WO2015140299A1 - Probiotiques naso-oro-pharyngés - Google Patents
Probiotiques naso-oro-pharyngés Download PDFInfo
- Publication number
- WO2015140299A1 WO2015140299A1 PCT/EP2015/055930 EP2015055930W WO2015140299A1 WO 2015140299 A1 WO2015140299 A1 WO 2015140299A1 EP 2015055930 W EP2015055930 W EP 2015055930W WO 2015140299 A1 WO2015140299 A1 WO 2015140299A1
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- WIPO (PCT)
- Prior art keywords
- lactobacillus
- oronasopharyngeal
- spray
- composition
- rhamnosus
- Prior art date
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
Definitions
- the present invention is directed to a novel probiotic oronasopharyngeal formulation of spray- dried bacteria comprising saccharides as protectants, for administration in the oronasopharyngeal cavity. Resuscitation of the formulation of the present invention gives rise to rapid deployable and viable microorganisms with preserved antimicrobial activity, and was found particularly useful for nasopharyngeal administration to treat respiratory conditions in humans and animals.
- the present invention is directed to the probiotic microorganisms used in the aforementioned formulation.
- spray-dried compositions comprising Lactobacillus strains, in particular comprising Lactobacillus rhamnosus proved optimal in the oronasopharyngeal applications of the present invention.
- the application also provides the use of Lactobacillus strains as anti-pathogenic and antibiofilm agents, in particular against the common nasopharyngeal pathogens (whereby produced acids such as lactic acid are important antimicrobial factors). Also disclosed are the processes associated with the manufacture of the spray-dried probiotic formulations of the present invention, as well as the use thereof in treating or preventing of infections of the oronasopharyngeal cavity.
- microbiota The human body is occupied by a vast number of microorganisms, which are collectively called microbiota. They inhabit the skin, oronasopharyngeal cavity, genital tract and gastrointestinal tract. The microbiota present in each of the niches provide to the host a vast number of health effects, including inhibition of pathogenic colonization, stimulation of barrier functions and promotion of immune regulation. Interest in the beneficial functions of the human microbiota has boomed within the last ten years, thanks to major advances in next generation sequencing technologies and so called 'metagenomic studies' (Qin et al., 2010; Arumugam et al., 201 1 ). In addition, the application of probiotics has increased significantly during the last decades.
- Probiotics are defined as microorganisms, which, upon application in adequate amounts, can provide a health benefit to the host (FAO/WHO, 2001 ).
- FAO/WHO a health benefit to the host
- probiotic applications have focused on the gastrointestinal tract, because of the ease to implement them in fermented foods, yogurts and oral/food supplements. Lactobacillus and Bifidobacterium are most commonly applied. For example:
- WO2006007526 relates to combinations of Bifidobacteria and Lactobacillus strains for use in the prevention and treatment of respiratory tract disorders such as acute otitis media in infants, by supplementing infant formulations with such combinations;
- US201 10020304 provides nutritional compositions comprising Lactobacillus sp and the use of such preparation in the prevention and treatment of pathogenic infections of the gastro-intestinal and upper respiratory tracts;
- US20120201798 relates to dietary supplements comprising probiotic Lactobacillus strains for the treatment or prevention of a respiratory infection
- EP2455092 relates to food compositions comprising non-replicating probiotic microorganisms for use in the prevention or treatment of upper respiratory tract infections
- Hojsak et al., 2010 relates to fermented milk products comprising Lactobacillus GG for use in the prevention of gastrointestinal and respiratory tract infections in children who attend day care centers;
- Various disorders involve a dysbalance of the microbiota of the nasopharyngeal cavity, such as rhinosinusitis (Abreu et al., 2012), pharyngitis, adenoiditis and tonsillitis (Swidsinksi et al., 2007; Jensen et al., 2013), and even middle ear infections (Revai et al., 2008).
- antibiotics are often applied, while they have many side effects such as accumulation of resistant microbes and disturbance of the symbiotic microbes.
- references all relate to the use of oral formulations or non-viable bacterial species or spores thereof, thereby aiming at improving the overall immune responses, and as such having an indirect beneficial effect on the oronasopharyngeal health.
- the inventors have now in contrast found that the oronasopharyngeal compositions of the present invention, when administered directly to the oronasopharyngeal cavity, have a direct effect on the oronasopharyngeal health by restoring or maintaining the natural microbiota of the oronasopharyngeal cavity, due to direct antimicrobial activity against the most common pathogenic microorganisms and recolonization of the cavity with beneficial bacteria.
- the present invention provides Lactobacillus probiotic strains with a strong activity against the common nasopharyngeal pathogens and their application in the treatment and prevention of infections of the oronasopharyngeal cavity.
- the present invention provides oronasopharyngeal formulations and the manufacture thereof.
