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WO2006070041A2 - Compounds with antifungal activity, which are produced by bifidobacterium spp - Google Patents

Compounds with antifungal activity, which are produced by bifidobacterium spp Download PDF

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Publication number
WO2006070041A2
WO2006070041A2 PCT/ES2005/070174 ES2005070174W WO2006070041A2 WO 2006070041 A2 WO2006070041 A2 WO 2006070041A2 ES 2005070174 W ES2005070174 W ES 2005070174W WO 2006070041 A2 WO2006070041 A2 WO 2006070041A2
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Prior art keywords
compounds
yeasts
against fungi
antifungal activity
bir
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Spanish (es)
French (fr)
Inventor
Yolanda Sanz Herranz
María del Carmen COLLADO AMORES
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Consejo Superior de Investigaciones Cientificas CSIC
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Consejo Superior de Investigaciones Cientificas CSIC
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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
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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

  • This patent belongs to the technical sector of the Agrifood and Pharmaceutical Industry. More specifically, this invention is framed within the technologies of control and conservation of crops and agri-food products destined for human or animal feed by biological methods, through the use of microorganisms with antimicrobial activity and its metabolites, and the field of food functional (probiotics).
  • mycotoxins are also important producers of toxic secondary metabolites, such as mycotoxins. These metabolites pose an important health risk due to their nephrotoxic, hapatotoxic, neurotoxin, teratogenic, estrogenic and carcinogenic properties.
  • the main fungi that produce mycotoxins belong to the genera Aspergillus, Penicillium, Fusarium and Alternar ⁇ a and the main toxins they produce are aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, fusaricines, moniliformin, patulin, cyclopiazonic acid and altemariol, among others.
  • Candida species are one of the main responsible for infections (candidiasis) of various types (intestinal, vaginal, urogenital, oral, skin, etc).
  • Candidiasis is frequently produced by C. albicans although it may be due to other species such as C. tropicalis and C. parapsilosis.
  • Bacteriocins Evolution, Ecology, and Application. Ann. Rev. Microbiol. 56: 117-137).
  • the synthesis of metabolites with antifungal activity has only been attributed to a limited number of strains of the genera Lactobacillus and Propionibacterium (Corsetti e ⁇ al., 1998.
  • Antimould activity of sourdough lactic acid bacteria identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Appl. Microbiol. Biotechnol. 50: 253-256; Lavermicocca e ⁇ al., 2000. Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21 B. Appl. Environ.
  • Therapeutic agent for dermatophathy having antifungal action JP9263539; Dohnalek e ⁇ al. 1999. Composition of treatment of candidiasis WO 99/17788; Khaziev e ⁇ al., 2002. Medical preparation for prophylaxis and treatment of urogenital infections, RU2185842).
  • the antifungal compounds produced by lactic bacteria and identified to date are divided into two large groups: (i) low molecular weight compounds (less than 1000 Da), including acetic, benzoic, phenyl-lactic acids, 4- lactic and caproic hydroxy-phenyl, methylidantoin, mevanololactone and cyclo-glycyl-leucine; and (ii) antimicrobial proteins (greater than 1000 Da) similar to bacteriocins.
  • These protein compounds have been identified only in Lactobacillus spp. and they are called "bacteriocin-like compounds", since they do not fit the criteria that define bacteriocins because they have a broader spectrum of action (Okkers ei al. 1999.
  • pentocin TV35b a bacteriocin-like peptide isolated from Lactobacillus pentosus with fungistatic effect on Candida albicans. J. Appl. Microbiol 87: 726-734; Magnusson et al., 2001. Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Appl. Environ. Microbiol 67: 1-5; Atanassova e ⁇ al., 2003. Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3. Int.
  • the microorganisms of the genus Bifidobacterium are natural inhabitants of the intestinal tract of man and animals. They are usually included within the group of lactic bacteria, physiologically defined by their ability to produce lactic acid as one of the main metabolites resulting from carbohydrate fermentation, although phylogenetically they are included within the Actinobacteria class (O ' Riordan, ei. 1997. Determination of genetic diversity within the genus Bifidobacterium and estimation of chromosomal size. FEMS Microbiol. Lett. 156: 259-264).
  • strains of this genus constitute one of the most important groups of probiotics for their recognized beneficial effects on health and are included in the elaboration of an increasingly large number of foods (fermented milks, cheeses, infant formulations, etc.). ) and pharmaceutical preparations.
  • This genus is considered, for example, essential in maintaining the balance of the intestinal ecosystem and the displacement of potentially harmful microorganisms, for which reason an important protective function is attributed.
  • Strains of this genus have been attributed beneficial effects in the treatment of traveler's diarrhea, rotavirus infections, diarrhea associated with the use of antibiotics and intestinal and extraintestinal infections caused by Candida spp. (Dohnalek e ⁇ al., 1999. Composition of treatment of candidiasis.
  • the present invention provides cellular and metabolic compounds with antifungal activity of strains of the genus Bifidobacterium.
  • the compounds resulting from the metabolism of strains of the genus Bifidobacterium, provided in the form of extracts, contain protein and non-protein compounds with antifungal activity and, therefore, expand the applications of this microbial group in the field of conservation of products intended for human consumption and animal and in the clinical and phytosanitary area.
  • These bacteria are producers of protein metabolites with a broad spectrum of antifungal activity (active against yeasts and fungi).
  • Another advantage of the present invention is the resistance of the producing strains to the conditions of gastrointestinal and technological stress that ensure its viability and stability.
  • the strains are capable of producing the antifungal compounds in intestinal conditions and in food, which guarantees their functionality and applications related to the agri-food and clinical sector.
  • antifungal activity has been detected in the genus Bifidobacterium.
  • the strains belonging to this genus are producers of compounds of a protein nature with characteristics similar to bacteriocins but with a spectrum of action that does not match the criteria that define them, since they are active against fungi and yeasts.
  • the strains produce other structural compounds and non-protein metabolic compounds, with antifungal activity at acidic pH, which act synergistically with the former increasing their effectiveness.
  • the present invention provides antifungal compounds derived from the metabolism of bifidobacteria, in the form of extracts. These antifungal compounds derived from the metabolism of bifidobacteria are the subject of the present invention and are claimed in the present invention.
  • the metabolites are secreted into the culture medium during the growth of the bifidobacteria and the extracts containing them are obtained from the cell-free supernatants of these cultures by lyophilization.
  • the spectrum of activity includes fungi and yeasts responsible for alterations in food and of zoo- and phytosanitary interest because they are potentially pathogenic or capable of synthesizing toxic metabolites belonging to the genera: Alternar ⁇ a, Aspergillus, Cladospor ⁇ um, Cryptococcus, Eurotium, Fusar ⁇ um, Penicillium, Tr ⁇ choderma, Brettanomyces, Candida, Cryptococcus, Debaryomyces, Dekkera, Hansenula, Hansenniaspora, Kloeckera, Kluyveromyces, Pichia, Rhodotorula, Torulaspora, Zygossacharomyces, etc.
  • the compounds responsible for the antifungal activity secreted to the culture media by the bifidobacteria and active in the neutralized extracts tested are substances of a protein nature and have biochemical characteristics similar to those of lactic bacteria bacteriocins, but do not meet the criteria of their definition since they inhibit fungi and yeasts.
  • the compounds responsible for the antifungal activity secreted to the culture media by the bifidobacteria and present in the non-neutralized and active extracts at acidic pH also include compounds of different nature, which enhance the effect of the antifungal proteins.
  • strains of the genus Bifidobacterium synthesized compounds with broad spectrum antifungal activity under the conditions to which they are exposed in the intestinal tract, they can act as probiotics with protective function at this level and be used for medical purposes.
  • the use of the strains of the present invention as probiotics is another object of the present invention.
  • Bifidobacteria grow and remain viable in various foods, thus constituting suitable vehicles for their contribution.
  • foods such as fermented and unfermented milks
  • the ability of bifidobacteria to secrete antifungal compounds to the culture medium during their growth was verified.
  • the strains of the genus Bifidobacterium, as well as their added metabolites in the form of extracts act as inhibitors of fungal and yeast development in food. That is, they act as bioprotective cultures in foods such as milk.
  • the use of these strains as bioprotective agents in agri-food products constitutes another object of the present invention.
  • the present invention provides cellular components and antifungal metabolites of strains of the genus Bifidobacterium.
  • the metabolites are secreted to the culture medium during the growth of the bifidobacteria and the extracts that contain them are obtained from the supernatants of these cultures by lyophilization.
  • Bacteria useful in this invention include representatives of the genus Bifidobacterium of human and animal origin, for example, strains of the species Bifidobacterium longum, B. infantis, B. catenulatum, B. pseudocatenulatum B. bifidum, B. breve, B. adolescentis, B. animalis-B. lactis and
  • the antimicrobial activity is detected in the presence of cells and in the extracts, neutralized (in which the effect of organic acids is eliminated) and without neutralizing (in which the effect of organic acids is added to that of protein compounds).
  • the spectrum of activity includes fungi and yeasts of zoo- and phytosanitary interest and responsible for alterations in foods belonging to the genera Alternaria, Aspergillus, Cladosporium, Cryptococcus, Eurotium, Fusarium, Penicillium, Trichoderma, Brettanomyces, Candida, Cryptococcus, Debaryomyces , Hansenniaspora, Hansenula, Kloeckera, Kluyveromyces, Pichia, Rhodotorula, Torulaspora, etc.
  • the global antifungal activity (of cellular components and metabolic products) of the strains of the genus Bifidobacterium was detected by the double layer technique against representatives of a large number of yeast and fungal genera mentioned above (Example 1; Figures 1 and 2).
  • the spectrum of inhibition was wide in all cases, but differences were observed between strains, with respect to their intensity (halos of inhibition between 0 and 30 mm) and spectrum of action.
  • the detection of antifungal activity due exclusively to the secretion of compounds of protein nature was carried out by means of inhibition tests of the neutralized extracts obtained from the cultures of the bifidobacteria against various indicator microorganisms by means of the agar diffusion technique.
  • fungi including important mycotoxin producers (Alternar ⁇ a alternata, Aspergillus parasiticus, Fusar ⁇ um spp. And Penicillium expansum), and yeast were inhibited by bifidobacteria, except Saccharomyces cerevisiae. This exception confers a great advantage to the bifidobacteria in its application as bioprotective cultures in products in which the S. cerevisiae species is used as the starter culture, since its addition would not compromise the success of the fermentation process.
  • the metabolic compounds produced by the bifidobacteria and responsible for the antifungal activity are substances of a protein and non-protein nature, whose production or release to the medium increases when MRS broth is used as a culture medium, containing a 4 mM concentration of cysteine and 1% of glycerol.
  • inhibitor compounds of a non-protein nature active at acidic pH, is confirmed because part of the antifungal activity is retained in non-neutralized extracts treated with proteases.
  • Protein responsible of the inhibition compounds are thermostable maintaining its activity after incubation at 100 0 C for at least 1 min on. They are also stable and active in a wide pH range (3-10). Therefore, its activity is compatible with the conditions of the gastrointestinal tract and with those of a large number of technological processes (fermentation and maturation of dairy products, meat, etc.).
  • the apparent molecular mass of the antifungal compounds is less than
  • the protein compounds responsible for the antimicrobial activity have biochemical characteristics similar to those of the bacteriocins of lactic bacteria, but do not meet the criteria of their definition since they have broad spectrum antifungal activity, including yeasts and fungi.
  • the antifungal activity of the extracts against fungi and yeasts is destroyed after treatment with various proteases (trypsin, pepsin and proteinase K).
  • strains of the genus Bifidobacterium grow and remain viable in various foods, thus constituting suitable vehicles for their contribution.
  • Fermented and unfermented milks are examples of foods that support the maintenance of their viability;
  • they are among the most suitable products for the administration of probiotic strains, because milk and its derivatives are habitual members of the diet in developed countries, have high nutritional value, are easy to digest and enjoy great acceptance in various population groups
  • Strains of the genus Bifidobacterium can ferment and coagulate foods such as milk; Therefore, they can be used for the production of fermented milks and other dairy products.
  • Bifidobacteria acted, for example, as bioprotective cultures against yeasts in milk, reducing the viability for example of Candida parapsilosis and Rhodotorula glutins, at least, between 1 and 3 logarithmic units and in L-EL-G, at least, between 2 and 3 logarithmic units (Figure 3).
  • the selected bifidobacteria acted as bioprotective cultures against fungi in milk, reducing the viability, for example, of Penicillium spp., At least, one logarithmic unit; and in L-EL-G, at least 2 logarithmic units ( Figure 3).
  • the protective effects resulting directly from the metabolites secreted by the bifidobacteria were confirmed by exclusive addition to the contaminated milk of their extracts.
  • the addition of non-neutralized extracts which would add the effects of organic acids and those of protein compounds, and neutralized produced a reduction in the levels of Penicillium spp. from at least two logarithmic units ( Figure 3).
  • the compounds of protein nature in the form of extracts as well as the cultures of the strains of the genus Bifidobacterium that produce them can act, in foods such as milk, as bioprotectors inhibiting the development of fungi and yeasts (Example 3).
  • the use of the aforementioned compounds as antifungal agents against fungi and yeasts that constitute a zoo- and phytosanitary risk and that are responsible for alterations in food, is claimed in the present invention.
  • Other uses of the present invention are the use of the extract containing the antifungal compounds against fungi and yeast alters of food and of zoo- and phytosanitary interest and in a particular case as a biological preservative and fungicide, and the use of the extract as a therapeutic compound against fungi and yeasts.
  • the use of these strains as bioprotective agents and biological fungicides of agri-food products and as probiotics for medical purposes (prophylactic or therapeutic) constitutes another object of the present invention.
  • FIGURES DESCRIPTIONS OF THE FIGURES
  • Figure 1 Representation of the spectrum of antifungal activity of strains of the genus Bifidobacterium by means of the double layer technique. The inhibitory effect against fungi and yeasts was determined by measures of inhibition halos and is expressed in millimeters (mm) *. The sign (-) indicates absence of inhibition.
  • Figure 2. An example of the degree of inhibition exerted by some strains of the genus Bifidobacterium on the growth of fungi and yeasts by means of the double layer technique is shown.
  • A Example of the inhibition of Bifidobacterium BIR -0307 and BIR-0326 grown in MRSC on Aspergillus parasiticus.
  • B Example of the inhibition of Bifidobacterium BIR-0326 grown in MRSC (B 1) and in Milk agar (B2) on Candida parapsilosis.
  • Figure 3 Representation of the inhibitory effect of some bifidobacteria on Candida parapsilosis, Rhodotorula glutinis and Penicillium sp. in co-cultures in milk (L) and in milk containing 0.5% yeast extract and 1% of glucose (L-EL-G) and by direct addition of the extracts, obtained from the supernatants of the bifidobacteria cultures, to cultures of the contaminating microorganisms.
  • EXAMPLE 1 PROCEDURE FOR DETECTION OF ANTIFUNGICAL ACTIVITY IN BIFIDOBACTERIES AND OBTAINING EXTRACTS WITH SUCH ACTIVITY.
  • the antifungal activity of the strains of the genus Bifidobacterium was determined by two methods: (i) by the double layer technique and (ii) by the agar diffusion technique.
  • the antifungal activity of each strain due to cellular and metabolic components, was evaluated globally by means of the double layer technique using as indicator microorganisms representatives of various genera of yeasts and fungi of health interest and food alterants, such as those shown in Figure 1.
  • the potentially active bifidobacteria were seeded on MRS plates, containing 0.05% cysteine (MRSC) in lines of about 2 cm and incubated in optimal conditions for 16 h and their subsequent development was inhibited with chloroform.
  • MRSC cysteine
  • Yeasts and indicator fungi were inoculated at a concentration of 10 4 -10 5 cells or spores / ml in 10 ml of suitable semi-solid agar, poured onto the agar layer of the protective microorganism and incubated under optimal conditions for the indicators. After 24-72 h of incubation the possible inhibition halos around the protective culture lines were measured.
  • the agar diffusion technique was used. 10 ml of MRSC broth were inoculated at 1% with a 24 h culture of each bifidobacteria and incubated for 16 h at 37 0 C. The supernatants were obtained by centrifugation (12,000 g, 15 min, 4 0 C) and concentrated by lyophilization The lyophiles were resuspended in 1 ml of 50 mM phosphate buffer at pH 6.5, neutralized with NaOH until reaching a pH of 6.5 to remove the effects of organic acids generated by fermentation, and were sterilized by filtration.
  • the indicator microorganism was inoculated at a concentration of 10 4 -10 5 cells or spores / ml in 10 ml of suitable semi-solid agar and poured onto a solid layer of agar of the same medium. After solidification, 5 mm wells were drilled, to which 40 ⁇ l of the neutralized extract of each bifidobacterium was added. It was allowed to diffuse 1 h at 4 0 C and, subsequently, it was incubated under the optimal conditions for each indicator microorganism. As indicator microorganisms, several fungi (Fusarium spp. And Penicillium sp.) And yeasts (Candida spp.) Were used.
  • yeast extract agar (Scharlau, Barcelona) was used for yeast growth and incubated in aerobiosis at 3O 0 C, except in the case of Cryptococcus albidus which was incubated at 26 0 C.
  • Malt extract agar (Scharlau, Barcelona) was used and incubated in aerobiosis at 26 0 C.
  • Yeast microorganisms Candida spp.
  • fungi Feusar ⁇ um spp. And Penicillium spp. Were used as indicator microorganisms.
  • 10% skim milk was used as a model of a food in which the selected strains could play a bioprotective role against yeasts and fungal yeasts or health risks.
  • the previously sterilized milk was inoculated with the bioprotective bifidobacteria, so that they will reach levels of 10 7 CFU / ml, and were contaminated with cultures of Candida parapsilosis, Rhodotorula glutinis, and Penicillium sp. so that they reach final levels of April 10 -10 5 CFU / ml, and incubated at 37 0 C for 24-48 h.
  • controls were analyzed in which only the contaminating fungus or yeast was inoculated.
  • Yeast and fungus counts were performed on Rosa de Bengal agar (Scharlau, Barcelona, Spain) by incubating in aerobiosis at 3O 0 C, for 48-72 h.

