WO2005033331A1 - Method for the identification of substances with prebiotic effect - Google Patents

Method for the identification of substances with prebiotic effect Download PDF

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Publication number
WO2005033331A1
WO2005033331A1 PCT/EP2004/010679 EP2004010679W WO2005033331A1 WO 2005033331 A1 WO2005033331 A1 WO 2005033331A1 EP 2004010679 W EP2004010679 W EP 2004010679W WO 2005033331 A1 WO2005033331 A1 WO 2005033331A1
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sample
microorganism
examined
microorganisms
active substance
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PCT/EP2004/010679
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German (de)
French (fr)
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Dirk Bockmühl
Regine Scholtyssek
Claudia Jassoy
Andrea SÄTTLER
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Henkel Kommanditgesellschaft Auf Aktien
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Priority to EP04765537A priority Critical patent/EP1664329A1/en
Publication of WO2005033331A1 publication Critical patent/WO2005033331A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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

Definitions

  • the present invention relates to a method for identifying prebiotically active substances, the prebiotically active substances obtainable with this method, as well as medicaments, cosmetics, foods and plant treatment compositions containing these substances.
  • microorganisms which are usually also found naturally on the healthy organism and may also be necessary for the optimal functioning of the organism and possibly even for the survival of the organism. Germs in the gastrointestinal tract of humans and mammals are required, for example, to ensure that food is used effectively. The more natural microflora of the skin acts as a natural protective barrier and prevents the settlement of pathogenic microorganisms.
  • microorganisms that are harmful to the skin can be microorganisms that do not naturally occur in the microflora of the healthy organism.
  • inflammatory conditions of the skin can be caused by an excessive proliferation of Propionibacterium acnes, which is a bacterium that can also be found on healthy skin.
  • pathogenicity In addition to pathogenicity, other factors may of course also play a role in why a microorganism is desirable or undesirable. For example, germs can also be undesirable, even though they are not pathogenic, such as germs that cause body odor.
  • Anti-bacterial agents are often used to combat unwanted germs.
  • Non-selectively antibacterial agents such as those used to prevent and combat acne in commercially available cosmetics, not only kill the unwanted skin germs but also the desired skin germs and thus lead to a disturbance in the biological balance of the microflora.
  • prabiotic and prabiotic active compositions contain the desired microorganisms, while prabiotic active compositions allow the growth of desired ones already at the application site Promote existing germs at the expense of the growth of unwanted germs without containing the desired germs themselves.
  • the object of the present invention was to provide a method which makes it possible to easily find substances which have a prebiotic effect.
  • substances with a prebiotic effect can be identified in a simple manner by a method which comprises the following steps: a) Examination of a sample containing microorganisms which has been exposed to at least one substance to be tested for prebiotic activity, the sample at least before the addition of the at least one substance to be examined contains at least one microorganism to be examined for its influence by the substance to be examined, b) examination of a comparative sample which was not exposed to the at least one substance to be examined for its prebiotic activity, c) determination of differences in the appearance and / or in the number, in particular concentration, of the at least one microorganism to be examined between sample and reference sample.
  • a prebiotic effect means that the growth and / or survivability of the desired germs of the microflora of a particular habitat is promoted compared to the growth and / or survivability of the undesired germs of the microflora of the habitat in question.
  • This can be achieved both by the fact that the active ingredient has a beneficial effect on the growth of the desired germs, without having a direct influence on the growth of the undesirable germs, and in that the active ingredient has an inhibitory effect on the growth of the undesired germs, without any direct influence to have the growth of the desired germs.
  • the active ingredient has a beneficial effect on the growth of the desired germs and at the same time has an inhibitory effect on the growth of the undesired germs.
  • the habitat can be any location, in particular any location of any organ or tissue of an organism that can be colonized by microorganisms naturally or due to a disease.
  • the method according to the invention can be carried out by using a sample containing microorganisms for the examination, which sample was obtained by an in vitro method, in particular by cultivation, especially by cultivation in shake flasks.
  • a sample containing microorganisms for the examination which sample was obtained by an in vitro method, in particular by cultivation, especially by cultivation in shake flasks.
  • direct analysis of samples that should have been taken from the affected habitat can be dispensed with.
  • the microorganisms to be examined are used in an in vitro method. A large number of substances can thus be examined in a simple manner to determine whether they have an effect on a large number of microorganisms which can be examined independently of one another to have. In this way, substances that have potentially prebiotic effects can be easily identified.
  • the sample containing microorganisms contains no other organism than the one to be examined for its influence by the added substance. This is achieved by specifically cultivating this organism, preferably in liquid culture (in a shake flask, in reaction vessels or in cell culture plates) or on agar plates.
  • This has the advantage that the multiplication of the microorganism can be used directly as an indicator of the influence of the substance to be examined, which can be done directly optically, in particular microscopically, by plating on agar plates or by a simple technical method, such as, for example, spectrometric determination of the optical density , can be determined. A high technical and time expenditure can be dispensed with here.
  • cultivation is first used to check whether a substance to be tested for its prebiotic activity has a positive effect on a desired microorganism.
  • a second step it is then investigated what effect the substance found to be positive on the desired microorganism has on undesired microorganisms (or possibly on other desired microorganisms).
  • a substance to be tested for its prebiotic activity has a desired, ie negative, effect on an undesired microorganism. Then it can be examined what effect the substance, which has a negative effect on the undesired microorganism, has on desired microorganisms (or possibly also on other undesired microorganisms).
  • the sample containing microorganisms can also contain several organisms. It can do this for example, a collective of desired microorganisms of a certain habitat or a group of undesirable microorganisms of a certain habitat. However, this can also be a sample that was taken from a natural habitat and then cultivated, preferably in shake flasks, on agar plates or in cell culture.
  • a sample that has been taken from a natural habitat that is to say an in vivo sample
  • an in vivo sample can also be analyzed directly without having been cultured beforehand.
  • an in vivo sample is used as a comparative sample, which comes from the same habitat and was exposed to at least one substance to be tested for its prebiotic activity.
  • the comparative sample is obtained by culturing under the same conditions as culturing the sample which contains the at least one substance to be tested for its prebiotic effect, with the difference that no incubation with the at least one substance to be examined.
  • a culture can be divided into two equal volumes after inoculation and one of the two cultures thus obtained can be inoculated with the at least one substance to be tested for its prebiotic effect, while the other sample serves as a reference sample.
  • two media of the same size can be inoculated with the same amount of sample containing microorganisms and cultured under the same conditions, with one culture being exposed to the at least one substance to be tested for its prebiotic effect and the other not.
  • incubation takes place with exactly one substance to be examined for its prebiotic effect. This has the advantage that it is immediately apparent whether this substance has an influence on the growth or survivability of the sample containing at least one microorganism of the microorganisms.
  • incubation takes place with a mixture of several substances to be tested for their prebiotic effect. This method can be used in particular if it is assumed that the number of hits, ie the number of substances that can be identified as effective, will be small.
  • a preliminary screening can then first be carried out in order to determine whether the mixture contains substances which have a prebiotic effect.
  • an individual screening can then be used to investigate the substances to which the proven effect can be attributed.
  • the at least one microorganism to be examined can be any microorganism that occurs naturally in a habitat or that is known for its pathogenic and / or undesirable activity with respect to a specific habitat.
  • the sample containing microorganisms can be a sample which was taken from any habitat of any organism and then optionally cultivated in vitro, the habitat in question from which the sample was taken preferably then naturally the at least one microorganism to be examined contains or may contain due to a pathogenic change.
  • the sample containing microorganisms can also contain, as the only microorganism, the at least one microorganism to be examined or, if appropriate, further microorganisms which have preferably been cultivated specifically with the at least one microorganism to be examined, preferably in vitro.
  • the organism from which the sample can be taken can be a plant, an animal or a human.
  • microflora from which the microorganism has been removed or the microflora sample itself can be, for example, the microflora of the The mouth, stomach or intestines of vertebrates, especially mammals, especially humans.
  • the microflora sample is a sample of an organ or tissue surface, in particular a sample of the leaf surface of plants, the scales or the skin, in particular of vertebrates and mammals.