- the present invention is based on the finding that certain viable Lactobacillus spp. strains, in particular Lactobacillus rhamnosus, Lactobacillus plantarum/pentosus and Lactobacillus casei, and their secreted products such as lactic acid, show strong growth inhibitory activity against the common nasopharyngeal pathogens.
- the present invention provides the use of said Lactobacillus spp. in the treatment and prevention of infections of the oronasopharyngeal cavity.
- the present invention provides an oronasopharyngeal composition comprising viable, spray-dried Lactobacillus species, for use in the prevention and/or treatment of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity.
- the Lactobacillus spp. strains identified in the present application in having growth inhibitory activity against nasopharyngeal pathogens are selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus salivahus, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus diolivorans, Lactobacillus buchneri, Lactobacillus gasseri, and Lactobacillus bulgaricus; more in particular Lactobacillus rhamnosus.
- the present invention accordingly provides;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus rhamnosus for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus casei for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus reuteri for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity; or
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus plantarum for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus helveticus for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus parabuchneri for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus pentosus for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus salivarius for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus brevis for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity;
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus buchneh for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus sakei for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus diolivorans for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus gasseri for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus bulgahcus for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity
- An oronasopharyngeal composition comprising combinations of said viable spray-dried Lactobacillus spp., for use in the treatment or prevention of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity.
- the present invention also provides the oronasopharyngeal compositions as defined hereinabove, wherein the Lactobacillus strains are replaced or complemented with Lactobacillus secreted products such as lactic acids.
- the present invention provides an oronasopharyngeal composition comprising viable, spray-dried Lactobacillus rhamnosus for use in the treatment or prevention of infections of the oronasopharyngeal cavity.
- the other probiotic bacteria preferably consist of Lactobacillus spp. and lactic acid bacteria; more in particular selected from the group comprising Lactobacillus casei, L. plantarum, L. paracasei, L. salivarius, L. sakei, L. pentosus, L. reuteri, L. brevis, L. parabuchneri, L. buchneri. L. sakei, L. diolivorans, L. bulgaricus, L.
- Lactobacillus casei Lactobacillus plantarum, Lactobacillus salivahus, Lactobacillus brevis and Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus sakei, Lactobacillus buchneri, Lactobacillus diolivorans, Lactobacillus bulgaricus, Lactobacillus gasseri and Lactobacillus pentosus.
- GRAS generally recognized as safe
- QPS presumption of safety
- the present invention provides an oronasopharyngeal composition
- viable, spray-dried Lactobacillus casei for use in the treatment or prevention of infections of the oronasopharyngeal cavity.
- other probiotic bacteria preferably consist of further Lactobacillus spp.; more in particular selected from the group comprising Lactobacillus rhamnosus, L. plantarum, L. paracasei, L. salivarius, L. sakei, L. pentosus, L. reuteri, L. brevis, L. parabuchneri, L. buchneri. L. sakei, L. diolivorans, L. bulgaricus, L.
- gasseri and other Lactobacillus species with a GRAS generally recognized as safe
- QPS presumption of safety
- Lactobacillus rhamnosus L. plantarum
- Lactobacillus reuteri Lactobacillus helveticus
- Lactobacillus parabuchneri L. salivarius
- L. sakei L. brevis
- L. buchneri L. parabuchneri
- L. bulgaricus L. gasseri and Lactobacillus pentosus.
- the present invention provides an oronasopharyngeal composition
- viable, spray-dried Lactobacillus reuteri for use in the treatment or prevention of infections of the oronasopharyngeal cavity.
- other probiotic bacteria preferably consist of further Lactobacillus spp.; more in particular selected from the group comprising Lactobacillus casei, L. plantarum, L. paracasei, L. salivarius, L. sakei, L. pentosus, L. rhamnosus, Lactobacillus helveticus, Lactobacillus parabuchneri, L. brevis, L. buchneri, L. bulgaricus, L.
- gasseri and other Lactobacillus species with a GRAS generally recognized as safe
- QPS presumption of safety
- the present invention is based on the identification of Lactobacillus species having growth inhibitory activity against common nasopharyngeal pathogens.
- the common nasopharyngeal pathogens inhibited by the aforementioned Lactobacillus species consists of the group comprising Moraxella catarrhalis, Haemophilus influenzae, and Corynebacterium tuberculostearicum, Corynebacterium accolens Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus (including MRSA).
- the present invention provides an oronasopharyngeal composition comprising viable, spray-dried Lactobacillus spp. strains in each of the aforementioned embodiments for use in the treatment or prevention of infections of the oronasopharyngeal cavity caused by an infection comprising one or more of the pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus pneumonia, Staphylococcus aureus (including MRSA), Corynebacterium tuberculostearicum and Corynebacterium accolens.