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Abstract

The invention relates to metabolic cellular compounds with antifungal activity from strains of the genus Bifidobacterium.The compounds resulting from the metabolism of strains of the genus Bifidobacterium, which take the form of extracts, contain protein and non-protein compounds with antifungal activity. The compounds produced by bifidobacteria have a wide range of activity including yeasts and fungi and, among same, important mycotoxin producers. The aforementioned strains and metabolic products can be used as bioprotectors, preservatives and biological fungicides against alterative yeasts and fungi of zoo- or phyto-sanitary interest in agri-food products and in pharmaceutical formulations.

Description

TITULOTITLE

COMPUESTOS CON ACTIVIDAD ANTIFÚNGICA PRODUCIDOS PORCOMPOUNDS WITH ANTIFUNGICAL ACTIVITY PRODUCED BY

BIFIDOBACTERIUM SPP.BIFIDOBACTERIUM SPP.

SECTOR DE LA TÉCNICASECTOR OF THE TECHNIQUE

Esta patente pertenece al sector de Ia técnica de Ia Industria Agroalimentaria y Farmacéutica. Más concretamente, esta invención se enmarca dentro de las tecnologías de control y conservación de cultivos y productos agroalimentarios destinados a alimentación humana o animal por métodos biológicos, a través del uso de microorganismos con actividad antimicrobiana y de sus metabolitos, y del campo de Ia alimentación funcional (probióticos).This patent belongs to the technical sector of the Agrifood and Pharmaceutical Industry. More specifically, this invention is framed within the technologies of control and conservation of crops and agri-food products destined for human or animal feed by biological methods, through the use of microorganisms with antimicrobial activity and its metabolites, and the field of food functional (probiotics).

ESTADO DE LA TÉCNICA Los hongos y levaduras son microorganismos responsables de Ia alteración de un gran número de productos destinados a alimentación humana (cereales, vinos, encurtidos, zumos y derivados de frutas, derivados lácteos, etc) y animal (Legan, 1993. Mould spoilage of bread: the problem and some solutions. Int. Biodeterior. Biodegrad. 32: 33-53. Loureiro et al., 2003. Spoilage yeast in the wine industry. Int. J. Food Microbiol. 86: 23-5.). Esta contaminación puede surgir en los primeros estadios de Ia producción en campo, con Ia consiguiente alteración y contaminación de granos y frutos, y es causa de grandes pérdidas económicas en diversos sectores industriales (Llorens eí al., 2004. Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops. Int. J. Food Protec. 94: 43-54).STATE OF THE TECHNIQUE Fungi and yeasts are microorganisms responsible for the alteration of a large number of products intended for human consumption (cereals, wines, pickles, juices and fruit derivatives, dairy products, etc.) and animals (Legan, 1993. Mold spoilage of bread: the problem and some solutions. Int. Biodeterior. Biodegrad. 32: 33-53. Loureiro et al., 2003. Spoilage yeast in the wine industry. Int. J. Food Microbiol. 86: 23-5.) . This contamination may arise in the first stages of field production, with the consequent alteration and contamination of grains and fruits, and is the cause of large economic losses in various industrial sectors (Llorens eí al., 2004. Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops. Int. J. Food Protec. 94: 43-54).