  • the sample containing microorganisms is a microflora sample that was taken from human skin.
  • skin is preferably the skin itself, in particular the human skin, but also also the mucous membrane and skin appendages, provided that they comprise living cells, in particular hair follicles, hair roots, hair bulbs, the ventral epithelium of the nail bed (lectulus) and sebaceous glands and Understand sweat glands.
  • skin here in particular also includes the skin or mucous membrane of the mouth, throat and pharynx, the esophagus, the stomach, the intestine and the rectum.
  • Desired skin germs on the facial skin of humans which could be promoted in their growth by a prabiotic active substance, are, for example, coagulase-negative staphylococci, in particular S. epidermidis, S. hominis, S. warn, S. saprophyticus, S. xylosus, S. capitis or S. simulans.
  • Unwanted skin germs on the facial skin of humans, which could be inhibited in their growth by a prebiotic active substance are, for example, coagulase-positive staphylococci, in particular S. aureus, or germs selected from the group consisting of Propionibacterium acnes, Candida albicans, Malassezia furfur or Corynebacterium spp.
  • Undesirable germs of the oral mucosa are selected for example from Streptococcus sp., In particular S. mutans, S. salivarius, S. gordonii, S. mitis, S. oralis, S. sang ⁇ is or S.
  • sobrinus from Actinomyces sp., In particular Actinomyces naesiundii, Actinobacillus sp., Especially Actinobacillus actinomycetemcomitans, Prevotella sp., in particular Prevotella intermedia, Porphyromonas sp., in particular Porphyromonas gingivalis, Treponema sp., in particular Treponema denticola, Neisseria sp., Fusobacterium sp., in particular Fusobacterium nucleatum, Candida, in particular Candida albicans or Candida glabrata.
  • the analysis of the effect of the at least one substance to be tested for its prebiotic activity can be carried out in addition to the spectrometry, microscopy and plating methods, for example also by methods such as FISH, DGGE, TGGE (Electrophoresis (1989) 10 (5-6), 377- 89), RT-PCR (Real time-PCR) (Genome Res. (1996) 6 (10), 986-94), DNA microarrays (Applied and Environmental Microbiology (2001) 67 (8), 3677-82) or Antibody assays, in particular ELISA, are carried out.
  • FISH Fluorescence in situ hybridization
  • microorganism species, species or groups can be identified directly in the sample in a highly specific manner and, if necessary, also visualized or quantified. In this way, a distortion-free representation of the actual in situ conditions of the biocenosis possible. Even microorganisms that have not yet been cultivated and therefore not described can be identified.
  • probes penetrate the cells present in the sample under investigation. If a microorganism of the type, genus or group for which the probes were developed is present in the sample under investigation, the probes in the microorganism cell bind to their target sequence and the cells can be detected on the basis of the labeling of the probes.
  • DGGE stands for "denaturing gradient gel electrophoresis". It is a separation process that takes advantage of the difference in melting behavior of different double-stranded DNA fragments. This method was originally developed to detect point mutations by gel electrophoresis, so that resolution is a very sensitive method (Fisher and Lerman (1979) Cell 16, 191-200; Myers et al. (1987) Methods in Enzymology 212, 71 -104). Recently, DGGE has been used very frequently to study natural habitats (Muyzer et al. (1996) Molecular Microbial Ecology Manual 3.4.4: 1-23: Eds: Akkermans et al., Holland 1996; Diez et al. (2001) Appl Environ. Microbiol. 67 (7), 2942-2951).
  • TGGE stands for "temperature gradient gel electrophoresis". In contrast to the DGGE, a temperature gradient is used instead of a denaturing agent. The methods are otherwise equivalent.
  • the present invention furthermore relates to prebiotically active substances which can be obtained with the aid of a process according to the invention, the use of these prebiotically active substances and medicaments, cosmetics, foods and crop protection agents containing these prabiotically active substances.
  • the present invention furthermore relates to the use of a prebiotically active substance according to the invention for the production of a Medicament to promote the growth of undesirable germs and / or to inhibit the growth of undesirable germs.
  • the medicaments and cosmetics according to the invention are those for topical application. These may accordingly contain further components as are customary for such agents, in particular selected from the group consisting of vitamins, provitamins or vitamin precursors of the vitamin B group or their derivatives and derivatives of 2-furanone, panthenol, pantolactone, nicotinamide and biotin, plant extracts , MMP-1 inhibiting substances, esters of retinol (vitamin A- ⁇ ) with a C 2 -i 8 carboxylic acid, surface-active substances as emulsifiers or dispersants, amino acids and their zinc salts and their acid addition salts, film-forming and / or emulsion-stabilizing and / or thickening and / or adhesive polymers, fatty substances, surfactants, anti-perspirants and polyols, organic, mineral and modified mineral light protection filters, protein hydrolyzates and their derivatives, mono-, oligo- and polysaccharides as well as

Abstract

The invention relates to a method for the identification of substances with prebiotic effect, the prebiotic substances obtained by the above method and medicaments, cosmetics, foodstuffs and plant treatment agents comprising said substances.

Description

Verfahren zur Identifizierung prabiotisch wirksamer Substanzen Methods for identifying prabiotic substances
Die vorliegende Erfindung betrifft ein Verfahren zur Identifizierung prabiotisch wirksamer Substanzen, die mit diesem Verfahren erhältlichen prabiotisch wirksamen Substanzen sowie Arzneimittel, Kosmetika, Lebensmittel und Pflanzenbehandlungsmittel enthaltend diese Substanzen.The present invention relates to a method for identifying prebiotically active substances, the prebiotically active substances obtainable with this method, as well as medicaments, cosmetics, foods and plant treatment compositions containing these substances.
Verschiedene Organe und Gewebe des menschlichen, tierischen und pflanzlichen Organismus sind durch Mikroorganismen besiedelt, die in der Regel auch natürlicherweise auf dem gesunden Organismus anzutreffen sind und darüber hinaus zum optimalen Funktionieren des Organismus und gegebenenfalls sogar zum Überleben des Organismus erforderlich sein können. Keime im Gastrointestinaltrakt von Menschen und Säugetieren sind so beispielsweise erforderlich um eine effektive Verwertung der Nahrung sicherzustellen. Die natürlichere Mikroflora der Haut wirkt als natürliche Schutzbarriere und verhindert das Ansiedeln pathogener Mikroorganismen.Various organs and tissues of the human, animal and plant organism are colonized by microorganisms, which are usually also found naturally on the healthy organism and may also be necessary for the optimal functioning of the organism and possibly even for the survival of the organism. Germs in the gastrointestinal tract of humans and mammals are required, for example, to ensure that food is used effectively. The more natural microflora of the skin acts as a natural protective barrier and prevents the settlement of pathogenic microorganisms.
Andererseits gibt es auch Mikroorganismen, die für die Haut schädlich sind. Es kann sich hierbei um Mikroorganismen handeln, die natürlicherweise in der Mikroflora des gesunden Organismus nicht vorkommen. Andererseits kann es jedoch auch sein, dass das natürliche Gleichgewicht zwischen den verschiedenen Mikroorganismen der natürlichen Mikroflora gestört ist und der pathogene Effekt auf eine zu starke Vermehrung eines der normalerweise nicht-pathogenen natürlicherweise in der Mikroflora anzutreffenden Mikroorganismen zurückzuführen ist. So können beispielsweise entzündliche Zustände der Haut auf eine zu starke Vermehrung von Propionibacterium acnes hervorgerufen werden, was ein auch auf gesunder Haut anzutreffendes Bakterium darstellt.On the other hand, there are also microorganisms that are harmful to the skin. These can be microorganisms that do not naturally occur in the microflora of the healthy organism. On the other hand, however, it may also be that the natural balance between the different microorganisms of the natural microflora is disturbed and the pathogenic effect is due to an excessive proliferation of one of the normally non-pathogenic microorganisms naturally found in the microflora. For example, inflammatory conditions of the skin can be caused by an excessive proliferation of Propionibacterium acnes, which is a bacterium that can also be found on healthy skin.