- the pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus pneumonia, Staphylococcus aureus (including MRSA), Corynebacterium tuberculostearicum and Corynebacterium accolens.
- the present invention provides an oronasopharyngeal composition comprising viable, spray-dried Lactobacillus spp. strains in each of the aforementioned embodiments for use in the treatment or prevention of infections of the oronasopharyngeal cavity caused by an infection comprising one or more of the pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Corynebacterium tuberculostearicum, Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus (including MRSA) and Corynebacterium accolens.
- the pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Corynebacterium tuberculostearicum, Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus (including MRSA) and Corynebacterium accolens.
- the Lactobacillus species of the present invention were found particularly useful in the treatment of upper respiratory tract infections.
- said upper respiratory tract infections include acute otitis media, pharyngitis, chronic sinusitis, acute sinusitis, rhinitis, oral mucositis and the like.
- the Lactobacillus species of the present invention were found to have an antiinflammatory activity, and to have a healing effect for mucosal lesions of the oronasopharyngeal cavity. Consequently, in a further aspect the present invention provides an oronasopharyngeal composition comprising viable, spray-dried Lactobacillus species in each of its embodiments, for use in;
- upper respiratory tract infections wherein said upper respiratory tract infections are selected from the group comprising acute otitis media, pharyngitis, chronic sinusitis, acute sinusitis, rhinitis, oral mucositis and the like;
- infections are caused by an infection comprising one or more of the pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Corynebacterium tuberculostearicum, Corynebacterium accolens, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus (including MRSA);
- pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Corynebacterium tuberculostearicum, Corynebacterium accolens, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus (including MRSA);
- upper respiratory tract infections are selected from the group comprising acute otitis media, pharyngitis, chronic sinusitis, acute sinusitis, rhinitis, oral mucositis and the like;
- a composition more in particular an oronasopharyngeal composition comprising an effective amount of the Lactobacillus spp. strains of the present invention.
- Possible embodiments for this method of treatment include;
- Lactobacillus rhamnosus strains are meant to include Lactobacillus rhamnosus AMB-GG, Lactobacillus rhamnosus AMB-GR-1 or a combination thereof;
- Lactobacillus casei strains are meant to include Lactobacillus casei AMB- 334, Lactobacillus casei AMB-Sh, Lactobacillus casei AMB-Act, natural isolates of Lactobacillus casei or a combination thereof.
- Lactobacillus plantarum strains are meant to include Lactobacillus plantarum AMB-8014, natural isolates of Lactobacillus plantarum or a combination thereof.
- Lactobacillus pentosus strains are meant to include L. pentosus AMB-pent1 , natural isolates of Lactobacillus pentosus or a combination thereof.
- Lactobacillus helveticus strains are meant to include Lactobacillus helveticus AMB-1807, natural isolates of Lactobacillus helveticus or a combination thereof.
- Lactobacillus reuteri strains are meant to include Lactobacillus reuteri AMB-RC-14, natural isolates of Lactobacillus helveticus or a combination thereof.
- Lactobacillus parabuchneri strains are meant to include Lactobacillus parabuchneri AMB- AB17, natural isolates or a combination thereof.
- Lactobacillus salivahus strains are meant to include natural isolates or a combination thereof.
- Lactobacillus brevis strains are meant to include natural isolates or a combination thereof.
- Lactobacillus sakei strains are meant to include natural isolates or a combination thereof
- Lactobacillus diolivorans strains are meant to include natural isolates or a combination thereof
- Lactobacillus buchneh strains are meant to include natural isolates or a combination thereof
- Lactobacillus gasseri strains are meant to include natural isolates or a combination thereof
- Lactobacillus bulgaricus strains are meant to include natural isolates or a combination thereof
- said method comprising administering into the oronasopharyngeal cavity of the subject to be treated, a composition comprising an effective amount of Lactobacillus rhamnosus.
- Lactobacillus spp. are in particular selected from the group comprising Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus salivahus, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus diolivorans, Lactobacillus buchneh, Lactobacillus gasseri, Lactobacillus bulgaricus and other Lactobacillus species with a GRAS (generally recognized as safe) or QPS (qualified presumption of safety) status; even more in particular selected from the group consisting of Lactobacillus casei, Lactobacillus rhamnosus Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus pentosus, Lactobacillus helveti
- each of the embodiments mentioned in relation to the use of the Lactobacillus spp. in the treatment and prevention of infections of the oronasopharyngeal cavity are equally applicable in the methods for the prevention or treatment of infections of the oronasopharyngeal cavity, and accordingly considered in being disclosed herein.