Entre las especies de hongos que se desarrollan en los cultivos y productos agroalimentarios se encuentran además importantes productores de metabolitos secundarios tóxicos, como las micotoxinas. Estos metabolitos suponen un riesgo importante para Ia salud por sus propiedades nefrotóxicas, hapatotóxicas, neurotóxincas, teratogénicas, estrogénicas y cancerígenas. Los principales hongos productores de micotoxinas pertenecen a los géneros Aspergillus, Penicillium, Fusarium y Alternaría y las principales toxinas que producen son las aflatoxinas, ocratoxinas, tricotecenos, zearalenona, fumonisinas, fusaricinas, moniliformina, patulina, ácido ciclopiazónico y altemariol, entre otras. Algunos hongos y levaduras también son responsables de infecciones y se consideran patógenos o patógenos oportunistas (Candida spp., Cryptococcus spp., Rhodotorula spp., etc.). Las especies de Candida son unas de las principales responsables de infecciones (candidiasis) de diversos tipos (intestinales, vaginales, urogenitales, orales, cutáneas, etc). La candidiasis es frecuentemente producida por C. albicans aunque puede ser debida a otras especies como C. tropicalis y C. parapsilosis.Among the species of fungi that develop in crops and food products are also important producers of toxic secondary metabolites, such as mycotoxins. These metabolites pose an important health risk due to their nephrotoxic, hapatotoxic, neurotoxin, teratogenic, estrogenic and carcinogenic properties. The main fungi that produce mycotoxins belong to the genera Aspergillus, Penicillium, Fusarium and Alternaría and the main toxins they produce are aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, fusaricines, moniliformin, patulin, cyclopiazonic acid and altemariol, among others. Some fungi and yeasts are also responsible for infections and are considered opportunistic pathogens (Candida spp., Cryptococcus spp., Rhodotorula spp., Etc.). Candida species are one of the main responsible for infections (candidiasis) of various types (intestinal, vaginal, urogenital, oral, skin, etc). Candidiasis is frequently produced by C. albicans although it may be due to other species such as C. tropicalis and C. parapsilosis.

Los mecanismos aplicados actualmente para evitar el desarrollo de hongos y levaduras no deseables en cultivos y productos destinados a alimentación humana y animal incluyen tratamientos físicos, químicos y biológicos. En este ámbito, las técnicas de conservación por métodos biológicos, mediante el uso de microorganismos productores de compuestos antifúngicos o de sus metabolitos son unas de las más atractivas (Stiles, 1996. Biopreservation by lactic acid bacteria. Antón. Leeuw. 70: 331-345). En las últimas décadas, el uso de bacterias lácticas y sus metabolitos como conservantes biológicos ha despertado un gran interés en Ia industria alimentaria ya que los integrantes de este grupo microbiano se consideran, en su mayor parte, seguros (GRAS). No obstante, Ia mayor parte de estudios y aplicaciones se basan exclusivamente en su actividad antibacteriana (Riley eí al., 2002. Bacteriocins: Evolution, Ecology, and Application. Ann. Rev. Microbiol. 56:117-137). La síntesis de metabolitos con actividad antifúngica sólo se ha atribuido a un número limitado de cepas de los géneros Lactobacillus y Propionibacterium (Corsetti eí al., 1998. Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Appl. Microbiol. Biotechnol. 50:253-256; Lavermicocca eí al., 2000. Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21 B. Appl. Environ. Microbiol. 66: 4048-4090; Magnusson et al., 2001. Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Appl. Environ. Microbiol. 67: 1-5; Sjógren eí al., 2003. Antifungal 3-Hydroxy Fatty Acids from Lactobacillus plantarum MiLAB 14. Appl. Environ. Microbiol. 69: 7554-7557; Schwenninger eí al., 2004. A mixed culture of Propionibacterium jensenii and Lactobacillus subsp. paracasei inhibits food spoilage yeast. Syst. Appl. Microbiol. 27: 229-237). En cuanto a las aplicaciones desarrolladas en el campo de Ia conservación de alimentos y otros materiales propensos a sufrir alteraciones por desarrollo de hongos y levaduras se pueden citar: un cultivo de Lactobacillus casei var. rhamnosus productor de una sustancia antifúngica (King et al., 1999. Method for inhibiting fungi. US Patent Number 4,956,177), un co-cultivo de Lactobacillus casei subsp. rhamnosus y Propionibacterium freudenreichii susp. shermanii (BioProfit; Valió Ltd. Helsinki, Finlandia; Wiesby, GMBH&Co., Niebúl, Alemania) y una mezcla de Propionibacterium jensenii y Lactobacillus con actividad antifúngica y antibacteriana (Miescher Schwenninger eí al., 2003. Mixtures of Propionibacterium jensenii and Lactobacillus sp. with antimicrobial activities for use as a natural preservation system. EP 1 308 506 A1 ). En el campo clínico, con fines terapéuticos o profilácticos, se han patentado diversas combinaciones de cepas de bacterias lácticas activas frente a Candida para infecciones gastrointestinales, dérmicas, orales, vaginales y urogenitales (Tetsuo eí al. 1997. Therapeutic agent for dermatophathy having antifungal action, JP9263539; Dohnalek eí al. 1999. Composition of treatment of candidiasis WO 99/17788; Khaziev eí al., 2002. Medical preparation for prophylaxis and treatment of urogenital infections, RU2185842).The mechanisms currently applied to prevent the development of undesirable fungi and yeasts in crops and products intended for human and animal feed include physical, chemical and biological treatments. In this area, conservation techniques by biological methods, through the use of microorganisms that produce antifungal compounds or their metabolites, are some of the most attractive (Stiles, 1996. Biopreservation by lactic acid bacteria. Anton. Leeuw. 70: 331- 3. 4. 5). In the last decades, the use of lactic bacteria and their metabolites as biological preservatives has aroused great interest in the food industry since the members of this microbial group are considered, for the most part, safe (GRAS). However, most studies and applications are based exclusively on their antibacterial activity (Riley eí al., 2002. Bacteriocins: Evolution, Ecology, and Application. Ann. Rev. Microbiol. 56: 117-137). The synthesis of metabolites with antifungal activity has only been attributed to a limited number of strains of the genera Lactobacillus and Propionibacterium (Corsetti eí al., 1998. Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Appl. Microbiol. Biotechnol. 50: 253-256; Lavermicocca eí al., 2000. Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21 B. Appl. Environ. Microbiol. 66: 4048-4090; Magnusson et al., 2001. Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Environ. Microbiol 67: 1-5; Sjógren eí al., 2003. Antifungal 3-Hydroxy Fatty Acids from Lactobacillus plantarum MiLAB 14. Appl. Environ. Microbiol 69: 7554-7557; Schwenninger eí al., 2004. A mixed culture of Propionibacterium jensenii and Lactobacillus subsp. paracasei inhibits food spoilage yeast. Syst Appl. Microbiol 27: 229-237). Regarding the applications developed in the field of food preservation and other materials prone to alterations due to fungal and yeast development, we can mention: a culture of Lactobacillus casei var. rhamnosus producer of an antifungal substance (King et al., 1999. Method for inhibiting fungi. US Patent Number 4,956,177), a co-culture of Lactobacillus casei subsp. rhamnosus and Propionibacterium freudenreichii susp. shermanii (BioProfit; Valió Ltd. Helsinki, Finland; Wiesby, GMBH & Co., Niebul, Germany) and a mixture of Propionibacterium jensenii and Lactobacillus with antifungal and antibacterial activity (Miescher Schwenninger eí al., 2003. Mixtures of Propionibacterium jensenii and. Lactobacillus sp. with antimicrobial activities for use as a natural preservation system EP 1 308 506 A1). In the clinical field, for therapeutic or prophylactic purposes, various combinations of active lactic bacterial strains against Candida have been patented for gastrointestinal, dermal, oral, vaginal and urogenital infections (Tetsuo eí al. 1997. Therapeutic agent for dermatophathy having antifungal action , JP9263539; Dohnalek eí al. 1999. Composition of treatment of candidiasis WO 99/17788; Khaziev eí al., 2002. Medical preparation for prophylaxis and treatment of urogenital infections, RU2185842).

Los compuestos antifúngicos producidos por bacterias lácticas e identificados hasta Ia fecha se dividen en dos grandes grupos: (i) compuestos de bajo peso molecular (menores de 1000 Da) entre los que se incluyen los ácidos acético, benzoico, fenil-láctico, 4-hidroxi-fenil láctico y capróico, metilidantoína, mevanololactona y ciclo-glicil-leucina; y (ii) proteínas antimicrobianas (mayores de 1000 Da) similares a las bacteriocinas. Estos compuestos proteicos se han identificado, tan sólo, en Lactobacillus spp. y se denominan "bacteriocin-like compounds", ya que no encajan con los criterios que definen las bacteriocinas porque tienen un espectro de acción más amplio (Okkers eí al. 1999. Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with fungistatic effect on Candida albicans. J. Appl. Microbiol. 87: 726-734; Magnusson et al., 2001. Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Appl. Environ. Microbiol. 67: 1-5 ; Atanassova eí al., 2003. Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3. Int. J. Food Microbiol. 87: 63-73). No obstante, los compuestos proteicos con actividad antifúngica identificados hasta ahora tienen actividad destacada sólo frente a levaduras o sólo frente a hongos, sin que se hayan descrito cepas ni compuestos con actividad inhibitoria importante frente a los dos grupos de microorganismos simultáneamente.The antifungal compounds produced by lactic bacteria and identified to date are divided into two large groups: (i) low molecular weight compounds (less than 1000 Da), including acetic, benzoic, phenyl-lactic acids, 4- lactic and caproic hydroxy-phenyl, methylidantoin, mevanololactone and cyclo-glycyl-leucine; and (ii) antimicrobial proteins (greater than 1000 Da) similar to bacteriocins. These protein compounds have been identified only in Lactobacillus spp. and they are called "bacteriocin-like compounds", since they do not fit the criteria that define bacteriocins because they have a broader spectrum of action (Okkers ei al. 1999. Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with fungistatic effect on Candida albicans. J. Appl. Microbiol 87: 726-734; Magnusson et al., 2001. Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Appl. Environ. Microbiol 67: 1-5; Atanassova eí al., 2003. Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3. Int. J. Food Microbiol. 87: 63-73). However, protein compounds with antifungal activity identified so far have outstanding activity only against yeasts or only against fungi, without strains or compounds with significant inhibitory activity against both groups of microorganisms have been described simultaneously.