In diesem Sinne ist auch eine Klassifizierung in pathogene und nicht-pathogene Mikroorganismen nur schwer vorzunehmen bzw. können in diesem Sinne Mikroorganismen aufgrund ihres Krankheiten auslösenden Potentials als pathogen eingestuft werden, obwohl sie als Mitglieder der natürlichen Mikroflora auf der Haut natürlich erwünscht sind. Die Frage, ob bestimmte Mikroorganismen pathogen oder nicht-pathogen sind, ist daher zu unterscheiden von der Frage ob diese Organismen bzw. eine Vermehrung dieser Organismen erwünscht oder unerwünscht sind. Während erstere Frage lediglich das pathogene Potential der Mikroorganismen berücksichtigt, berücksichtigt letztere Frage auch die Konzentration der betreffenden Mikroorganismen, insbesondere in Bezug auf die Zusammensetzung der Mikroflora des jeweiligen Habitats zu einem gegebenen Zeitpunkt.In this sense, a classification into pathogenic and non-pathogenic microorganisms is difficult or can be done in this sense Microorganisms are classified as pathogenic due to their disease-causing potential, although they are naturally desired as members of the natural microflora on the skin. The question of whether certain microorganisms are pathogenic or non-pathogenic must therefore be distinguished from the question of whether these organisms or an increase in these organisms are desired or undesirable. While the former question only takes into account the pathogenic potential of the microorganisms, the latter question also takes into account the concentration of the microorganisms in question, in particular with regard to the composition of the microflora of the respective habitat at a given point in time.
Außer dem Gesichtspunkt der Pathogenität können natürlich auch andere Faktoren eine Rolle spielen, weshalb ein Mikroorganismus erwünscht oder unerwünscht ist. So können etwa auch Keime unerwünscht sein, obwohl sie nicht pathogen sind, wie beispielsweise Keime, die Körpergeruch verursachen.In addition to pathogenicity, other factors may of course also play a role in why a microorganism is desirable or undesirable. For example, germs can also be undesirable, even though they are not pathogenic, such as germs that cause body odor.
Zur Bekämpfung unerwünschter Keime werden häufig unselektiv antibakterielle Wirkstoffe eingesetzt. Unselektiv antibakterielle Wirkstoffe, wie sie beispielsweise zur Akneprävention und -bekämpfung in handelsüblichen Kosmetika eingesetzt werden, töten jedoch nicht nur die unerwünschten Hautkeime sondern auch die erwünschten Hautkeime ab und führen so zu einer Störung des biologischen Gleichgewichts der Mikroflora.Anti-bacterial agents are often used to combat unwanted germs. Non-selectively antibacterial agents, such as those used to prevent and combat acne in commercially available cosmetics, not only kill the unwanted skin germs but also the desired skin germs and thus lead to a disturbance in the biological balance of the microflora.
Es besteht daher ein Bedarf, Mittel zur Verfügung zu haben, die selektiv das Wachstum und/oder die Überlebensfähigkeit der wünschenswerten Keime eines Habitats gegenüber dem Wachstum und/oder der Überlebensfähigkeit der unerwünschten Keime des betreffenden Habitats fördern. Solche Substanzen werden auch als "prabiotisch" bezeichnet.There is therefore a need to have means available which selectively promote the growth and / or viability of the desirable germs of a habitat over the growth and / or survivability of the undesirable germs of the habitat in question. Such substances are also called "prabiotic".
Der Unterschied zwischen prabiotisch und prabiotisch wirksamen Zusammensetzungen besteht darin, dass probiotische Zusammensetzungen selbst die erwünschten Mikroorganismen enthalten, während prabiotisch wirksame Zusammensetzungen das Wachstum erwünschter, bereits am Applikationsort vorhandener, Keime auf Kosten des Wachstums unerwünschter Keime fördern, ohne selbst die erwünschten Keime zu enthalten.The difference between prabiotic and prabiotic active compositions is that probiotic compositions themselves contain the desired microorganisms, while prabiotic active compositions allow the growth of desired ones already at the application site Promote existing germs at the expense of the growth of unwanted germs without containing the desired germs themselves.
Der Nachweis eines präbiotischen Effektes von Substanzen ist bislang im wesentlichen beschränkt auf das Intestum. So ist in verschiedenen Publikationen die Verwendung von Substanzen, die förderlich auf das Wachstum wünschenswerter Darmbakterien sind, beschrieben (siehe z.B. Ahn et al. (1990) Microbial Ecology in Health and Disease 3, 223-229). Auch für die Haut wurde bereits über Substanzen mit präbiotischer Wirkung berichtet. (Werbebroschüre über BioEcolia® der Solabia Group, Frankreich; EP1050300).The detection of a prebiotic effect of substances has so far been essentially restricted to the intestine. The use of substances which promote the growth of desirable intestinal bacteria is described in various publications (see, for example, Ahn et al. (1990) Microbial Ecology in Health and Disease 3, 223-229). Substances with a prebiotic effect have also been reported for the skin. (Advertising brochure about BioEcolia® from the Solabia Group, France; EP1050300).
Aufgabe der vorliegenden Erfindung war es nun, ein Verfahren bereitzustellen, das es erlaubt, auf einfache Weise Substanzen ausfindig zu machen, die einen präbiotischen Effekt bewirken.The object of the present invention was to provide a method which makes it possible to easily find substances which have a prebiotic effect.
Überraschenderweise wurde nun gefunden, dass auf einfache Weise Substanzen mit präbiotischer Wirkung identifiziert werden können durch ein Verfahren, das folgende Schritte umfasst: a) Untersuchung einer Mikroorganismen enthaltenden Probe, die mindestens einer auf ihre präbiotische Wirksamkeit hin zu untersuchenden Substanz ausgesetzt wurde, wobei die Probe zumindest vor Zugabe der mindestens einen zu untersuchenden Substanz mindestens einen auf seine Beeinflussung durch die zu untersuchende Substanz hin zu untersuchenden Mikroorganismus enthält, b) Untersuchung einer Vergleichsprobe, die nicht der mindestens einen auf ihre präbiotische Wirksamkeit hin zu untersuchenden Substanz ausgesetzt wurde, c) Ermittlung von Unterschieden im Erscheinungsbild und/oder in der Anzahl, insbesondere Konzentration, des mindestens einen zu untersuchenden Mikroorganismus zwischen Probe und Vergleichsprobe. Unter präbiotischem Effekt ist erfindungsgemäß zu verstehen, dass das Wachstum und/oder die Überlebensfähigkeit der erwünschten Keime der Mikroflora eines bestimmten Habitats gegenüber dem Wachstum und/oder der Überlebensfähigkeit der unerwünschten Keime der Mikroflora des betreffenden Habitats gefördert wird. Dies kann sowohl dadurch erreicht werden, dass der Wirkstoff förderlich auf das Wachstum der erwünschten Keime wirkt, ohne unmittelbar Einfluss auf das Wachstum der unerwünschten Keime zu haben, als auch dadurch, dass der Wirkstoff hemmend auf das Wachstum der unerwünschten Keime wirkt, ohne unmittelbar Einfluss auf das Wachstum der erwünschten Keime zu haben. In einer erfindungsgemäß besonders bevorzugten Ausführungsform jedoch wirkt der Wirkstoff förderlich auf das Wachstum der erwünschten Keime und wirkt zugleich hemmend auf das Wachstum der unerwünschten Keime.Surprisingly, it has now been found that substances with a prebiotic effect can be identified in a simple manner by a method which comprises the following steps: a) Examination of a sample containing microorganisms which has been exposed to at least one substance to be tested for prebiotic activity, the sample at least before the addition of the at least one substance to be examined contains at least one microorganism to be examined for its influence by the substance to be examined, b) examination of a comparative sample which was not exposed to the at least one substance to be examined for its prebiotic activity, c) determination of differences in the appearance and / or in the number, in particular concentration, of the at least one microorganism to be examined between sample and reference sample. According to the invention, a prebiotic effect means that the growth and / or survivability of the desired germs of the microflora of a particular habitat is promoted compared to the growth and / or survivability of the undesired germs of the microflora of the habitat in question. This can be achieved both by the fact that the active ingredient has a beneficial effect on the growth of the desired germs, without having a direct influence on the growth of the undesirable germs, and in that the active ingredient has an inhibitory effect on the growth of the undesired germs, without any direct influence to have the growth of the desired germs. In a particularly preferred embodiment according to the invention, however, the active ingredient has a beneficial effect on the growth of the desired germs and at the same time has an inhibitory effect on the growth of the undesired germs.
