WO2006070041A2 - Composes a activite antifongique obtenus a partir de bifidobacterium spp - Google Patents

Composes a activite antifongique obtenus a partir de 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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PCT/ES2005/070174
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English (en)
Spanish (es)
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

L'invention concerne des composés cellulaires et métaboliques à activité antifongique obtenus à partir de souches du genre Bifidobacterium. Ces composés dérivés du métabolisme des souches du genre Bifidobacterium, obtenus sous forme d'extraits, contiennent des composés protéiques et non protéiques à activité antifongique. Lesdits composés obtenus à partir des bifidobactéries présentent un large spectre d'activité comprenant levures et champignons et, parmi ces derniers, d'importants producteurs de mycotoxines. Ces champignons et leurs produits métaboliques peuvent être utilisés en tant que bioprotecteurs, conservateurs et fongicides biologiques contre des levures et des champignons nuisibles et d'intérêt zoosanitaire ou phytosanitaire, dans des produits agroalimentaires et des formulations pharmaceutiques.
PCT/ES2005/070174 2004-12-28 2005-12-07 Composes a activite antifongique obtenus a partir de bifidobacterium spp Ceased WO2006070041A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111206004A (zh) * 2020-03-10 2020-05-29 江南大学 一株长双歧杆菌及其在抑制丝状真菌中的应用
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 (zh) * 2020-03-10 2020-05-29 江南大学 一株长双歧杆菌及其在抑制丝状真菌中的应用

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