Los microorganismos del género Bifidobacterium son habitantes naturales de tracto intestinal del hombre y animales. Se incluyen habitualmente dentro del grupo de bacterias lácticas, definido fisiológicamente por su capacidad para producir ácido láctico como uno de los principales metabolitos resultantes de Ia fermentación de carbohidratos, aunque filogenéticamente se incluyen dentro de Ia clase Actinobactería (O'Riordan, eí al. 1997. Determination of genetic diversity within the genus Bifidobacterium and estimation of chromosomal size. FEMS Microbiol. Lett. 156: 259-264). Actualmente, las cepas de este género constituyen uno de los grupos más importantes de probióticos por sus reconocidos efectos beneficiosos sobre Ia salud y se incluyen en Ia elaboración de un número cada vez más amplio de alimentos (leches fermentadas, quesos, formulaciones infantiles, etc.) y preparaciones farmacéuticas. Este género se considera, por ejemplo, esencial en el mantenimiento del equilibrio del ecosistema intestinal y el desplazamiento de microorganismos potencialmente perjudiciales, por Io que se Ie atribuye una importante función protectora. A cepas de este género se les ha atribuido efectos beneficiosos en el tratamiento de Ia diarrea del viajero, las infecciones por rotavirus, Ia diarrea asociada al uso de antibióticos y las infecciones intestinales y extraintestinales causadas por Candida spp. (Dohnalek eí al., 1999. Composition of treatment of candidiasis. WO 99/17788; Levchenko, et al. 2001. Strain bifidobacterium breve 79-88 used for preparing curative-prophylactic fermented-milk, nonfermented-milk food- stuffs, biologically active supplements and bacterial preparations. RU2176270; Neeser, eí al. 2001. New bifidobacteria preventing diarrhea caused by pathogenic bacteria. WO 01/11015 A1 ; McNaught, eí al. 2001. Probiotics in clinical practice: a critical review of the evidence. Nutr. Res. 21 : 343-353). No obstante, los efectos antagónicos de las bifidobacterias frente a Ia flora competitiva se han atribuido a Ia competición por sitios de adhesión, a sus propiedades inmunomoduladoras y a Ia producción de ácidos orgánicos, sin que existan evidencias claras de Ia implicación de compuestos antimicrobianos de naturaleza proteica (Asahara, eí al. 2001. Increased resistance of minee to Salmonella entérica serovar Thyphimuríum infection by symbiotic administration of Bifidobacteria and transgalactosylated oligosaccharides. J. Appl. Microbiol. 91 : 985-996; Bruno, eí al. 2002. Inhibition of pathogenic and putrefactive microorganisms by Bifidobacterium sp. Milchwissenschaft 57: 617-62). Tan sólo se ha demostrado que algunas bifidobacterias aisladas de heces de niños producen péptidos antimicrobianos activos frente a Listeria monocytogenes y sólo se ha purificado una bacteriocina (Bifidocina B) de B. bifídum NCFB 1454, que posee un espectro de inhibición restringido a bacterias Gram-positivas (Yildirim, eí al. 1998. Characterization and antimicrobial spectrum of Bifidocin B, a bacteriocin produced by Bifidobacterium bifídum NCFB 1454. J. Food Prot. 61 : 47-51 ; Yildirim, eí al. 1999. Purification, amino acid sequence and mode of action of Bifidocin B produced by Bifidobacterium bifídum NCFB 1454. J. Appl. Microbiol. 86: 45-54. Touré, eí al. 2003. Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes. J. Appl. Microbiol. 95: 1058-1069).The microorganisms of the genus Bifidobacterium are natural inhabitants of the intestinal tract of man and animals. They are usually included within the group of lactic bacteria, physiologically defined by their ability to produce lactic acid as one of the main metabolites resulting from carbohydrate fermentation, although phylogenetically they are included within the Actinobacteria class (O ' Riordan, ei. 1997. Determination of genetic diversity within the genus Bifidobacterium and estimation of chromosomal size. FEMS Microbiol. Lett. 156: 259-264). Currently, the strains of this genus constitute one of the most important groups of probiotics for their recognized beneficial effects on health and are included in the elaboration of an increasingly large number of foods (fermented milks, cheeses, infant formulations, etc.). ) and pharmaceutical preparations. This genus is considered, for example, essential in maintaining the balance of the intestinal ecosystem and the displacement of potentially harmful microorganisms, for which reason an important protective function is attributed. Strains of this genus have been attributed beneficial effects in the treatment of traveler's diarrhea, rotavirus infections, diarrhea associated with the use of antibiotics and intestinal and extraintestinal infections caused by Candida spp. (Dohnalek eí al., 1999. Composition of treatment of candidiasis. WO 99/17788; Levchenko, et al. 2001. Strain bifidobacterium breve 79-88 used for preparing curative-prophylactic fermented-milk, nonfermented-milk food-stuffs, biologically active supplements and bacterial preparations RU2176270; Neeser, eí al. 2001. New bifidobacteria preventing diarrhea caused by pathogenic bacteria. WO 01/11015 A1; McNaught, eí al. 2001. Probiotics in clinical practice: a critical review of the evidence. Nutr. Res. 21: 343-353). However, the antagonistic effects of bifidobacteria against competitive flora have been attributed to competition for adhesion sites, their immunomodulatory properties and the production of organic acids, without clear evidence of the involvement of antimicrobial compounds of a protein nature. (Asahara, eí al. 2001. Increased resistance of minee to Salmonella enterica serovar Thyphimuríum infection by symbiotic administration of Bifidobacteria and transgalactosylated oligosaccharides. J. Appl. Microbiol. 91: 985-996; Bruno, eí al. 2002. Inhibition of pathogenic and putrefactive microorganisms by Bifidobacterium sp. Milchwissenschaft 57: 617-62). It has only been shown that some bifidobacteria isolated from the faeces of children produce active antimicrobial peptides against Listeria monocytogenes and only one bacteriocin (Bifidocin B) has been purified from B. bifidum NCFB 1454, which has a spectrum of inhibition restricted to Gram bacteria. positive (Yildirim, eí al. 1998. Characterization and antimicrobial spectrum of Bifidocin B, a bacteriocin produced by Bifidobacterium bifidum NCFB 1454. J. Food Prot. 61: 47-51; Yildirim, eí al. 1999. Purification, amino acid sequence and mode of action of Bifidocin B produced by Bifidobacterium bifidum NCFB 1454. J. Appl. Microbiol. 86: 45-54. Touré, ei. 2003. Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes. J. Appl Microbiol. 95: 1058-1069).

La presente invención aporta compuestos celulares y metabólicos con actividad antifúngica de cepas del género Bifidobacterium. Los compuestos resultantes del metabolismo de cepas del género Bifidobacterium, aportados en forma de extractos, contienen compuestos proteicos y no proteicos con actividad antifúngica y, por consiguiente, amplía las aplicaciones de este grupo microbiano en el campo de Ia conservación de productos destinados a alimentación humana y animal y en el área clínica y fitosanitaria. Estas bacterias son productoras de metabolitos proteicos con amplio espectro de actividad antifúngica (activos frente a levaduras y hongos). Otra ventaja de Ia presente invención es Ia resistencia de las cepas productoras a las condiciones de estrés gastrointestinal y tecnológico que aseguran su viabilidad y estabilidad. Además, las cepas son capaces de producir los compuestos antifúngicos en condiciones intestinales y en alimentos Io que garantiza su funcionalidad y aplicaciones relacionadas con el sector agroalimentario y clínico.The present invention provides cellular and metabolic compounds with antifungal activity of strains of the genus Bifidobacterium. The compounds resulting from the metabolism of strains of the genus Bifidobacterium, provided in the form of extracts, contain protein and non-protein compounds with antifungal activity and, therefore, expand the applications of this microbial group in the field of conservation of products intended for human consumption and animal and in the clinical and phytosanitary area. These bacteria are producers of protein metabolites with a broad spectrum of antifungal activity (active against yeasts and fungi). Another advantage of the present invention is the resistance of the producing strains to the conditions of gastrointestinal and technological stress that ensure its viability and stability. In addition, the strains are capable of producing the antifungal compounds in intestinal conditions and in food, which guarantees their functionality and applications related to the agri-food and clinical sector.

DESCRIPCIÓN DE LA INVENCIÓN Descripción breveDESCRIPTION OF THE INVENTION Brief Description

En Ia presente invención se ha detectado actividad antifúngica en el género Bifidobacterium. Las cepas pertenecientes a este género son productoras de compuestos de naturaleza proteica con características similares a las bacteriocinas pero con un espectro de acción que no coincide con los criterios que las definen, ya que son activos frente a hongos y levaduras. Además, las cepas producen otros compuestos estructurales y compuestos metabólicos no proteicos, con actividad antifúngica a pH ácido, que actúan de forma sinérgica con los primeros incrementado su efectividad.In the present invention antifungal activity has been detected in the genus Bifidobacterium. The strains belonging to this genus are producers of compounds of a protein nature with characteristics similar to bacteriocins but with a spectrum of action that does not match the criteria that define them, since they are active against fungi and yeasts. In addition, the strains produce other structural compounds and non-protein metabolic compounds, with antifungal activity at acidic pH, which act synergistically with the former increasing their effectiveness.

La presente invención proporciona compuestos antifúngicos derivados del metabolismo de las bifidobacterias, en forma de extractos. Estos compuestos antifúngicos derivados del metabolismo de las bifidobacterias constituyen objeto de Ia presente invención y son reivindicados en Ia presente invención.The present invention provides antifungal compounds derived from the metabolism of bifidobacteria, in the form of extracts. These antifungal compounds derived from the metabolism of bifidobacteria are the subject of the present invention and are claimed in the present invention.

Los metabolitos son secretados al medio de cultivo durante el crecimiento de las bifidobacterias y los extractos que los contienen se obtienen a partir de los sobrenadantes libres de células de estos cultivos mediante liofilización. El espectro de actividad incluye a hongos y levaduras responsables de alteraciones en alimentos y de interés zoo- y fitosanitario por ser potencialmente patógenos o capaces de sintetizar metabolitos tóxicos pertenecientes a los géneros: Alternaría, Aspergillus, Cladosporíum, Cryptococcus, Eurotium, Fusaríum, Penicillium, Tríchoderma, Brettanomyces, Candida, Cryptococcus, Debaryomyces, Dekkera, Hansenula, Hansenniaspora, Kloeckera, Kluyveromyces, Pichia, Rhodotorula, Torulaspora, Zygossacharomyces, etc.The metabolites are secreted into the culture medium during the growth of the bifidobacteria and the extracts containing them are obtained from the cell-free supernatants of these cultures by lyophilization. The spectrum of activity includes fungi and yeasts responsible for alterations in food and of zoo- and phytosanitary interest because they are potentially pathogenic or capable of synthesizing toxic metabolites belonging to the genera: Alternaría, Aspergillus, Cladosporíum, Cryptococcus, Eurotium, Fusaríum, Penicillium, Tríchoderma, Brettanomyces, Candida, Cryptococcus, Debaryomyces, Dekkera, Hansenula, Hansenniaspora, Kloeckera, Kluyveromyces, Pichia, Rhodotorula, Torulaspora, Zygossacharomyces, etc.