Bei dem Habitat kann es sich hierbei um jeden beliebigen Ort, insbesondere um jeden beliebigen Ort jedes beliebigen Organs oder Gewebes eines Organismus handeln, der von Mikroorganismen natürlicherweise oder aufgrund einer Erkrankung besiedelt werden kann.The habitat can be any location, in particular any location of any organ or tissue of an organism that can be colonized by microorganisms naturally or due to a disease.
In einer erfindungsgemäß besonders vorteilhaften Weise kann das erfindungsgemäße Verfahren hierbei durchgeführt werden, indem eine Mikroorganismen enthaltende Probe für die Untersuchung verwendet wird, die durch ein in-vitro-Verfahren, insbesondere durch Kultivierung, vor allem durch Kultivierung in Schüttelkolben, erhalten wurde. Auf diese Weise kann auf eine direkte Analyse von Proben, die dem betroffenen Habitat entnommen hätten werden müssen, verzichtet werden. Ebenso kann darauf verzichtet werden, die auf ihre präbiotische Aktivität hin zu untersuchende Substanz dem Habitat selbst auszusetzen, was insbesondere angesichts möglicher Nebenwirkungen problematisch sein kann. Stattdessen werden die zu untersuchenden Mikroorganismen in einem in-vitro-Verfahren herangezogen. So kann auf einfache Weise eine Vielzahl von Stoffen darauf hin untersucht werden, ob sie einen Effekt auf eine Vielzahl unabhängig voneinander untersuchbarer Mikroorganismen haben. Auf diese Weise können auf einfache Weise Stoffe, die potentiell präbiotische Wirkung besitzen, ausfindig gemacht werden.In a particularly advantageous manner according to the invention, the method according to the invention can be carried out by using a sample containing microorganisms for the examination, which sample was obtained by an in vitro method, in particular by cultivation, especially by cultivation in shake flasks. In this way, direct analysis of samples that should have been taken from the affected habitat can be dispensed with. It is also possible to dispense with exposing the substance to be tested for its prebiotic activity to the habitat itself, which can be problematic in particular in view of possible side effects. Instead, the microorganisms to be examined are used in an in vitro method. A large number of substances can thus be examined in a simple manner to determine whether they have an effect on a large number of microorganisms which can be examined independently of one another to have. In this way, substances that have potentially prebiotic effects can be easily identified.
In einer bevorzugten Ausführungsform enthält die Mikroorganismen enthaltende Probe hierbei keinen anderen Organismus als den auf seine Beeinflussung durch die zugegebene Substanz hin zu untersuchenden. Dies wird dadurch erreicht, dass gezielt dieser Organismus, vorzugsweise in Flüssigkultur (im Schüttelkolben, in Reaktionsgefäßen oder in Zellkulturplatten) oder auf Agarplatten kultiviert wird. Dies hat den Vorteil, dass unmittelbar die Vermehrung des Mikroorganismus als Indikator für die Beeinflussung durch die zu untersuchende Substanz verwendet werden kann, die unmittelbar optisch, insbesondere mikroskopisch, durch Ausplattieren auf Agarplatten oder durch eine einfache technische Methode, wie beispielsweise spektrometrische Bestimmung der optischen Dichte, bestimmt werden kann. Auf einen hohen technischen und zeitlichen Aufwand kann hierbei verzichtet werden.In a preferred embodiment, the sample containing microorganisms contains no other organism than the one to be examined for its influence by the added substance. This is achieved by specifically cultivating this organism, preferably in liquid culture (in a shake flask, in reaction vessels or in cell culture plates) or on agar plates. This has the advantage that the multiplication of the microorganism can be used directly as an indicator of the influence of the substance to be examined, which can be done directly optically, in particular microscopically, by plating on agar plates or by a simple technical method, such as, for example, spectrometric determination of the optical density , can be determined. A high technical and time expenditure can be dispensed with here.
In einer bevorzugten Ausführungsform wird hierbei durch Kultivierung zunächst überprüft, ob eine auf ihre präbiotische Wirksamkeit hin zu untersuchende Substanz einen positiven Effekt auf einen erwünschten Mikroorganismus hat. In einem zweiten Schritt wird dann untersucht, welchen Effekt die als positiv auf den erwünschten Mikroorganismus ausfindig gemachte Substanz auf unerwünschte Mikroorganismen (bzw. gegebenenfalls auf andere erwünschte Mikroorganismen) hat.In a preferred embodiment, cultivation is first used to check whether a substance to be tested for its prebiotic activity has a positive effect on a desired microorganism. In a second step it is then investigated what effect the substance found to be positive on the desired microorganism has on undesired microorganisms (or possibly on other desired microorganisms).
Umgekehrt kann auch zunächst überprüft werden, ob eine auf ihre präbiotische Wirksamkeit hin zu untersuchende Substanz einen erwünschten, also negativen, Effekt auf einen unerwünschten Mikroorganismus hat. Anschließend kann dann untersucht werden, welchen Effekt die als negativ auf den unerwünschten Mikroorganismus einwirkende Substanz auf erwünschte Mikroorganismen (bzw. gegebenenfalls auch auf andere unerwünschte Mikroorganismen) hat.Conversely, it can also first be checked whether a substance to be tested for its prebiotic activity has a desired, ie negative, effect on an undesired microorganism. Then it can be examined what effect the substance, which has a negative effect on the undesired microorganism, has on desired microorganisms (or possibly also on other undesired microorganisms).
In einer anderen bevorzugten Ausführungsform kann die Mikroorganismen enthaltende Probe auch mehrere Organismen enthalten. Es kann sich hierbei beispielsweise um ein Kollektiv erwünschter Mikroorganismen eines bestimmten Habitats oder aber um ein Kollektiv unerwünschter Mikroorganismen eines bestimmten Habitats handeln. Es kann sich hierbei aber auch um eine Probe handeln, die einem natürlichen Habitat entnommen und anschließend, vorzugsweise in Schüttelkolben, auf Agarplatten oder in Zellkultur, kultiviert wurde.In another preferred embodiment, the sample containing microorganisms can also contain several organisms. It can do this for example, a collective of desired microorganisms of a certain habitat or a group of undesirable microorganisms of a certain habitat. However, this can also be a sample that was taken from a natural habitat and then cultivated, preferably in shake flasks, on agar plates or in cell culture.
Alternativ kann auch direkt eine Probe, die einem natürlichen Habitat entnommen wurde, also eine in-vivo-Probe, analysiert werden, ohne dass diese zuvor kultiviert wurde. Als Vergleichsprobe wird in diesem Fall entsprechend eine in-vivo-Probe verwendet, die demselben Habitat entstammt und mindestens einer auf ihre präbiotische Wirksamkeit hin zu untersuchenden Substanz ausgesetzt wurde.Alternatively, a sample that has been taken from a natural habitat, that is to say an in vivo sample, can also be analyzed directly without having been cultured beforehand. In this case, an in vivo sample is used as a comparative sample, which comes from the same habitat and was exposed to at least one substance to be tested for its prebiotic activity.