- the method of treatments are equally applicable in;
- upper respiratory tract infections are selected from the group comprising acute otitis media, pharyngitis, chronic sinusitis, acute sinusitis, rhinitis, oral mucositis and the like;
- infections are caused by an infection comprising one or more of the pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, and Corynebactehum tuberculosteahcum, Corynebactehum accolens, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus (including MRSA) ;
- pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, and Corynebactehum tuberculosteahcum, Corynebactehum accolens, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus (including MRSA) ;
- upper respiratory tract infections are selected from the group comprising acute otitis media, pharyngitis, chronic sinusitis, acute sinusitis, rhinitis, oral mucositis and the like;
- pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, and Corynebactehum tuberculosteahcum, Corynebactehum accolens, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus (including MRSA) .
- an effective amount of bacteria equals at least 1 .10 5 CFU ; in particular from about and between 1 .10 5 to 1 .10 9 CFU per day.
- Administering said amount can be done in any form known to the skilled artisan and suitable for administration to the oronasopharyngeal cavity, such as nasal sprays, buccal tablets, buccal sprays, aerosols and throat lozenges, ear drops and the like. It is accordingly a further aspect of the present invention to provide pharmaceutical compositions for use in the treatment or prevention of infections of the oronasopharyngeal cavity, comprising the Lactobacillus spp. in any one of the different embodiments as described herein.
- the Lactobacillus spp. used in said formulations are Lactobacillus spp. spray-dried in the presence of mono- and/or disaccharides as protectants.
- Stable and dry powders of the Lactobacillus spp. were obtained by spray-drying the bacteria using a saccharide protectant selected from the group consisting of glucose, mannose, mannitol, dextran, lactose, trehalose, or combinations thereof.
- the saccharide protectant used includes trehalose.
- the mono- and/or disaccharide protectant is added to the bacteria in a ratio of and between 1 :1 to 1 :7 (bacteria:saccharide); in particular 1 :2 to 1 :5; more in particular 1 :3 to 1 :4; even more particular 1 :2 to 1 :6.
- Viable spray-dried Lactobacillus species more in particular Lactobacillus rhamnosus for use in the oronasopharyngeal treatment of infections of the oronasopharyngeal cavity.
- Lactobacillus rhamnosus is selected from Lactobacillus rhamnosus AMB-GG, Lactobacillus rhamnosus AMB-GR-1 or a combination thereof. 3. Use according to statement 1 , wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus AMB-GG.
- Lactobacillus spp. are selected from the group comprising Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivahus, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus diolivorans, Lactobacillus buchneri, Lactobacillus gasseri, and Lactobacillus bulgahcus.
- infections of the oronasopharyngeal cavity are caused by one or more of the pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Corynebactehum tuberculostearicum, Corynebactehum accolens, Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus (including MRSA).
- pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Corynebactehum tuberculostearicum, Corynebactehum accolens, Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus (including MRSA).
- the upper respiratory tract infections are selected from the group consisting of acute otitis media, pharyngitis, chronic sinusitis, rhinitis, oral mucositis and the like.
- oronasopharyngeal formulation comprises at least 1 .10 5 CFU of Lactobacillus species, as defined in any one of statements 1 to 3, or at least
- a method for the prevention or treatment of infections of the oronasopharyngeal cavity comprising administering into the oronasopharyngeal cavity of the subject to be treated, a composition comprising an effective amount of Lactobacillus species. .
- an effective amount of Lactobacillus species comprises at least 1 .10 5 CFU of Lactobacillus species; in particular from about and between
- Lactobacillus species are selected from Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus salivahus, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus diolivorans, Lactobacillus buchneri, Lactobacillus gasseri, and Lactobacillus bulgahcus; more in particular Lactobacillus rhamnosus.
- Lactobacillus rhamnosus is selected from Lactobacillus rhamnosus AMB-GG, Lactobacillus rhamnosus AMB-GR-1 , natural isolates or a combination thereof.
- a method for the prevention or treatment of infections of the oronasopharyngeal cavity comprising administering into the oronasopharyngeal cavity of the subject to be treated a composition comprising an effective amount of Lactobacillus species combinations as defined in any one of statements 4 to 6.
- an effective amount of the Lactobacillus species combinations as defined in any one of statements 4 to 6, comprises at least 1 .10 5 CFU of bacteria; in particular from about and between 1 .10 6 to 1 .10 9 CFU of bacteria.
- the mono- and/or disaccharides are selected from the group comprising glucose, mannose, mannitol, dextran, lactose and trehalose.
- Lactobacillus species is Lactobacillus rhamnosus; more in particular selected from Lactobacillus rhamnosus AMB-GG, Lactobacillus rhamnosus AMB-GR-1 , natural isolates or a combination thereof
- composition further comprises spray-dried probiotic bacteria selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus salivahus, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus diolivorans, Lactobacillus buchneri, Lactobacillus gasseri, and Lactobacillus bulgaricus.