Los compuestos responsables de Ia actividad antifúngica secretados a los medios de cultivo por las bifidobacterias y activos en los extractos neutralizados ensayados son sustancias de naturaleza proteica y poseen características bioquímicas similares a las de las bacteriocinas de bacterias lácticas, pero no cumplen los criterios de su definición ya que inhiben a hongos y levaduras. Los compuestos responsables de Ia actividad antifúngica secretados a los medios de cultivo por las bifidobacterias y presentes en los extractos no neutralizados y activos a pH ácido incluyen también compuestos de distinta naturaleza, que potencian el efecto de las proteínas antifúngicas.The compounds responsible for the antifungal activity secreted to the culture media by the bifidobacteria and active in the neutralized extracts tested are substances of a protein nature and have biochemical characteristics similar to those of lactic bacteria bacteriocins, but do not meet the criteria of their definition since they inhibit fungi and yeasts. The compounds responsible for the antifungal activity secreted to the culture media by the bifidobacteria and present in the non-neutralized and active extracts at acidic pH also include compounds of different nature, which enhance the effect of the antifungal proteins.

Los compuestos celulares y metabólicos de estas bacterias con actividad antifúngica, así como su uso como agentes antifúngicos frente a hongos y levaduras de interés zoo- y fitosanitario y alterantes de alimentos, están reivindicados en Ia presente invención.The cellular and metabolic compounds of these bacteria with antifungal activity, as well as their use as antifungal agents against fungi and yeasts of zoo- and phytosanitary interest and food alters, are claimed in the present invention.

Puesto que las cepas del género Bifidobacterium sintetizaron compuestos con actividad antifúngica de amplio espectro, en las condiciones a las que están expuestas en el tracto intestinal, pueden actuar como probióticos con función protectora a este nivel y ser utilizados con fines médicos. El uso de las cepas de Ia presente invención como probióticos es otro objeto de Ia presente invención.Since strains of the genus Bifidobacterium synthesized compounds with broad spectrum antifungal activity, under the conditions to which they are exposed in the intestinal tract, they can act as probiotics with protective function at this level and be used for medical purposes. The use of the strains of the present invention as probiotics is another object of the present invention.

Las bifidobacterias crecen y se mantienen viables en diversos alimentos constituyendo, por tanto, vehículos idóneos para su aporte. Además, en alimentos como las leches fermentadas y no fermentadas se comprobó Ia capacidad de las bifidobacterias para secretar compuestos antifúngicos al medio de cultivo durante su crecimiento. Las cepas del género Bifidobacterium, así como sus metabolitos adicionados en forma de extractos, actúan como inhibidores del desarrollo de hongos y levaduras en alimentos. Es decir, actúan como cultivos bioprotectores en alimentos como Ia leche. El uso de estas cepas como agentes bioprotectores en productos agroalimentarios constituye otro objeto de Ia presente invención.Bifidobacteria grow and remain viable in various foods, thus constituting suitable vehicles for their contribution. In addition, in foods such as fermented and unfermented milks, the ability of bifidobacteria to secrete antifungal compounds to the culture medium during their growth was verified. The strains of the genus Bifidobacterium, as well as their added metabolites in the form of extracts, act as inhibitors of fungal and yeast development in food. That is, they act as bioprotective cultures in foods such as milk. The use of these strains as bioprotective agents in agri-food products constitutes another object of the present invention.

Descripción detallada La presente invención proporciona componentes celulares y metabolitos antifúngicos de cepas del género Bifidobacterium. Los metabolitos son secretados al medio de cultivo durante el crecimiento de las bifidobacterias y los extractos que los contienen se obtienen a partir de los sobrenadantes de estos cultivos mediante liofilización.Detailed Description The present invention provides cellular components and antifungal metabolites of strains of the genus Bifidobacterium. The metabolites are secreted to the culture medium during the growth of the bifidobacteria and the extracts that contain them are obtained from the supernatants of these cultures by lyophilization.

Las bacterias útiles en esta invención incluyen representantes del género Bifidobacterium de origen humano y animal, por ejemplo, cepas de las especies Bifidobacterium longum, B. infantis, B. catenulatum, B. pseudocatenulatum B. bifidum, B. breve, B. adolescentis, B. animalis-B. lactis yBacteria useful in this invention include representatives of the genus Bifidobacterium of human and animal origin, for example, strains of the species Bifidobacterium longum, B. infantis, B. catenulatum, B. pseudocatenulatum B. bifidum, B. breve, B. adolescentis, B. animalis-B. lactis and

B. globosum.B. Globesum

La actividad antimicrobiana se detecta en presencia de células y en los extractos, neutralizados (en los que se elimina el efecto de los ácidos orgánicos) y sin neutralizar (en los que el efecto de los ácidos orgánicos se suma al de los compuestos proteicos). El espectro de actividad incluye a hongos y levaduras de interés zoo- y fitosanitario y responsables de alteraciones en alimentos pertenecientes a los géneros Alternaría, Aspergillus, Cladosporium, Cryptococcus, Eurotium, Fusarium, Penicillium , Trichoderma, Brettanomyces, Candida, Cryptococcus, Debaryomyces, Dekkera, Hansenniaspora, Hansenula, Kloeckera, Kluyveromyces, Pichia, Rhodotorula, Torulaspora, etc.The antimicrobial activity is detected in the presence of cells and in the extracts, neutralized (in which the effect of organic acids is eliminated) and without neutralizing (in which the effect of organic acids is added to that of protein compounds). The spectrum of activity includes fungi and yeasts of zoo- and phytosanitary interest and responsible for alterations in foods belonging to the genera Alternaria, Aspergillus, Cladosporium, Cryptococcus, Eurotium, Fusarium, Penicillium, Trichoderma, Brettanomyces, Candida, Cryptococcus, Debaryomyces , Hansenniaspora, Hansenula, Kloeckera, Kluyveromyces, Pichia, Rhodotorula, Torulaspora, etc.

La actividad antifúngica global (de componentes celulares y productos metabólicos) de las cepas del género Bifidobacterium fue detectada por Ia técnica de Ia doble capa frente a representantes de un amplio numero de géneros de levaduras y hongos citados con anterioridad (Ejemplo 1 ; Figuras 1 y 2). El espectro de inhibición fue amplio en todos los casos, pero observándose diferencias entre cepas, respecto a su intensidad (halos de inhibición entre 0 y 30 mm) y espectro de acción. La detección de actividad antifúngica debida exclusivamente a Ia secreción de compuestos de naturaleza proteica, se realizó mediante ensayos de inhibición de los extractos neutralizados obtenidos a partir de los cultivos de las bifidobacterias frente diversos microorganismos indicadores mediante Ia técnica de difusión en agar. Mediante Ia misma técnica, pero utilizando extractos sin neutralizar, se detectó simultáneamente Ia actividad de otros compuestos, como los ácidos orgánicos, activos a pH ácido. De acuerdo con los resultados obtenidos por los dos procedimientos las cepas más activas fueron Bifidobacterium BIR-0307, BIR-0324 y BIR-0326, pertenecientes a las especies B. longum infantis, B. catenulatum y B. bifidum, respectivamente. Estas cepas han sido reivindicadas en Ia patente titulada: "Extractos proteicos con amplio espectro de actividad antibacteriana y cepas del género Bifidobacterium que los producen" cuyo titular es Consejo Superior de Investigaciones Científicas y sus autores Collado, M. C. y Sanz, Y. que se presenta en Ia misma fecha que Ia presente solicitud. La mayoría de las hongos, incluyendo importantes productores de micotoxinas (Alternaría alternata, Aspergillus parasiticus, Fusaríum spp. y Penicillium expansum), y de levaduras fueron inhibidos por las bifidobacterias, salvo Saccharomyces cerevisiae. Esta excepción confiere una gran ventaja a las bifidobacterias en su aplicación como cultivos bioprotectores en productos en los que se utilice Ia especie S. cerevisiae como cultivo iniciador, ya que su adición no comprometería el éxito del proceso de fermentación.The global antifungal activity (of cellular components and metabolic products) of the strains of the genus Bifidobacterium was detected by the double layer technique against representatives of a large number of yeast and fungal genera mentioned above (Example 1; Figures 1 and 2). The spectrum of inhibition was wide in all cases, but differences were observed between strains, with respect to their intensity (halos of inhibition between 0 and 30 mm) and spectrum of action. The detection of antifungal activity due exclusively to the secretion of compounds of protein nature, was carried out by means of inhibition tests of the neutralized extracts obtained from the cultures of the bifidobacteria against various indicator microorganisms by means of the agar diffusion technique. By means of the same technique, but using non-neutralized extracts, the activity of other compounds, such as organic acids, active at acidic pH was simultaneously detected. According to the results obtained by the two procedures, the most active strains were Bifidobacterium BIR-0307, BIR-0324 and BIR-0326, belonging to the species B. longum infantis, B. catenulatum and B. bifidum, respectively. These strains have been claimed in the patent entitled: "Protein extracts with a broad spectrum of antibacterial activity and strains of the genus Bifidobacterium that produce them" whose head is the Higher Council for Scientific Research and its authors Collado, MC and Sanz, Y. presented on the same date as the present application. The majority of fungi, including important mycotoxin producers (Alternaría alternata, Aspergillus parasiticus, Fusaríum spp. And Penicillium expansum), and yeast were inhibited by bifidobacteria, except Saccharomyces cerevisiae. This exception confers a great advantage to the bifidobacteria in its application as bioprotective cultures in products in which the S. cerevisiae species is used as the starter culture, since its addition would not compromise the success of the fermentation process.

Los compuestos metabólicos producidos por las bifidobacterias y responsables de Ia actividad antifúngica son sustancias de naturaleza proteica y no proteica, cuya producción o liberación al medio aumenta cuando se utiliza como medio de cultivo caldo MRS, conteniendo una concentración 4 mM de cisteína y un 1% de glicerol.The metabolic compounds produced by the bifidobacteria and responsible for the antifungal activity are substances of a protein and non-protein nature, whose production or release to the medium increases when MRS broth is used as a culture medium, containing a 4 mM concentration of cysteine and 1% of glycerol.

La naturaleza proteica de algunos de los compuestos responsables de Ia actividad antifúngica se confirma por Ia destrucción de Ia misma tras el tratamiento de extractos neutralizados con todas las proteinasas ensayadas.The protein nature of some of the compounds responsible for the antifungal activity is confirmed by its destruction after the treatment of neutralized extracts with all the proteinases tested.

La existencia de compuestos inhibidores de naturaleza no proteica, activos a pH ácido, se confirma porque parte de Ia actividad antifúngica se retiene en extractos no neutralizados tratados con proteasas.The existence of inhibitor compounds of a non-protein nature, active at acidic pH, is confirmed because part of the antifungal activity is retained in non-neutralized extracts treated with proteases.