Im Falle der Kultivierung der Mikroorganismen enthaltenden Probe in vitro wird die Vergleichsprobe dadurch erhalten, dass eine Kultivierung unter den selben Bedingungen erfolgt wie Kultivierung der Probe, die die mindestens eine auf ihre präbiotische Wirkung hin zu untersuchende Substanz enthält, mit dem Unterschied, dass keine Inkubation mit der mindestens einen zu untersuchenden Substanz erfolgt. Hierzu kann eine Kultur nach der Animpfung in zwei gleich große Volumina aufgeteilt werden und eine von den beiden so erhaltenen Kulturen mit der mindestens einen auf ihre präbiotische Wirkung hin zu untersuchende Substanz angeimpft werden, während die andere Probe als Vergleichsprobe dient. Alternativ können beispielsweise auch zwei gleich große Medien mit der gleichen Menge an Mikroorganismen enthaltenden Probe angeimpft und unter denselben Bedingungen kultiviert werden, wobei die eine Kultur der mindestens einen auf ihre präbiotische Wirkung hin zu untersuchenden Substanz ausgesetzt wird und die andere nicht.In the case of culturing the sample containing microorganisms in vitro, the comparative sample is obtained by culturing under the same conditions as culturing the sample which contains the at least one substance to be tested for its prebiotic effect, with the difference that no incubation with the at least one substance to be examined. For this purpose, a culture can be divided into two equal volumes after inoculation and one of the two cultures thus obtained can be inoculated with the at least one substance to be tested for its prebiotic effect, while the other sample serves as a reference sample. Alternatively, for example, two media of the same size can be inoculated with the same amount of sample containing microorganisms and cultured under the same conditions, with one culture being exposed to the at least one substance to be tested for its prebiotic effect and the other not.
In einer bevorzugten Ausführungsform des Verfahrens erfolgt Inkubation mit genau einer auf ihre präbiotische Wirkung hin zu untersuchenden Substanz. Dies hat den Vorteil, dass direkt ersichtlich wird, ob diese Substanz einen Einfluss auf das Wachstum bzw. die Überlebensfähigkeit des mindestens einen Mikroorganismus der Mikroorganismen enthaltenden Probe hat. In einer anderen bevorzugten Ausführungsform erfolgt Inkubation mit einer Mischung aus mehreren auf ihre präbiotische Wirkung hin zu untersuchenden Substanzen. Dieses Verfahren ist insbesondere dann anwendbar, wenn davon ausgegangen wird, dass die Anzahl der Treffer, also die Anzahl der als wirksam identifizierbaren Substanzen, klein sein wird. Es kann dann zunächst ein Vorscreening erfolgen, um zu ermitteln, ob die Mischung Substanzen enthält, die präbiotische Wirkung zeigen. In einem zweiten Schritt kann dann durch ein Einzel- Screening untersucht werden, auf welche Substanzen der nachgewiesene Effekt zurückzuführen ist.In a preferred embodiment of the method, incubation takes place with exactly one substance to be examined for its prebiotic effect. This has the advantage that it is immediately apparent whether this substance has an influence on the growth or survivability of the sample containing at least one microorganism of the microorganisms. In another preferred embodiment, incubation takes place with a mixture of several substances to be tested for their prebiotic effect. This method can be used in particular if it is assumed that the number of hits, ie the number of substances that can be identified as effective, will be small. A preliminary screening can then first be carried out in order to determine whether the mixture contains substances which have a prebiotic effect. In a second step, an individual screening can then be used to investigate the substances to which the proven effect can be attributed.
Grundsätzlich kann es sich bei dem mindestens einen zu untersuchenden Mikroorganismus um jeden beliebigen Mikroorganismus handeln, der in einem Habitat natürlicherweise vorkommt oder aber der für seine pathogene und/oder unerwünschte Aktivität bezüglich eines bestimmten Habitats bekannt ist.In principle, the at least one microorganism to be examined can be any microorganism that occurs naturally in a habitat or that is known for its pathogenic and / or undesirable activity with respect to a specific habitat.
Ebenso kann es sich bei der Mikroorganismen enthaltenden Probe um eine Probe handeln, die jedem beliebigem Habitat jedes beliebigen Organismus entnommen und anschließend gegebenenfalls in vitro kultiviert wurde, wobei dann das betreffende Habitat, der die Probe entnommen wurde, vorzugsweise den mindestens einen zu untersuchenden Mikroorganismus natürlicherweise enthält oder aufgrund einer pathogenen Veränderung enthalten kann. Andererseits kann die Mikroorganismen enthaltenden Probe auch als einzigen Mikroorganismus den mindestens einen zu untersuchenden Mikroorganismus oder gegebenenfalls weitere Mikroorganismen, die vorzugsweise gezielt mit dem mindestens einen zu untersuchenden Mikroorganismus vorzugsweise in vitro kultiviert wurden, enthalten.Likewise, the sample containing microorganisms can be a sample which was taken from any habitat of any organism and then optionally cultivated in vitro, the habitat in question from which the sample was taken preferably then naturally the at least one microorganism to be examined contains or may contain due to a pathogenic change. On the other hand, the sample containing microorganisms can also contain, as the only microorganism, the at least one microorganism to be examined or, if appropriate, further microorganisms which have preferably been cultivated specifically with the at least one microorganism to be examined, preferably in vitro.
Bei dem Organismus, dem die Probe entnommen werden kann, kann es sich hierbei um eine Pflanze, ein Tier oder um einen Menschen handeln.The organism from which the sample can be taken can be a plant, an animal or a human.
Bei der Mikroflora, der der Mikroorganismus entnommen wurde bzw. der Mikroflora-Probe selbst kann es sich beispielsweise um die Mikroflora des Mundes, Magens oder Darms von Wirbeltieren, insbesondere von Säugetieren, vor allem des Menschen handeln.The microflora from which the microorganism has been removed or the microflora sample itself can be, for example, the microflora of the The mouth, stomach or intestines of vertebrates, especially mammals, especially humans.
In einer bevorzugten Ausführungsform handelt es sich bei der Mikroflora-Probe um eine Probe einer Organ- bzw. Gewebe-Oberfläche, insbesondere um eine Probe der Blattoberfläche von Pflanzen, der Schuppen bzw. der Haut, insbesondere von Wirbel- und Säugetieren.In a preferred embodiment, the microflora sample is a sample of an organ or tissue surface, in particular a sample of the leaf surface of plants, the scales or the skin, in particular of vertebrates and mammals.
In einer bevorzugten Ausführungsform handelt es sich hierbei bei der Mikroorganismen enthaltenden Probe um eine Mikroflora-Probe, die der menschlichen Haut entnommen wurde. Erfindungsgemäß sind unter dem Begriff "Haut" bevorzugt die Haut selbst, insbesondere die menschliche Haut, daneben aber auch die Schleimhaut sowie Hautanhangsgebilde, sofern sie lebende Zellen umfassen, insbesondere Haarfollikel, Haarwurzel, Haarzwiebel, das ventrale Epithel des Nagelbetts (Lectulus) sowie Talgdrüsen und Schweißdrüsen zu verstehen. Der Begriff Haut umfasst hierbei insbesondere auch die Haut bzw. Schleimhaut des Mund-, Hals- und Rachenraums, der Speiseröhre, des Magens, des Darms und des Rektums.In a preferred embodiment, the sample containing microorganisms is a microflora sample that was taken from human skin. According to the invention, the term "skin" is preferably the skin itself, in particular the human skin, but also also the mucous membrane and skin appendages, provided that they comprise living cells, in particular hair follicles, hair roots, hair bulbs, the ventral epithelium of the nail bed (lectulus) and sebaceous glands and Understand sweat glands. The term “skin” here in particular also includes the skin or mucous membrane of the mouth, throat and pharynx, the esophagus, the stomach, the intestine and the rectum.
Erwünschte Hautkeime der Gesichtshaut des Menschen, die durch eine prabiotisch wirksame Substanz in ihrem Wachstum gefördert werden könnten, sind beispielsweise Koagulase-negative Staphylokokken, insbesondere S. epidermidis, S. hominis, S. warnen, S. saprophyticus, S. xylosus, S. capitis oder S. simulans. Unerwünschte Hautkeime der Gesichtshaut des Menschen, die durch eine prabiotisch wirksame Substanz in ihrem Wachstum gehemmt werden könnten, sind beispielsweise Koagulase-positive Staphylokokken, insbesondere S. aureus, oder Keime ausgewählt aus der Gruppe bestehend aus Propionibacterium acnes, Candida albicans, Malassezia furfur oder Corynebacterium spp.Desired skin germs on the facial skin of humans, which could be promoted in their growth by a prabiotic active substance, are, for example, coagulase-negative staphylococci, in particular S. epidermidis, S. hominis, S. warn, S. saprophyticus, S. xylosus, S. capitis or S. simulans. Unwanted skin germs on the facial skin of humans, which could be inhibited in their growth by a prebiotic active substance, are, for example, coagulase-positive staphylococci, in particular S. aureus, or germs selected from the group consisting of Propionibacterium acnes, Candida albicans, Malassezia furfur or Corynebacterium spp.