- probiotic bacteria selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus salivahus, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus diolivorans, Lactobacillus buchner
- composition is selected from nasal sprays, buccal tablets, buccal sprays, aerosols, throat lozenges, ear drops and the like
- An oronasopharyngeal composition comprising viable, spray-dried Lactobacillus species, for use in the prevention and/or treatment of infections of the oronasopharyngeal cavity, by administration of said composition in said cavity.
- compositions for use according to statement 1 wherein said composition is selected from the list comprising nasal sprays, buccal tablets, buccal sprays, aerosols, throat lozenges and ear drops.
- composition for use according to statement 3 wherein said Lactobacillus rhamnosus is selected from Lactobacillus rhamnosus GG, Lactobacillus rhamnosus GR-1 or a combination thereof.
- Lactobacillus spp selected from the list comprising Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus salivarius, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacill
- pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Staphylococcus aureus (including MRSA), Streptococcus pneumoniae, Streptococcus pyogenes, Corynebacterium tuberculostearicum and Corynebacterium accolens.
- composition for use according to anyone of statements 1 to 8, wherein the oronasopharyngeal composition comprises at least 1 .10 5 CFU of Lactobacillus species; in particular from about and between 1 .10 6 to 1 .10 9 CFU of said Lactobacillus species or Lactobacillus species combinations.
- a method for the prevention and/or treatment of infections of the oronasopharyngeal cavity comprising administering a composition as defined in any one of statements 1 to 8, into the oronasopharyngeal cavity of a subject in need thereof.
- Lactobacillus rhamnosus is selected from Lactobacillus rhamnosus GG, Lactobacillus rhamnosus GR-1 or a combination thereof.
- the composition further comprises other viable spray-dried probiotic bacteria; in particular Lactobacillus spp selected from the list comprising Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus salivahus, Lactobacillus helveticus, Lactobacillus parabuchneri, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus diolivorans, Lactobacillus buchneh, Lactobacillus gasseri, and Lactobacillus bulgahcus.
- the Lactobacillus strains may be replaced or complemented with Lactobacillus secreted products such as lactic acids.
- Fig. 1 Time course analysis of the antipathogenic activity of spent culture supernatant and secreted products of different Lactobacillus strains.
- Fig. 2 Antibiofilm capacity of the spent culture supernatant and secreted products of different Lactobacillus strains against Moraxella catarrhalis
- Fig. 3 Antipathogenic activity of (lactic) acid in spent culture supernatant of different Lactobacillus strains based on (A) time course analysis of spent culture supernatant of L. rhamnosus AMB-GG against Moraxella catarrhalis, (B) antibiofilm activity of spent culture supernatant of L. rhamnosus AMB-GG against Moraxella catarrhalis
- Fig. 4 Effect of different saccharides on survival after spray-drying.
- the saccharides were added in a ratio of 1 :1 .
- the content of the vials was 200 mg, this was dissolved in 10 mL MRS medium for resuscitation before plating on agar.
- Fig. 5 Viability test of L. rhamnosus AMB-GG spray-dried after adding saccharides - lactose (A), trehalose (B), dextran (C) and mannitol (D) - in different concentrations (1 :1 proportion, 1 :2 proportion, 1 :5 proportion) to the bacterial suspension.
- Fig. 6 (A) Comparison of the 1 :5 proportion of lactose, trehalose and dextran to the bacterial suspension. The viability was calculated after resuscitation in 10 ml MRS and incubation at 37°C for 3 days; (B) Addition of lactose or mannitol to the growth medium of L. rhamnosus AMB-GG. After an overnight incubation at 37°C a PBS-feed suspension was made, which was spray-dried.
- Fig 7 Cumulative effect of the addition of mannitol - the saccharide with the best result for the growth medium, and trehalose - the saccharide with the best result for the bacterial (feed) suspension just before spray-drying.
- Fig 9 (A-B): Growth curves (A) and viability (B) of L. rhamnosus AMB-GG after tablet compression, comparing samples without addition of trehalose and those with addition of 2 parts trehalose
- Fig 10 Viability test after resuscitation in different conditions.
- MRS deMan, Rogosa and Sharpe broth
- PBS phosphate buffered saline
- GLC glucose
- RT room temperature.
- Fig 11 Water activity of spray-dried powder as such, with addition of 5 parts trehalose and with addition of 5 parts lactose.
- Fig 12 Viability of L. rhamnosus AMB-GG after short term storage at 4°C. Comparison between as such spray-dried powder and with addition of different concentrations of trehalose and lactose. Results are given for 0 days and 40 days of storage.
- Fig 13 Viability of L. rhamnosus AMB-GG after long term storage (1 year) at 4°C. Comparison between as such spray-dried powder and with addition of different 2 parts trehalose.