Los compuestos proteicos responsables de Ia inhibición son termoestables manteniendo su actividad tras su incubación a 100 0C durante al menos 1 min. También son estables y activos en un amplío intervalo de pH (3-10). Por tanto, su actividad es compatible con las condiciones propias del tracto gastrointestinal y con las de un amplio número de procesos tecnológicos (fermentación y maduración de productos lácteos, cárnicos, etc.). La masa molecular aparente de los compuestos antifúngicos es menor deProtein responsible of the inhibition compounds are thermostable maintaining its activity after incubation at 100 0 C for at least 1 min on. They are also stable and active in a wide pH range (3-10). Therefore, its activity is compatible with the conditions of the gastrointestinal tract and with those of a large number of technological processes (fermentation and maturation of dairy products, meat, etc.). The apparent molecular mass of the antifungal compounds is less than

100 kDa, determinada por ultrafiltración.100 kDa, determined by ultrafiltration.

Por tanto, los compuestos proteicos responsables de Ia actividad antimicrobiana poseen características bioquímicas similares a las de las bacteriocinas de bacterias lácticas, pero no cumplen los criterios de su definición ya que poseen actividad antifúngica de amplio espectro, incluyendo levaduras y hongos. Así, Ia actividad antifúngica de los extractos frente a hongos y levaduras se destruye tras su tratamiento con diversas proteasas (tripsina, pepsina y proteinasa K). El hecho de que el espectro de inhibición incluya hongos y levaduras es una característica inusual en las bacteriocinas convencionales de bacterias Gram-positivas y en las similares ("bacteriocin- like") con actividad antifúngica identificadas hasta ahora sólo en Lactobacillus spp., confiriendo a este género y sus metabolitos grandes ventajas como probióticos, bioprotectores, conservantes biológicos y productos fitosanitarios biológicos.Therefore, the protein compounds responsible for the antimicrobial activity have biochemical characteristics similar to those of the bacteriocins of lactic bacteria, but do not meet the criteria of their definition since they have broad spectrum antifungal activity, including yeasts and fungi. Thus, the antifungal activity of the extracts against fungi and yeasts is destroyed after treatment with various proteases (trypsin, pepsin and proteinase K). The fact that the spectrum of inhibition includes fungi and yeasts is an unusual characteristic in conventional bacteriocins of Gram-positive bacteria and similar ("bacteriocin-like") with antifungal activity identified so far only in Lactobacillus spp., Conferring to This genus and its metabolites have great advantages such as probiotics, bioprotectors, biological preservatives and biological phytosanitary products.

Las cepas del género Bifidobacterium sintetizaron compuestos con actividad anti bacteriana de amplio espectro, frente a hongos y levaduras, en las condiciones a las que están expuestas en el tracto intestinal, presencia de sales biliares (0,5-5,0 %) y pancreatina (0,1-3,0 g/l) por Io que pueden actuar como probióticos con función protectora a este nivel y ser utilizados con fines médicos.The strains of the genus Bifidobacterium synthesized compounds with broad-spectrum anti-bacterial activity, against fungi and yeasts, under the conditions to which they are exposed in the intestinal tract, presence of bile salts (0.5-5.0%) and pancreatin (0.1-3.0 g / l) so that they can act as probiotics with protective function at this level and be used for medical purposes.

Se determinó el posible uso de alimentos como Ia leche y medios basados en leche como sustrato para el crecimiento de las bifidobacterias y vehículos para su aporte en forma de células viables. En leche, todas las bifidobacterias seleccionadas e inoculadas al 1% fueron capaces de crecer, alcanzando niveles de 107-109 UFC/ml, tras períodos de incubación de entre 24 y 48 h. El pH de Ia leche se redujo de valores iniciales de 6,4 a valores entre 4,0 y 5,2 y se observó cierto grado de coagulación, especialmente importante en las muestras inoculadas con Ia cepa Bifidobacterium BIR-0324 y su aroma fue el típico del yogur. En L-EL-G, todas las bifidobacterias seleccionadas fueron capaces de crecer, y el aumento en el número de viables fue ligeramente superior (entre 1 y 0,5 unidades logarítmicas) al obtenido en L. El grado de acidificación alcanzado también fue mayor, obteniéndose valores finales de pH entre 3,0 y 4,6. Se observó también un mayor grado de coagulación y, este fue especialmente importante, en las muestras inoculadas con las cepas Bifidobacterium BIR -0312, BIR-0324 y BIR-0326 y su aroma fue el típico de leches fermentadas azucaradas. Las bifidobacterias fueron capaces de sintetizar compuestos con actividad antifúngica en alimentos como Ia leche y medios basados en leche. La capacidad para secretar compuestos antimicrobianos al medio de cultivo durante su crecimiento en estos medios se verificó mediante ensayos de inhibición por Ia técnica de Ia doble capa (ver Figura 2.B2) y por Ia técnica de difusión en agar mediante el análisis de Ia actividad de sus extractos.The possible use of foods such as milk and milk-based media was determined as a substrate for the growth of bifidobacteria and vehicles for their contribution in the form of viable cells. In milk, all bifidobacteria selected and inoculated at 1% were able to grow, reaching levels of 10 7 -10 9 CFU / ml, after incubation periods of between 24 and 48 h. The pH of the milk was reduced from initial values of 6.4 to values between 4.0 and 5.2 and some degree of coagulation was observed, especially important in the samples inoculated with the Bifidobacterium BIR-0324 strain and its aroma was the typical of yogurt. In L-EL-G, all the bifidobacteria selected were able to grow, and the increase in the number of viable was slightly higher (between 1 and 0.5 logarithmic units) to that obtained in L. The degree of acidification achieved was also higher , obtaining final pH values between 3.0 and 4.6. A higher degree of coagulation was also observed and, this was especially important, in the samples inoculated with the Bifidobacterium BIR -0312, BIR-0324 and BIR-0326 strains and its aroma was typical of sugary fermented milks. Bifidobacteria were able to synthesize compounds with antifungal activity in foods such as milk and milk-based media. The ability to secrete antimicrobial compounds to the culture medium during their growth in these media was verified by inhibition tests by the double layer technique (see Figure 2.B2) and by the agar diffusion technique by analyzing the activity of his extracts.

Por Io tanto, las cepas del género Bifídobacterium crecen y se mantienen viables en diversos alimentos constituyendo, por consiguiente, vehículos idóneos para su aporte. Las leches fermentadas y no fermentadas, constituyen ejemplos de alimentos que sirven de soporte para el mantenimiento de su viabilidad; además, se encuentran entre los productos más adecuados para Ia administración de cepas probióticas, debido a que Ia leche y sus derivados son integrantes habituales de Ia dieta en países desarrollados, tienen alto valor nutritivo, son fáciles de digerir y gozan de gran aceptación en los diversos grupos de población.Therefore, strains of the genus Bifidobacterium grow and remain viable in various foods, thus constituting suitable vehicles for their contribution. Fermented and unfermented milks are examples of foods that support the maintenance of their viability; In addition, they are among the most suitable products for the administration of probiotic strains, because milk and its derivatives are habitual members of the diet in developed countries, have high nutritional value, are easy to digest and enjoy great acceptance in various population groups

Las cepas del género Bifídobacterium pueden fermentar y coagular alimentos como Ia leche; por tanto, pueden ser utilizadas para Ia elaboración de leches fermentadas y otros derivados lácteos.Strains of the genus Bifidobacterium can ferment and coagulate foods such as milk; Therefore, they can be used for the production of fermented milks and other dairy products.

Las cepas del género Bifídobacterium, así como sus metabolitos adicionados en forma de extractos, actúan como inhibidores del desarrollo de hongos y levaduras en productos agroalimentarios. Las bifidobacterias actuaron, por ejemplo, como cultivos bioprotectores frente a levaduras en leche, reduciendo Ia viabilidad por ejemplo de Candida parapsilosis y Rhodotorula glutins, al menos, entre 1 y 3 unidades logarítmicas y en L-EL-G, al menos, entre 2 y 3 unidades logarítmicas (Figura 3). Las bifidobacterias seleccionadas actuaron como cultivos bioprotectores frente a hongos en leche reduciendo Ia viabilidad por ejemplo de Penicillium spp., al menos, una unidad logarítmica; y en L-EL-G, al menos, 2 unidades logarítmicas (Figura 3). Los efectos protectores consecuencia directa de los metabolitos secretados por las bifidobacterias, se confirmaron por adición exclusiva a Ia leche contaminada de sus extractos. La adición de extractos no neutralizados, en los que se sumaría los efectos de los ácidos orgánicos y los de compuestos de naturaleza proteica, y neutralizados produjeron una reducción de los niveles de Penicillium spp. de al menos dos unidades logarítmicas (Figura 3). Por tanto, los compuestos de naturaleza proteica en forma de extractos así como los cultivos de las cepas del género Bifidobacterium que los producen pueden actuar, en alimentos como Ia leche, como bioprotectores inhibiendo el desarrollo de hongos y levaduras (Ejemplo 3).The strains of the genus Bifidobacterium, as well as their added metabolites in the form of extracts, act as inhibitors of the development of fungi and yeasts in agri-food products. Bifidobacteria acted, for example, as bioprotective cultures against yeasts in milk, reducing the viability for example of Candida parapsilosis and Rhodotorula glutins, at least, between 1 and 3 logarithmic units and in L-EL-G, at least, between 2 and 3 logarithmic units (Figure 3). The selected bifidobacteria acted as bioprotective cultures against fungi in milk, reducing the viability, for example, of Penicillium spp., At least, one logarithmic unit; and in L-EL-G, at least 2 logarithmic units (Figure 3). The protective effects resulting directly from the metabolites secreted by the bifidobacteria, were confirmed by exclusive addition to the contaminated milk of their extracts. The addition of non-neutralized extracts, which would add the effects of organic acids and those of protein compounds, and neutralized produced a reduction in the levels of Penicillium spp. from at least two logarithmic units (Figure 3). Therefore, the compounds of protein nature in the form of extracts as well as the cultures of the strains of the genus Bifidobacterium that produce them can act, in foods such as milk, as bioprotectors inhibiting the development of fungi and yeasts (Example 3).

El uso de los mencionados compuestos como agentes antifúngicos frente a hongos y levaduras que constituyen un riesgo zoo- y fitosanitaro y que son responsables de alteraciones en alimentos, está reivindicado en Ia presente invención. Otros usos de Ia presente invención son el uso del extracto que contiene los compuestos antifúngicos frente a hongos y levaduras alterantes de alimentos y de interés zoo- y fitosanitaro y en un caso particular como conservante y fungicida biológico, y el uso del extracto como compuesto terapéutico frente a hongos y levaduras. El uso de estas cepas como agentes bioprotectores y fungicidas biológicos de productos agroalimentarios y como probióticos con fines médicos (profilácticos o terapéuticos) constituye otro objeto de Ia presente invención.The use of the aforementioned compounds as antifungal agents against fungi and yeasts that constitute a zoo- and phytosanitary risk and that are responsible for alterations in food, is claimed in the present invention. Other uses of the present invention are the use of the extract containing the antifungal compounds against fungi and yeast alters of food and of zoo- and phytosanitary interest and in a particular case as a biological preservative and fungicide, and the use of the extract as a therapeutic compound against fungi and yeasts. The use of these strains as bioprotective agents and biological fungicides of agri-food products and as probiotics for medical purposes (prophylactic or therapeutic) constitutes another object of the present invention.