Unerwünschte Keime der Mundschleimhaut sind beispielsweise ausgewählt aus Streptococcus sp., insbesondere S. mutans, S. salivarius, S. gordonii, S. mitis, S. oralis, S. sangυis oder S. sobrinus, aus Actinomyces sp., insbesondere Actinomyces naesiundii, Actinobacillus sp., insbesondere Actinobacillus actinomycetemcomitans, Prevotella sp., insbesondere Prevotella intermedia, Porphyromonas sp., insbesondere Porphyromonas gingivalis, Treponema sp., insbesondere Treponema denticola, Neisseria sp., Fusobacterium sp., insbesondere Fusobacterium nucleatum, Candida, insbesondere Candida albicans oder Candida glabrata.Undesirable germs of the oral mucosa are selected for example from Streptococcus sp., In particular S. mutans, S. salivarius, S. gordonii, S. mitis, S. oralis, S. sangυis or S. sobrinus, from Actinomyces sp., In particular Actinomyces naesiundii, Actinobacillus sp., Especially Actinobacillus actinomycetemcomitans, Prevotella sp., in particular Prevotella intermedia, Porphyromonas sp., in particular Porphyromonas gingivalis, Treponema sp., in particular Treponema denticola, Neisseria sp., Fusobacterium sp., in particular Fusobacterium nucleatum, Candida, in particular Candida albicans or Candida glabrata.
Die Analyse des Effektes der mindestens einen auf ihre präbiotische Aktivität hin zu untersuchenden Substanz kann erfindungsgemäß außer durch die Methoden Spektrometrie, Mikroskopie und Ausplattieren beispielsweise auch durch Verfahren wie FISH, DGGE, TGGE (Electrophoresis (1989) 10(5-6), 377-89), RT- PCR (Real time-PCR) (Genome Res. (1996) 6(10), 986-94), DNA-Microarrays (Applied and Environmental Microbiology (2001) 67(8), 3677-82) oder Antikörper- Assays, insbesondere ELISA, erfolgen. Diese Verfahren sind vorteilhafterweise vor allem dann anwendbar, wenn die Mikroorganismen enthaltende Probe unterschiedliche Mikroorganismen enthält, so dass ein rein quantitatives Verfahren, das eine Differenzierung zwischen den Mikroorganismen nicht erlaubt, wie etwa die spektrometrische Bestimmung der optischen Dichte, nicht geeignet ist, den Effekt der zu untersuchenden Substanz differenziert nach den unterschiedlichen Mikroorganismen wiederzugeben.According to the invention, the analysis of the effect of the at least one substance to be tested for its prebiotic activity can be carried out in addition to the spectrometry, microscopy and plating methods, for example also by methods such as FISH, DGGE, TGGE (Electrophoresis (1989) 10 (5-6), 377- 89), RT-PCR (Real time-PCR) (Genome Res. (1996) 6 (10), 986-94), DNA microarrays (Applied and Environmental Microbiology (2001) 67 (8), 3677-82) or Antibody assays, in particular ELISA, are carried out. These methods are advantageously applicable especially when the sample containing microorganisms contains different microorganisms, so that a purely quantitative method which does not permit differentiation between the microorganisms, such as the spectrometric determination of the optical density, is not suitable for the effect of reproduce substance to be examined differentiated according to the different microorganisms.
FISH steht hierbei für "Fluoreszenz-in-situ-Hybridisierung". Dieses Verfahren wurde zu Beginn der 90er Jahre entwickelt. Als Sonden werden fluoreszenzmarkierte Oligonukleotide eingesetzt. Das Verfahren ist bereits zur Untersuchung vieler Umweltproben erfolgreich zum Einsatz gekommen (Amann et al. (1990) J. Bacteriol. 172, 762). Das Verfahren macht sich den Umstand zunutze, dass die in jeder Zelle vorkommenden ribosomalen Ribonukleinsäuren (RNAs) sowohl hochkonservierte als auch variable, d.h. gattungs- oder sogar artspezifische Sequenzen aufweisen. Gegen diese Sequenzbereiche können komplementäre Oligonukleotide hergestellt werden, die zusätzlich mit einem detektierbaren Marker versehen werden können. Mittels dieser sogenannten Nukleinsäuresonden können Mikroorganismenarten, -gattungen oder -gruppen hochspezifisch direkt in der Probe identifiziert und bei Bedarf auch visualisiert oder quantifiziert werden. Auf diese Weise ist eine verzerrungsfreie Darstellung der tatsächlichen in situ-Verhältnisse der Biozönose möglich. Sogar bisher nichtkultivierte und demnach nicht beschriebene Mikroorganismen können identifiziert werden. Bei der FISH-Methode dringen Sonden in die in der untersuchten Probe vorhandenen Zellen ein. Sofern ein Mikroorganismus der Art, Gattung oder Gruppe, für welche die Sonden entwickelt wurden, in der untersuchten Probe vorhanden ist, binden die Sonden in der Mikroorganismenzelle an ihre Zielsequenz, und die Zellen können aufgrund der Markierung der Sonden detektiert werden.FISH stands for "fluorescence in situ hybridization". This process was developed in the early 1990s. Fluorescence-labeled oligonucleotides are used as probes. The method has already been successfully used to examine many environmental samples (Amann et al. (1990) J. Bacteriol. 172, 762). The method takes advantage of the fact that the ribosomal ribonucleic acids (RNAs) occurring in each cell have both highly conserved and variable, ie genus or even species-specific sequences. Complementary oligonucleotides can be produced against these sequence regions, which can additionally be provided with a detectable marker. Using these so-called nucleic acid probes, microorganism species, species or groups can be identified directly in the sample in a highly specific manner and, if necessary, also visualized or quantified. In this way, a distortion-free representation of the actual in situ conditions of the biocenosis possible. Even microorganisms that have not yet been cultivated and therefore not described can be identified. With the FISH method, probes penetrate the cells present in the sample under investigation. If a microorganism of the type, genus or group for which the probes were developed is present in the sample under investigation, the probes in the microorganism cell bind to their target sequence and the cells can be detected on the basis of the labeling of the probes.
DGGE steht für "denaturierende Gradienten-Gelelektrophorese". Es handelt sich hierbei um ein Trennverfahren, dass sich Unterschied im Schmelzverhalten unterschiedlicher doppelsträngiger DNA-Fragmente zunutze macht. Ursprünglich wurde diese Methode entwickelt um Punktmutationen gelelektrophoretisch nachzuweisen, so dass es sich hinsichtlich der Auflösung um eine sehr empfindliche Methode handelt (Fisher und Lerman (1979) Cell 16, 191-200; Myers et al. (1987) Methods in Enzymology 212, 71-104). DGGE wird in jüngerer Zeit sehr häufig zur Untersuchung natürlicher Habitate eingesetzt (Muyzer et al. (1996) Molecular Microbial Ecology Manual 3.4.4:1-23: Eds: Akkermans et al., Holland 1996; Diez et al. (2001 ) Appl. Environ. Microbiol. 67(7), 2942-2951 ).DGGE stands for "denaturing gradient gel electrophoresis". It is a separation process that takes advantage of the difference in melting behavior of different double-stranded DNA fragments. This method was originally developed to detect point mutations by gel electrophoresis, so that resolution is a very sensitive method (Fisher and Lerman (1979) Cell 16, 191-200; Myers et al. (1987) Methods in Enzymology 212, 71 -104). Recently, DGGE has been used very frequently to study natural habitats (Muyzer et al. (1996) Molecular Microbial Ecology Manual 3.4.4: 1-23: Eds: Akkermans et al., Holland 1996; Diez et al. (2001) Appl Environ. Microbiol. 67 (7), 2942-2951).