- Fig. 14 Mean inhibition zones of spray-dried Lactobacillus after a resuscitation step of 0,5 h in competition with Moraxella catarrhalis.
- LGG susp LGG spray-dried as such;
- LGG susp TRH LGG spray-dried after adding trehalose based on the spot antipathogenic activity.
- the present invention is based on the discovery that certain viable species, i.e. Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus parabuchneri and Lactobacillus reuteri, of the genus of Lactobacillus, and their secreted products in the supernatant, are particularly useful in the treatment and or prevention of infections of the nasopharyngeal cavity.
- all strains for the aforementioned species are meant in being suitable for the therapeutic applications of the present invention.
- Preferred strains are selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus reuteri and Lactobacillus plantarum.
- the known isolates are included within the context of the present invention and meant in being suitable for the therapeutic applications of the present invention.
- the Lactobacillus isolates used are selected from the group consisting of L. rhamnosus, L. casei, L. plantarum, L. reuteri.
- the Lactobacillus rhamnosus species used in the different embodiments of the present invention is L. rhamnosus AMB-GG and L. rhamnosus AMB-GR-1 .
- the Lactobacillus casei strain used in the different embodiments of the present invention is L. casei AMB- 334 and natural isolates.
- Lactobacillus spp. requires isolating said bacteria from eventual growth culture media.
- the skilled artisan is well aware of the techniques available for isolating the viable bacteria from a growth culture media such as centrifugation, filtration, micro manipulation, and the like.
- the isolated bacteria are preferably maintained in a dry state, such as for example achieved using freeze drying or spray- drying.
- Lactobacillus spp. obtained by spray-drying the bacteria using a saccharide protectant, such as for example glucose, mannose, mannitol, dextran, lactose or trehalose; in particular trehalose.
- a saccharide protectant such as for example glucose, mannose, mannitol, dextran, lactose or trehalose; in particular trehalose.
- An exemplary process suitable for spray-drying the bacteria of the present invention is for instance available from Sunny-Roberts and Knorr (lnt. Diary J., 19 (2009) 209-2014).
- the Lactobacillus spp. of the present invention can be prepared by any known or otherwise effective method for pharmaceutically formulating or manufacturing the selected product form. Methods for preparing the pharmaceutical compositions according to the present invention can be found in "Remington ' s Pharmaceutical Sciences", Mid. Publishing Co., Easton, Pa., USA.
- the compositions comprising the spray-dried bacteria and the saccharide protectants as defined herein can be formulated along with common excipients, diluents, or carriers, and formed into oral tablets, capsules, sprays, mouth washes, lozenges, treated substrates (e. g.
- oral or topical swabs, pads, or disposable, non-digestible substrate treated with the compositions of the present invention ; oral liquids (e. g. , suspensions, solutions, emulsions), powders, or any other suitable dosage form; in as long as said formulation does not interfere with the viability of the spray-dried bacteria.
- Non-limiting examples of suitable excipients, diluents, and carriers can be found in "Handbook of Pharmaceutical Excipients", Second edition, American Pharmaceutical Association, 1994 and include: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as acetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; carriers such as propylene glycol and ethyl alcohol, and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
- fillers and extenders such as starch
- EXAMPLE 1 IDENTIFICATION OF THE ANTI PATHOGENIC ACTIVITY AIM OF EXPERIMENT
- a streak-line based assay and spot-based antipathogenic assay were performed to investigate the anti-pathogenic activity of different living Lactobacillus species against common nasopharyngeal pathogens, while a radial diffusion antipathogenic assay, a time course analysis of the antipathogenic activity and an antibiofilm assay were performed to investigate the antipathogenic activity of spent culture supernatant and thus the secreted products of different Lactobacillus strains.
- Corynebacterium ATCC35529 Tryptic Soy broth Tryptic Soy agar tuberculostearicum
- Streptococcus ATCC49619 Tryptic Soy broth + 5% Chocolate agar / pneumoniae * blood Blood agar
- Lactobacillus single colony isolate obtained in our lab from a stock culture of AMB- casei ATCC334 ATCC334
- Lactobacillus single colony isolate obtained in our lab from a commercially AMB-Sh1 casei available fermented drink containing L. casei Shirota (Yakult®)
- Lactobacillus single colony isolate obtained in our lab from a commercially AMB-Act1 casei available fermented drink (Actimel®) containing L. casei DN- 1 14001
- Lactobacillus Single colony isolate from spontaneously fermented carrot AMB-MCJ casei juice
- Lactobacillus Single colony isolate from spontaneously fermented beet juice AMB-AB17 parabuchneri
- Lactobacillus Single colony isolate from spontaneously fermented carrot AMB-NM63- parabuchneri juice 3
- Lactobacillus single colony isolate obtained in our lab from a stock culture of AMB-pM plantarum CMPG5300
- Lactobacillus single colony isolate obtained in our lab from a commercially AMB-RC4 reuteri available probiotic supplement containing L. reuteri RC4
- Lactobacillus single colony isolate obtained in our lab from a commercially AMB-GR-1 rhamnosus available probiotic supplement containing L. rhamnosus GR-1
- Lactobacilli were streak inoculated from a colony on a starter plate (MRS) on a test plate (medium of pathogen + glucose is needed) and incubated at 37°C for 3 nights. Then, the pathogens were streak inoculated from a colony on a starter plate on the test plates in 3 repetitions. The plates were incubated at 37°C for 24h and the inhibition zone was measured.