DESCRIPCIONES DE LAS FIGURAS Figura 1. Representación del espectro de actividad antifúngica de cepas del género Bifidobacterium mediante Ia técnica de Ia doble capa. El efecto inhibidor frente a hongos y levaduras se determinó mediante medidas de los halos de inhibición y está expresado en milímetros (mm)*. El signo (-) indica ausencia de inhibición. Figura 2. Se muestra un ejemplo del grado de Ia inhibición que ejercen algunas cepas del género Bifidobacterium sobre el crecimiento de hongos y levaduras mediante Ia técnica de Ia doble capa. A. Ejemplo de Ia inhibición de Bifidobacterium BIR -0307 y BIR-0326 crecidos en MRSC sobre Aspergillus parasiticus. B. Ejemplo de Ia inhibición de Bifidobacterium BIR-0326 crecido en MRSC (B 1 ) y en agar Leche (B2) sobre Candida parapsilosis.DESCRIPTIONS OF THE FIGURES Figure 1. Representation of the spectrum of antifungal activity of strains of the genus Bifidobacterium by means of the double layer technique. The inhibitory effect against fungi and yeasts was determined by measures of inhibition halos and is expressed in millimeters (mm) *. The sign (-) indicates absence of inhibition. Figure 2. An example of the degree of inhibition exerted by some strains of the genus Bifidobacterium on the growth of fungi and yeasts by means of the double layer technique is shown. A. Example of the inhibition of Bifidobacterium BIR -0307 and BIR-0326 grown in MRSC on Aspergillus parasiticus. B. Example of the inhibition of Bifidobacterium BIR-0326 grown in MRSC (B 1) and in Milk agar (B2) on Candida parapsilosis.

Figura 3. Representación del efecto inhibidor de algunas bifidobacterias sobre Candida parapsilosis, Rhodotorula glutinis y Penicillium sp. en co-cultivos en leche (L) y en leche conteniendo un 0,5 % de extracto de levadura y un 1% de glucosa (L-EL-G) y por adición directa de los extractos, obtenidos a partir de los sobrenadantes de los cultivos de las bifidobacterias, a cultivos de los microorganismos contaminantes.Figure 3. Representation of the inhibitory effect of some bifidobacteria on Candida parapsilosis, Rhodotorula glutinis and Penicillium sp. in co-cultures in milk (L) and in milk containing 0.5% yeast extract and 1% of glucose (L-EL-G) and by direct addition of the extracts, obtained from the supernatants of the bifidobacteria cultures, to cultures of the contaminating microorganisms.

EJEMPLOS DE REALIZACIÓN DE LA INVENCIÓNEXAMPLES OF EMBODIMENT OF THE INVENTION

EJEMPLO 1. PROCEDIMIENTO DE DETECCIÓN DE ACTIVIDAD ANTIFÚNGICA EN BIFIDOBACTERIAS Y DE OBTENCIÓN DE EXTRACTOS CON DICHA ACTIVIDAD.EXAMPLE 1. PROCEDURE FOR DETECTION OF ANTIFUNGICAL ACTIVITY IN BIFIDOBACTERIES AND OBTAINING EXTRACTS WITH SUCH ACTIVITY.

Detección de actividad antifúngica en cultivos y extractos obtenidos a partir de ellos y análisis del espectro de inhibición.Detection of antifungal activity in cultures and extracts obtained from them and analysis of the inhibition spectrum.

La actividad antifúngica de las cepas del género Bifidobacterium, se determinó mediante dos métodos: (i) por Ia técnica de Ia doble capa y (ii) por Ia técnica de difusión en agar.The antifungal activity of the strains of the genus Bifidobacterium, was determined by two methods: (i) by the double layer technique and (ii) by the agar diffusion technique.

La actividad antifúngica de cada cepa, debida a componentes celulares y metabólicos, se evaluó globalmente mediante Ia técnica de Ia doble capa utilizando como microorganismos indicadores representantes de diversos géneros de levaduras y hongos de interés sanitario y alterantes de alimentos, como los que se muestran en Ia Figura 1. Las bifidobacterias potencialmente activas se sembraron en placas de MRS, conteniendo un 0.05% de cisteína (MRSC) en líneas de unos 2 cm y se incubaron en condiciones óptimas durante 16 h y su desarrollo posterior se inhibió con cloroformo. Las levaduras y hongos indicadores se inocularon a una concentración de 104-105 células o esporas/ mi en 10 mi de agar semisólido adecuado, se vertió sobre Ia capa de agar del microorganismo protector y se incubó en condiciones óptimas para los indicadores. Tras 24-72 h de incubación se midieron los posibles halos de inhibición alrededor de las líneas del cultivo protector.The antifungal activity of each strain, due to cellular and metabolic components, was evaluated globally by means of the double layer technique using as indicator microorganisms representatives of various genera of yeasts and fungi of health interest and food alterants, such as those shown in Figure 1. The potentially active bifidobacteria were seeded on MRS plates, containing 0.05% cysteine (MRSC) in lines of about 2 cm and incubated in optimal conditions for 16 h and their subsequent development was inhibited with chloroform. Yeasts and indicator fungi were inoculated at a concentration of 10 4 -10 5 cells or spores / ml in 10 ml of suitable semi-solid agar, poured onto the agar layer of the protective microorganism and incubated under optimal conditions for the indicators. After 24-72 h of incubation the possible inhibition halos around the protective culture lines were measured.

A fin de evaluar Ia actividad antifúngica debida a Ia secreción de compuestos de naturaleza proteica se utilizó Ia técnica de difusión en agar. 10 mi de caldo MRSC se inocularon al 1% con un cultivo de 24 h de cada bifidobacteria y se incubaron durante 16 h a 370C. Los sobrenadantes se obtuvieron por centrifugación (12.000 g, 15 min, 40C) y se concentraron por liofilización. Los liófilos se resuspendieron en 1 mi de tampón fosfato 50 mM a pH 6,5, se neutralizaron con NaOH hasta alcanzar un pH de 6,5 para eliminar los efectos de los ácido orgánicos generados por fermentación, y se esterilizaron por filtración. Estas muestras constituyeron los extractos crudos en los que se determinó Ia posible actividad de proteínas antibacterianas producidas por las bifidobacterias. El microorganismo indicador se inoculó a una concentración de 104-105 células o esporas/ml en 10 mi de agar semisólido adecuado y se vertió sobre una capa sólida de agar del mismo medio. Tras solidificar, se perforaron pocilios de 5 mm, a los que se añadieron 40 μl del extracto neutralizado de cada bifidobacteria. Se dejó difundir 1 h a 40C y, posteriormente, se incubó en las condiciones óptimas para cada microorganismo indicador. Como microorganismos indicadores, se utilizaron varios hongos (Fusarium spp. y Penicillium sp.) y levaduras (Candida spp.).In order to evaluate the antifungal activity due to the secretion of protein compounds, the agar diffusion technique was used. 10 ml of MRSC broth were inoculated at 1% with a 24 h culture of each bifidobacteria and incubated for 16 h at 37 0 C. The supernatants were obtained by centrifugation (12,000 g, 15 min, 4 0 C) and concentrated by lyophilization The lyophiles were resuspended in 1 ml of 50 mM phosphate buffer at pH 6.5, neutralized with NaOH until reaching a pH of 6.5 to remove the effects of organic acids generated by fermentation, and were sterilized by filtration. These samples constituted the crude extracts in which the possible activity of antibacterial proteins produced by the bifidobacteria was determined. The indicator microorganism was inoculated at a concentration of 10 4 -10 5 cells or spores / ml in 10 ml of suitable semi-solid agar and poured onto a solid layer of agar of the same medium. After solidification, 5 mm wells were drilled, to which 40 μl of the neutralized extract of each bifidobacterium was added. It was allowed to diffuse 1 h at 4 0 C and, subsequently, it was incubated under the optimal conditions for each indicator microorganism. As indicator microorganisms, several fungi (Fusarium spp. And Penicillium sp.) And yeasts (Candida spp.) Were used.

En ambos casos, para el crecimiento de levaduras se utilizó agar extracto de levadura (Scharlau, Barcelona) y se incubó en aerobiosis a 3O0C, excepto en el caso de Cryptococcus albidus que se incubó a 26 0C. Para el crecimiento de hongos se utilizó agar Extracto de Malta (Scharlau, Barcelona) y se incubó en aerobiosis a 26 0C.In both cases, yeast extract agar (Scharlau, Barcelona) was used for yeast growth and incubated in aerobiosis at 3O 0 C, except in the case of Cryptococcus albidus which was incubated at 26 0 C. For fungal growth Malt extract agar (Scharlau, Barcelona) was used and incubated in aerobiosis at 26 0 C.

Caracterización de los compuestos responsables de Ia actividad antifúngica La caracterización de los compuestos metabólicos responsables de Ia actividad antifúngica de los extractos neutralizados y no neutralizados, obtenidos por el procedimiento descrito en el apartado anterior, se realizó mediante los siguientes ensayos:Characterization of the compounds responsible for the antifungal activity The characterization of the metabolic compounds responsible for the antifungal activity of the neutralized and non-neutralized extracts, obtained by the procedure described in the previous section, was carried out by means of the following tests:

(i) Sensibilidad a diversos enzimas proteolíticos (tripsina, proteinasa K y pepsina, suministrados por Sigma (St. Louis, MO). Los extractos se incubaron en presencia de 2 mg/ml de cada enzima y fueron incubados en condiciones óptimas de actuación para cada una de ellas, durante 2 h. Tras cada tratamiento se determinó Ia actividad residual utilizando como control los extractos incubados en ausencia de enzimas. (ii) Estabilidad a Ia temperatura y pH. Los extractos se incubaron a distintas temperaturas (80 y 100 0C) durante 10-30 min y a distintos pH (3-10), durante 1 h. Tras cada tratamiento se determinó Ia actividad residual utilizando controles no tratados. (iii) Masa molecular aparente. Se determinó mediante el uso de sistemas de filtración-centrifugación (Millipore, Bedford, Mass) con membranas de límites de exclusión entre 3 y 100 kDa. Los extractos se fraccionaron de forma progresiva con filtros de distinto límite de exclusión y se determinó en que fracciones (retenido y filtrado) se concentraba Ia actividad.(i) Sensitivity to various proteolytic enzymes (trypsin, proteinase K and pepsin, supplied by Sigma (St. Louis, MO). The extracts were incubated in the presence of 2 mg / ml of each enzyme and were incubated under optimal conditions for each of them, for 2 h After each treatment the residual activity was determined using as control the extracts incubated in the absence of enzymes. (ii) Stability at the temperature and pH The extracts were incubated at different temperatures (80 and 100 0 C) for 10-30 min and at different pH (3-10), for 1 h After each treatment the residual activity was determined using untreated controls. (iii) Apparent molecular mass. It was determined by the use of filtration-centrifugation systems (Millipore, Bedford, Mass) with membranes of exclusion limits between 3 and 100 kDa. The extracts were fractionated progressively with filters of different exclusion limits and it was determined in which fractions (retained and filtered) the activity was concentrated.

