• Aumento progresivo en los niveles de células NKs, implicadas fundamentalmente en la defensa anti-viral y anti-tumoral.  • Progressive increase in the levels of NK cells, mainly involved in anti-viral and anti-tumor defense.
· Aumento en la producción de β-defensinas, lo que implicaría un aumento defensivo frente a bacterias y virus. A la vista de los resultados, la cepa de Lactobacillus delbrueckii subsp. bulgaricus, NBIMCC 8481 ha demostrado tener efectos a nivel sistémico, mejorando la respuesta del sistema inmune, el cual sufre un importante deterioro como consecuencia del envejecimiento cronológico. · Increase in the production of β-defensins, which would imply a defensive increase against bacteria and viruses. In view of the results, the strain of Lactobacillus delbrueckii subsp. bulgaricus, NBIMCC 8481 has been shown to have effects at a systemic level, improving the response of the immune system, which suffers a significant deterioration as a result of chronological aging.

Claims

Reivindicaciones Claims
1. - Uso de la cepa de Lactobacillus delbrueckii subsp. bulgaricus, depositada en el "Nacional Bank for Industrial Microorganisms and Cell Cultures" (NBI CC) bajo el n° 8481 para la preparación de un fármaco o complemento alimenticio administrable por vía oral para el tratamiento de la inmunosenescencia o deterioro de la capacidad defensiva del sistema inmunitario como consecuencia del envejecimiento cronológico.  1. - Use of the strain of Lactobacillus delbrueckii subsp. bulgaricus, deposited in the "National Bank for Industrial Microorganisms and Cell Cultures" (NBI CC) under n ° 8481 for the preparation of an orally administrable drug or food supplement for the treatment of immunosenescence or deterioration of the defensive capacity of the immune system as a consequence of chronological aging.
2. - Uso según la reivindicación 1 en el que el tratamiento de la inmunosenescencia o deterioro de la capacidad defensiva del sistema inmunitario como consecuencia del envejecimiento cronológico implica una serie de modificaciones tanto en la respuesta inmunitaria innata como en la adaptativa consistentes en:  2. Use according to claim 1, in which the treatment of immunosenescence or deterioration of the defensive capacity of the immune system as a consequence of chronological aging implies a series of modifications in both the innate and adaptive immune responses consisting of:
-disminución de la frecuencia de la subpoblación de linfocitos T CD8+CD28  -decrease in the frequency of the CD8 + CD28 T lymphocyte subpopulation
-aumento tanto en linfocitos CD4+ como CD8+ de la subpoblación na'íve-increase in both CD4 + and CD8 + lymphocytes of the subpopulation na ' íve
CD45RA+CCR7+ CD45RA + CCR7 +
- aumento en la proporción de linfocitos T CD4+ de reciente migración tímica  - increase in the proportion of CD4 + T lymphocytes of recent thymic migration
-incremento de los niveles de mRNA de sirtuína-1 (SIRT1)  -increment of sirtuin-1 mRNA levels (SIRT1)
-aumento de los niveles de células NK  -increase in NK cell levels
-aumento en la producción de β-defensinas  -Increase in the production of β-defensins
3. -Uso según la reivindicación 1 caracterizado porque el fármaco es una cápsula que contiene dicha cepa Lactobacillus delbrueckii subsp. bulgaricus liofilizado.  3. Use according to claim 1 characterized in that the drug is a capsule containing said strain Lactobacillus delbrueckii subsp. lyophilized bulgaricus.
4. - Uso según la reivindicación 3 caracterizado porque cada cápsula contiene 400 mg de Lactobacillus delbrueckii subsp. Bulgaricus.  4. - Use according to claim 3 characterized in that each capsule contains 400 mg of Lactobacillus delbrueckii subsp. Bulgaricus.
PCT/ES2011/000329 2011-02-09 2011-11-11 Use of a strain of lactobacillus bulgaricus for treating immunosenescence WO2012107601A1 (en)

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