- MRS starter plate
- test plate medium of pathogen + glucose is needed
- Moraxella catarrhalis ATCC25238, Staphylococcus aureus (3 strains: ATCC29213, methicillin resistant (MRSA) and methicillin sensitive (MSSA)) and Corynebacterium tuberculostearicum are stored in a glycerol stock at -80°C. Staphylococcus aureus (ATCC29213, methicillin resistant (MRSA) and methicillin sensitive (MSSA)) and M.
- catarrhalis bacteria were grown on a Mueller Hinton (MH) plates.A colony was inoculated in 5 mL MH broth and incubated for 24 h, then 2% culture was added to MH soft agar (0.5 % agar) and poured over the plates with Lactobacillus spots. The plates were incubated overnight at 37°C.
- C. tuberculostearicum bacteria were grown on a Tryptic soy (TS) plate, a colony was inoculated in 5 mL TS broth and incubated for 24 h, then 2% culture was added to MH soft agar (0.5 % agar) and poured over the plates with Lactobacillus spots. The plates were incubated overnight at 37°C. 0.1 % hexetidine was added to the spot plate before the soft agar was poured as a positive control.
- a pathogen suspension is made in the appropriate medium and incubated for 1 night at 37°C.
- the CFU is calculated by plating different dilutions of starter suspension on an appropriate plate. Different dilutions of the pathogen are incubated on the plate by adding 25 mL of (not too hot) agar to a different volume of suspension. The plates are dried and wholes are made (diameter 0.4 cm, height 0.5 cm) punched in the agar. Supernatant of lactobacilli, positive and negative control are added to the holes (30 ⁇ ). When the test material was fully diffused into the agar, an overlay of 20 mL agar was poured over the agar plate and the plate was incubated for 24h at 37°C.
- Inhibition zones were measured afterwards. 0.1 % hexetidine was used as a positive control. Sterile MRS with pH 4,3 was used as a negative control. Supernatant was made by incubating overnight lactobacilli in MRS broth. SN was obtained by centrifugation for 30 min. at 6797 g (8000 rpm) at 4°C. The SN was then filter sterilized (0.20 ⁇ cellulose acetate, VWR) to remove remaining cells and the pH was measured.
- Every well of the 100-well plate contained a volume of 300 ⁇ test material (mostly 240 ⁇ culture + 60 ⁇ L test material or sterile dH 2 0).
- As a negative control different dilutions (240 ⁇ L culture + 60 ⁇ L sterile dH 2 0) of a culture of Moraxella catarrhalis ( ⁇ 10 8 CFU/mL) was tested: 10 "2 until 10 " 5 .
- MH broth and demineralized water were tested.
- the positive controls were different concentrations of hexetidine (final concentrations: 0,01 %; 0,005% and 0,001 %) and lactic acid (final concentrations: 100 mM; 50 mM; 25 mM; 2,5 mM and 0,25 mM).
- the concentration of the supernatant tested was 1 /20.
- MRS shows inhibition to the growth of the pathogen. Bacteria were grown, and the optical density at 600 nm was measured automatically after shaking each 20 min during 168 h.
- Biofilm formation of the pathogens in coculture with SN was as previously described by Pearson et al. (2006).
- M. catarrhalis was inoculated into 5 mL BH I broth. This culture was incubated overnight at 37°C ( ⁇ 10 5 CFU/mL) and diluted 1 :100 in BHI. A volume of 190 ⁇ of this suspension was loaded into a 96-well microplate and incubated at 37°C for 19 h.
- 10 ⁇ portions of SN were loaded, in 8 repetitions, into the 96- well microplate. The broth was then removed from each well and replaced by 200 ⁇ PBS plus 10 ⁇ of 0.7% (wt/vol) crystal violet.
- the supernatant was prepared as explained above.