Como microorganismos indicadores se utilizaron levaduras (Candida spp.) y hongos (Fusaríum spp. y Penicillium spp.).Yeast microorganisms (Candida spp.) And fungi (Fusaríum spp. And Penicillium spp.) Were used as indicator microorganisms.

EJEMPLO 2. FUNCIÓN BIOPROTECTORA DE LAS CEPAS DEL GÉNERO BIFIDOBACTERIUM Y DE SUS EXTRACTOS EN ALIMENTOS.EXAMPLE 2. BIOPROTECTING FUNCTION OF THE BIFIDOBACTERIUM GENDER CEPAS AND ITS EXTRACTS IN FOODS.

Se utilizó leche descremada al 10% como modelo de un alimento en el que las cepas seleccionadas podrían desempeñar una función bioprotectora frente a levaduras y hongos alterantes o de riesgo para Ia salud. La leche, previamente esterilizada, se inoculó con las bifidobacterias bioprotectoras, de modo que alcanzarán niveles de 107 UFC/ml, y se contaminaron con cultivos de Cándida parapsilosis, Rhodotorula glutinis, y Penicillium sp. de modo que alcanzaran niveles finales de 104-105 UFC/ml, y se incubaron a 37 0C, durante 24-48 h. Paralelamente, se analizaron controles en los que sólo se inoculó el hongo o levadura contaminante. A distintos tiempos se tomaron alícuotas y se evaluó comparativamente Ia viabilidad de los microorganismos contaminantes en presencia o ausencia de Ia bifidobacteria bioprotectora. Los recuentos de levaduras y hongos se realizaron en agar Rosa de Bengala (Scharlau, Barcelona, España) incubando en aerobiosis a 3O0C, durante 48-72 h. A fin de estudiar el efecto bioprotector de los compuestos antimicrobianos secretados al medio por las bifidobacterias durante su crecimiento en medios que constituyen modelos de alimentos como L y L-EL-G, 0,2 volúmenes de los extractos correspondientes, neutralizados y sin neutralizar, se adicionaron a Ia leche previamente inoculada con el microorganismo contaminante (Penicillium sp. 104 UFC/ml). Se incubó a 37 0C durante 48 h y se analizó Ia viabilidad a distintos tiempos mediante recuentos en placa. Paralelamente, se analizaron controles en los que se inoculó Ia bacteria contaminante y un volumen equivalente de medio no fermentado. 10% skim milk was used as a model of a food in which the selected strains could play a bioprotective role against yeasts and fungal yeasts or health risks. The previously sterilized milk was inoculated with the bioprotective bifidobacteria, so that they will reach levels of 10 7 CFU / ml, and were contaminated with cultures of Candida parapsilosis, Rhodotorula glutinis, and Penicillium sp. so that they reach final levels of April 10 -10 5 CFU / ml, and incubated at 37 0 C for 24-48 h. In parallel, controls were analyzed in which only the contaminating fungus or yeast was inoculated. At different times, aliquots were taken and the viability of the contaminating microorganisms in the presence or absence of the bioprotective bifidobacterium was evaluated comparatively. Yeast and fungus counts were performed on Rosa de Bengal agar (Scharlau, Barcelona, Spain) by incubating in aerobiosis at 3O 0 C, for 48-72 h. In order to study the bioprotective effect of the antimicrobial compounds secreted to the environment by bifidobacteria during their growth in media that constitute food models such as L and L-EL-G, 0.2 volumes of the corresponding extracts, neutralized and not neutralized, They were added to the milk previously inoculated with the contaminating microorganism (Penicillium sp. 10 4 CFU / ml). It incubated at 37 0 C for 48 h and the viability was analyzed at different times by plate counts. In parallel, controls were analyzed in which the contaminating bacteria were inoculated and an equivalent volume of unfermented medium.

Claims

REIVINDICACIONES 1. Compuestos con actividad antifúngica caracterizados porque: a) están producidos por el género Bifidobacterium, b) son componentes celulares y metabólicos de las bifidobacterias c) los componentes metabólicos son secretados al medio y obtenidos en forma de extractos. d) los componentes metabólicos son de naturaleza proteica y no proteica1. Compounds with antifungal activity characterized in that: a) they are produced by the genus Bifidobacterium, b) they are cellular and metabolic components of bifidobacteria c) the metabolic components are secreted into the medium and obtained in the form of extracts. d) the metabolic components are of a protein and non-protein nature 2. Compuestos con actividad antifúngica según las reivindicación 1 caracterizados porque los compuestos proteicos: a) tienen una masa molecular aparente menor de 100 kDa b) son termoestables c) presentan estabilidad y actividad en un intervalo de pH de 3-10 d) son sensibles a enzimas proteolíticas2. Compounds with antifungal activity according to claim 1 characterized in that the protein compounds: a) have an apparent molecular mass of less than 100 kDa b) are thermostable c) have stability and activity in a pH range of 3-10 d) are sensitive to proteolytic enzymes 3. Compuestos con actividad antifúngica según Ia reivindicación 1 caracterizados porque los compuestos no proteicos son activos a pH ácido.3. Compounds with antifungal activity according to claim 1 characterized in that the non-protein compounds are active at acidic pH. 4. Compuestos con actividad antifúngica según las reivindicaciones de Ia 1 a Ia 3 caracterizados porque son producidos por cepas de, entre otras, las especies, Bifidobacterium longum, B. infantis, B. catenulatum, B. pseudocatenulatum B. bifidum, B. breve, B. adolescentis, B. animalis/lactis y B. globosum.4. Compounds with antifungal activity according to claims 1 to 3, characterized in that they are produced by strains of, among others, the species, Bifidobacterium longum, B. infantis, B. catenulatum, B. pseudocatenulatum B. bifidum, B. breve , B. adolescentis, B. animalis / lactis and B. balloonsum. 5. Compuestos con actividad antifúngica según las reivindicaciones de Ia 1 a Ia 4 caracterizados porque están producidos por las cepas Bifidobacterium BIR-0304, BIR-0307, BIR-0312, BIR-0324, BIR-0326 y5. Compounds with antifungal activity according to claims 1 to 4, characterized in that they are produced by the Bifidobacterium BIR-0304, BIR-0307, BIR-0312, BIR-0324, BIR-0326 and BIR-0349.BIR-0349. 6. Uso de los compuestos según las reivindicaciones de Ia 1 a Ia 5 como agentes antifúngicos frente a hongos y levaduras alterantes y de interés fito- y zoosanitario. 6. Use of the compounds according to claims 1 to 5 as antifungal agents against fungi and yeasts altering and of phyto- and zoosanitary interest. 7. Uso de los compuestos según Ia reivindicación 6 como conservante biológico o fungicida en productos agroalimentarios frente a hongos y levaduras alterantes y patógenos de vegetales y animales. 7. Use of the compounds according to claim 6 as a biological preservative or fungicide in agri-food products against fungi and yeasts and plant and animal pathogens. 8. Uso de los compuestos según Ia reivindicación 6 como agente terapéutico frente a hongos y levaduras.8. Use of the compounds according to claim 6 as a therapeutic agent against fungi and yeasts. 9. Uso del extracto que contiene los compuestos de las reivindicaciones de Ia 1 a Ia 5 frente a hongos y levaduras alterantes y de interés fito- y zoosanitario.9. Use of the extract containing the compounds of claims 1 to 5 against fungi and yeasts altering and of phyto- and zoosanitary interest. 10. Uso del extracto según Ia reivindicación 9 como conservante biológico o fungicida en productos agroalimentarios frente a hongos y levaduras alterantes y patógenos de vegetales y animales.10. Use of the extract according to claim 9 as a biological preservative or fungicide in agri-food products against fungi and yeasts and plant and animal pathogens. 11. Uso del extracto según Ia reivindicación 9 como compuesto terapéutico frente a hongos y levaduras.11. Use of the extract according to claim 9 as a therapeutic compound against fungi and yeasts. 12. Uso de los microorganismos productores de los compuestos activos frente a hongos y levaduras de las reivindicaciones de Ia 1 a Ia 5 como agentes antifúngicos frente a hongos y levaduras alterantes y de interés fito- y zoosanitario. 12. Use of the microorganisms producing the active compounds against fungi and yeasts of claims 1 to 5 as antifungal agents against fungi and yeasts altering and of phyto- and zoosanitary interest. 13. Uso de los microorganismos productores de los compuestos según Ia reivindicación 12 como conservantes biológicos y fungicidas en productos agroalimentarios frente a hongos y levaduras alterantes y patógenos de vegetales y animales.13. Use of the microorganisms producing the compounds according to claim 12 as biological preservatives and fungicides in agro-food products against fungi and yeasts and plant and animal pathogens. 14. Uso de los microorganismos productores de los compuestos según Ia reivindicación 12 como probióticos frente a hongos y levaduras con fines médicos (profilácticos o terapéuticos). 14. Use of the microorganisms producing the compounds according to claim 12 as probiotics against fungi and yeasts for medical purposes (prophylactic or therapeutic).
PCT/ES2005/070174 2004-12-28 2005-12-07 Compounds with antifungal activity, which are produced by bifidobacterium spp Ceased WO2006070041A2 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111206004A (en) * 2020-03-10 2020-05-29 江南大学 Bifidobacterium longum and application thereof in inhibiting filamentous fungi
US20210345658A1 (en) * 2012-04-11 2021-11-11 R.J. Reynolds Tobacco Company Method for treating plants with probiotics
US20220022460A1 (en) * 2018-11-29 2022-01-27 Rhodia Operations Use of guar derivatives in biofungicide compositions

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210345658A1 (en) * 2012-04-11 2021-11-11 R.J. Reynolds Tobacco Company Method for treating plants with probiotics
US20220022460A1 (en) * 2018-11-29 2022-01-27 Rhodia Operations Use of guar derivatives in biofungicide compositions
CN111206004A (en) * 2020-03-10 2020-05-29 江南大学 Bifidobacterium longum and application thereof in inhibiting filamentous fungi

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