TGGE steht für „Temperatur-Gradienten-Gelelektrophorese". Im Gegensatz zur DGGE wird statt mit einem denaturierenden Agens mit einem Temperaturgradienten gearbeitet. Die Methoden sind ansonsten äquivalent.TGGE stands for "temperature gradient gel electrophoresis". In contrast to the DGGE, a temperature gradient is used instead of a denaturing agent. The methods are otherwise equivalent.
Gegenstand der vorliegenden Erfindung sind ferner prabiotisch wirksame Substanzen, die mit Hilfe eines erfindungsgemäßen Verfahrens erhältlich sind, die Verwendung dieser prabiotisch wirksamen Substanzen sowie Arzneimittel, Kosmetika, Lebensmittel und Pflanzenschutzmittel enthaltend diese prabiotisch wirksamen Substanzen.The present invention furthermore relates to prebiotically active substances which can be obtained with the aid of a process according to the invention, the use of these prebiotically active substances and medicaments, cosmetics, foods and crop protection agents containing these prabiotically active substances.
Gegenstand der vorliegenden Erfindung ist ferner die Verwendung einer erfindungsgemäßen prabiotisch wirksamen Substanz zur Herstellung eines Arzneimittels zur Förderung des Wachstums unerwünschter Keime und/oder zur Hemmung des Wachstums unerwünschter Keime.The present invention furthermore relates to the use of a prebiotically active substance according to the invention for the production of a Medicament to promote the growth of undesirable germs and / or to inhibit the growth of undesirable germs.
In einer erfindungsgemäß bevorzugten Ausführungsform handelt es sich bei den erfindungsgemäßen Arzneimitteln und Kosmetika um solche zur topischen Applikation. Diese können entsprechend weitere Komponenten enthalten wie sie für solche Mittel üblich sind, insbesondere ausgewählt aus der Gruppe bestehend aus Vitaminen, Provitaminen oder Vitaminvorstufen der Vitamin B-Gruppe oder deren Derivate sowie Derivate von 2-Furanon, Panthenol, Pantolacton, Nicotinsäureamid und Biotin, Pflanzenextrakte, MMP-1 -inhibierende Substanzen, Ester von Retinol (Vitamin A-ι) mit einer C2-i8-Carbonsäure, oberflächenaktive Substanzen als Emulgator oder Dispergiermittel, Aminosäuren und deren Zinksalze und deren Säureadditionssalze, filmbildende und/oder emulsionsstabilisierende und/oder verdickende und/oder adhäsive Polymere, Fettstoffe, Tenside, Anti-Transpirantien und Polyole, organische, mineralische und modifizierte mineralische Lichtschutzfilter, Proteinhydrolysate und deren Derivate, Mono-, Oligo- und Polysaccharide sowie deren Derivate, α-Hydroxycarbonsäuren und α-Ketocarbonsäuren sowie deren Ester-, Lacton- oder Salzformen. In a preferred embodiment according to the invention, the medicaments and cosmetics according to the invention are those for topical application. These may accordingly contain further components as are customary for such agents, in particular selected from the group consisting of vitamins, provitamins or vitamin precursors of the vitamin B group or their derivatives and derivatives of 2-furanone, panthenol, pantolactone, nicotinamide and biotin, plant extracts , MMP-1 inhibiting substances, esters of retinol (vitamin A-ι) with a C 2 -i 8 carboxylic acid, surface-active substances as emulsifiers or dispersants, amino acids and their zinc salts and their acid addition salts, film-forming and / or emulsion-stabilizing and / or thickening and / or adhesive polymers, fatty substances, surfactants, anti-perspirants and polyols, organic, mineral and modified mineral light protection filters, protein hydrolyzates and their derivatives, mono-, oligo- and polysaccharides as well as their derivatives, α-hydroxycarboxylic acids and α-ketocarboxylic acids and their Ester, lactone or salt forms.

Claims

Patentansprüche: claims:
1. Verfahren zur Identifizierung einer prabiotisch wirksamen Substanz, folgende Schritte umfassend: a) Untersuchung einer Mikroorganismen enthaltenden Probe, die mindestens einer auf ihre präbiotische Wirksamkeit hin zu untersuchenden Substanz ausgesetzt wurde, wobei die Probe zumindest vor Zugabe der mindestens einen zu untersuchenden Substanz mindestens einen auf seine Beeinflussung durch die zu untersuchende Substanz hin zu untersuchenden Mikroorganismus enthält, b) Untersuchung einer Vergleichsprobe, die nicht der mindestens einen auf ihre präbiotische Wirksamkeit hin zu untersuchenden Substanz ausgesetzt wurde, c) Ermittlung von Unterschieden im Erscheinungsbild und/oder in der Anzahl des mindestens einen zu untersuchenden Mikroorganismus zwischen Probe und Vergleichsprobe.1. A method for identifying a prebiotically active substance, comprising the following steps: a) Examination of a sample containing microorganisms which has been exposed to at least one substance to be tested for prebiotic activity, the sample being at least one before adding the at least one substance to be examined b) Examination of a comparative sample which was not exposed to the at least one substance to be examined for its prebiotic effectiveness, c) Determination of differences in the appearance and / or the number of the microorganism to be investigated at least one microorganism to be examined between sample and reference sample.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass die Mikroorganismen enthaltenden Proben durch ein in-vitro-Verfahren erhalten wurden.2. The method according to claim 1, characterized in that the samples containing microorganisms were obtained by an in vitro method.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass die Mikroorganismen enthaltenden Proben durch Kultivierung erhalten wurden.3. The method according to claim 1 or 2, characterized in that the samples containing microorganisms were obtained by cultivation.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, dass die Kultivierung in Schüttelkolben erfolgte.4. The method according to claim 3, characterized in that the cultivation was carried out in shake flasks.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass die durch Kultivierung erhaltene Mikroorganismen enthaltende Probe keinen anderen Mikroorganismus als den mindestens einen zu untersuchenden Mikroorganismus enthält. 5. The method according to any one of claims 1 to 4, characterized in that the sample containing microorganisms obtained by cultivation contains no other microorganism than the at least one microorganism to be examined.
6. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass die durch Kultivierung erhaltene Mikroorganismen enthaltende Probe mehrere Mikroorganismen enthält.6. The method according to any one of claims 1 to 4, characterized in that the sample containing microorganisms obtained by cultivation contains several microorganisms.
7. Verfahren nach Anspruch 6, dadurch gekennzeichnet, dass es sich bei der Mikroorganismen enthaltenden Probe um eine Probe handelt, die durch Kultivierung einer Mikroflora-Probe der Haut erhalten wurde.7. The method according to claim 6, characterized in that the sample containing microorganisms is a sample obtained by cultivating a microflora sample of the skin.
8. Verfahren nach Anspruch 7, dadurch gekennzeichnet, dass es sich bei der Haut um menschliche Haut handelt.8. The method according to claim 7, characterized in that the skin is human skin.
9. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass die Mikroorganismen enthaltenden Proben einem Organ oder Gewebe oder der Oberfläche eines Organs oder Gewebes entnommen wurden.9. The method according to claim 1, characterized in that the microorganism-containing samples were taken from an organ or tissue or the surface of an organ or tissue.
10.Verfahren nach Anspruch 9, dadurch gekennzeichnet, dass es sich bei der Oberfläche eines Organs oder Gewebes um die Hautoberfläche handelt.10. The method according to claim 9, characterized in that the surface of an organ or tissue is the skin surface.
11.Verfahren nach Anspruch 10, dadurch gekennzeichnet, dass es sich bei den Mikroorganismen um solche der Mikroflora der menschlichen Haut handelt.11. The method according to claim 10, characterized in that the microorganisms are those of the microflora of human skin.
12. Verfahren nach einem der Ansprüche 1 bis 11 , dadurch gekennzeichnet, dass es sich bei dem mindestens einen zu untersuchenden Mikroorganismus um einen erwünschten Mikroorganismus handelt.12. The method according to any one of claims 1 to 11, characterized in that the at least one microorganism to be examined is a desired microorganism.
13. Verfahren nach einem der Ansprüche 1 bis 11 , dadurch gekennzeichnet, dass es sich bei dem mindestens einen zu untersuchenden Mikroorganismus um einen unerwünschten Mikroorganismus handelt.13. The method according to any one of claims 1 to 11, characterized in that the at least one microorganism to be examined is an undesirable microorganism.
14. Verfahren nach einem der Ansprüche 1 bis 13, dadurch gekennzeichnet, dass zunächst eine Mischung von Substanzen auf ihre präbiotische Wirksamkeit hin untersucht werden und in einem zweiten Schritt die präbiotische wirksame Substanz durch Einzel-Screening ermittelt wird.14. The method according to any one of claims 1 to 13, characterized in that first a mixture of substances on their prebiotic Effectiveness are examined and in a second step the prebiotic active substance is determined by individual screening.
15. Verfahren nach einem der Ansprüche 1 bis 14, dadurch gekennzeichnet, dass die Untersuchung auf Unterschiede in der Konzentration oder des Erscheinungsbildes des mindestens einen zu untersuchenden Mikroorganismus zwischen Probe und Vergleichsprobe durch Ausplattieren, Mikroskopie, Spektroskopie oder Spektrometrie erfolgt.15. The method according to any one of claims 1 to 14, characterized in that the examination for differences in the concentration or appearance of the at least one microorganism to be examined between sample and reference sample is carried out by plating, microscopy, spectroscopy or spectrometry.
16. Verfahren nach einem der Ansprüche 1 bis 14, dadurch gekennzeichnet, dass die Untersuchung auf Unterschiede in der Anzahl des mindestens einen zu untersuchenden Mikroorganismus zwischen Probe und Vergleichsprobe durch FISH erfolgt.16. The method according to any one of claims 1 to 14, characterized in that the examination for differences in the number of at least one microorganism to be examined between sample and comparative sample is carried out by FISH.
17. Verfahren nach einem der Ansprüche 1 bis 14, dadurch gekennzeichnet, dass die Untersuchung auf Unterschiede in der Anzahl des mindestens einen zu untersuchenden Mikroorganismus zwischen Probe und Vergleichsprobe durch DGGE, TGGE, RT-PCR, DNA-Microarrays oder Antikörper-Assays erfolgt.17. The method according to any one of claims 1 to 14, characterized in that the examination for differences in the number of at least one microorganism to be examined between sample and comparative sample is carried out by DGGE, TGGE, RT-PCR, DNA microarrays or antibody assays.
18. Präbiotisch wirksame Substanz, erhältlich durch ein Verfahren nach einem der Ansprüche 1 bis 17.18. Prebiotically active substance obtainable by a process according to one of claims 1 to 17.
19. Verwendung einer prabiotisch wirksamen Substanz nach Anspruch 18 als Pflanzenschutzmittel.19. Use of a prebiotically active substance according to claim 18 as a crop protection agent.
20. Verwendung einer prabiotisch wirksamen Substanz nach Anspruch 18 als Lebensmittelzusatz.20. Use of a prebiotically active substance according to claim 18 as a food additive.
21. Verwendung einer prabiotisch wirksamen Substanz nach Anspruch 18 in einer topischen kosmetischen Zusammensetzung. 21. Use of a prebiotically active substance according to claim 18 in a topical cosmetic composition.
22. Arzneimittel, enthaltend eine prabiotisch wirksame Substanz nach Anspruch 18 in einem geeigneten Träger.22. Medicament containing a prebiotically active substance according to claim 18 in a suitable carrier.
23. Verwendung einer prabiotisch wirksamen Substanz nach Anspruch 18 zur Herstellung eines Arzneimittels zur Hemmung des Wachstums unerwünschter Keime.23. Use of a prebiotically active substance according to claim 18 for the manufacture of a medicament for inhibiting the growth of undesirable germs.
24. Verwendung einer prabiotisch wirksamen Substanz nach Anspruch 18 zur Herstellung eines Arzneitmittels zur Förderung des Wachstums erwünschter Keime.24. Use of a prebiotically active substance according to claim 18 for the manufacture of a medicament for promoting the growth of desired germs.
25. Verwendung nach Anspruch 23 oder 24, dadurch gekennzeichnet, dass es sich bei den Keimen um Keime der Hautflora handelt. 25. Use according to claim 23 or 24, characterized in that the germs are germs of the skin flora.
Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)
This international searchreport has not been established inrespect of certain claims under Article 17 (2) (a) for the following reasons 1 I I Claims Nos ' — ' because they relate to subject matter not required to be searched by this Authonty, namelyThis international searchreport has not been established inrespect of certain claims under Article 17 (2) (a) for the following reasons 1 I I Claims Nos '-' because they relate to subject matter not required to be searched by this Authonty, namely
Xj Claims Nos 18-25 because they relate to parts of the international application that do not comply with the prescπbed req rements to such an extent that no meaningful mternational search can be carπed out, specifically See BEIBLATT PCT/ISA/210Xj Claims Nos 18-25 because they relate to parts of the international application that do not comply with the prescπbed req rements to such an extent that no meaningful mternational search can be carπed out, specifically See BEIBLATT PCT / ISA / 210
D Claims Nos because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6 4(a) Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authonty found multiple inventions in this international application, as fohowsD Claims Nos because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6 4 (a) Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authonty found multiple inventions in this international application, as fohows
Ansprüche Nr weil sie sich auf Teile der internationalen Anmeldung beziehen, die den vorgeschriebenen Anforderungen so wenig entsprechen, daß eine sinnvolle internationale Recherche nicht durchgeführt werden kann nämlichClaims No because they relate to parts of the international application which do not meet the prescribed requirements so much that a meaningful international search cannot be carried out
I I As all required additional search fees were timely paid by the applicant, this mternational search report Covers all searchable claims 2 I I As all searchable claims couldbeseaxchedwιmoutefJortjusüfymganaddιüonalfee,thιsAumon1ydιdnot vιtepayment of any additional fee 3 I I As only so e of the required additional search fees were timely paid by the applicant, this mternational search report — Covers only t ose claims for which fees were paid, specifically claims NosII As all required additional search fees were timely paid by the applicant, this mternational search report Covers all searchable claims 2 II As all searchable claims couldbeseaxchedwιmoutefJortjusüfymganaddιüonalfee, thιsAumon1ydιdnot vιtepayment of any additional fee 3 II As only so e of the required additional search fees were timely paid by the applicant, this international search report - covers only to claims for which fees were paid, specifically claims Nos
D No required additional search fees were timely paid by the applicant Consequently, this mternational search report is r rpeαsttnnrc'+tperdt i tno t thripe i innvvpennttiinonn f fiirrsstt m meennttiioonneedd i inn t thhee r c.llaaiimmss, i itt i iss p c.πovvperrpedd by r c.llmaimmαs NTnoσsD No required additional search fees were timely paid by the applicant Consequently, this mternational search report is r rpeαsttnnrc '+ tperdt i tno t thripe i innvvpennttiinonn f fiirrsstt m meennttiioonneedd i inn t thhee r c.llaaiimmss, i itt i iss p c. πovvperrpedd by r c.llmaimmαs NTnoσs
Remark on Protest | | The additional search fees were accomparned by the applicant' s protest No protest accomparned the payment of additional search feesRemark on Protest | | The additional search fees were accompanied by the applicant's protest No protest accomparned the payment of additional search fees
Form PCT/ISA/210 (continuation of first sheet (1)) (July 1992)
Figure imgf000018_0001
Form PCT / ISA / 210 (continuation of first sheet (1)) (July 1992)
Figure imgf000018_0001
PCT/EP2004/010679 2003-09-27 2004-09-23 Method for the identification of substances with prebiotic effect WO2005033331A1 (en)

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US20130115317A1 (en) * 2011-11-08 2013-05-09 The Procter & Gamble Company Method for Identifying Pre-Biotics and Compositions Containing the Same
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