- the supernatant samples of the different lactobacilli were also treated by different methods and tested for antipathogenic activity as described above: (i) supernatant of lactobacilli was first heated at 1 10°C for 60 min and then treated with proteinase K and incubated for 60 min at 37°C (ii) SN of lactobacilli brought at pH 7 (iii) dialysis (cut-off 1000 Da) against a hepes-citrate-tis buffer (60-40- 20 mM) pH 4,3 was done to remove metabolites of the lactobacilli (such as lactic acid and other produced metabolic by products) that are smaller than this size.
- the positive and negative controls were 0,1 % hexetidin and MRS at pH 4,3 respectively. The latter was done to check whether the effect of the supernatant was not only caused by the low pH (due to addition of HCI) but by a specific, secreted molecule in the supernatant.
- the supernatant of lactobacilli after growth for 16 h-24 h to stationary phase has generally a mean pH of 4,3.
- Table 3 Mean inhibition zones of different Lactobacillus species in competition with pathogenic species
- HI Haemophilus influenzae
- MC Moraxella catarrhalis ATCC25238
- SA Staphylococcus aureus ATCC29213
- SPY Streptococcus pyogenes BM137.
- Streak inoculation tests showed a clear potential of the antipathogenic activity of lactobacilli against different pathogens.
- a strong activity is observed against H. influenzae, M. catarrhalis and S. pyogenes for all lactobacilli tested, highlighting that the observed activities are Lactobacillus genus-specific, but species- and strain-independent.
- a smaller activity is observed against S. aureus, but this activity is consistent.
- L. rhamnosus AMB-GG showed the strongest activity against all pathogens tested.
- MC Moraxella catarrhalis
- SA Staphylococcus aureus
- MRSA methicillin resistant Staphylococcus aureus
- MSSA methicillin sensitive Staphylococcus aureus
- CT Corynebacterium tuberculostearicum.
- lactobacilli that show less antimicrobial activity, also grow less fast compared to e.g. L. rhamnosus AMB-GG and therefore have produced less (lactic) acid or other antimicrobial molecules.
- lactobacilli tested also showed activity (with inhibition zones up to 0.2 cm) against Corynebactehum tuberculosteahcum ATCC35529, Streptococcus pyogenes BM137 and Haemophilus influenzae. In contrast, no Lactobacillus strains tested showed activity in this test against at least one of the S. aureus strains tested.
- Table 5 Mean inhibition zones of spent culture supernatant and secreted products of different Lactobacillus species in competition with pathogenic species
- CT Corynebacterium tuberculostearicum ATCC35529
- HI Haemophilus influenzae
- MC Moraxella catarrhalis ATCC25238
- SPN Streptococcus pneumoniae ATCC49619
- SPY Streptococcus pyogenes BM137
- FIG 1 the time course analysis of the antimicrobial activity of spent culture supernatant and secreted products of different lactobacilli is provided. Addition of different dilutions of the Lactobacillus supernatant samples to M. catarrhalis cultures was investigated. At a dilution of 1/20, a strain-specific effect of the inhibition of the growth of Moraxella by the Lactobacillus supernatant samples was observed. Addition of the supernatant of the L. rhamnosus AMB-GG, L. case; AM B-Sh, L. casei AMB-Act and L. plantarum AMB-pl1 inhibited the growth of Moraxella for a whole week (168 h). Addition of supernatant of L.
- casei AMB-ATCC334 and L. reuteri AMB- RC4 only showed a small delay of the growth of Moraxella. However, after 20h the pathogen can grow normal and even reaches a higher OD than the control. L. rhamnosus AMB-GR-1 can inhibit the growth of Moraxella longer but after ⁇ 30h Moraxella starts to grow and also reaches a higher OD than the control.
- Both L. rhamnosus AMB-GG, L. rhamnosus AMB-GR-1 , L. reuteri AMB-RC4, L. casei AMB-Act and L. casei AMB-Sh can reduce the biofilm formation of M. catarrhalis by up to 70%.
- L. rhamnosus AMB-pl1 and L. casei ATCC334 also have potential to reduce biofilm formation although their activity is more variable.
- a nasopharyngeal formulation should be stable, show fast activity in nasopharyngeal cavity (while intestinal applications have more time) and should preserve the antimicrobial activity and adhesion properties of the lactobacilli, so that they are able to temporarily colonize the nasopharynx.
- Step 1 Bacterial culture
- Lactobacillus suspension 500ml
- PBS PBS
- the spray-drying process was performed on a Buchi B-290 spray-dryer with a two-fluid nozzle type, having a 1 .5 mm nozzle size, using an inlet temperature of 120 °C, an outlet temperature of 56°C, a spray rate or feed flow (pump %) of 10 to 16%, an aspiration rate % (air flow) of about 80%, and a pressure-flow (mm flow) of 45 mm.
- a spray rate or feed flow pump % of 10 to 16%
- an aspiration rate % air flow
- mm flow pressure-flow
- Spray rate (pump %) 10% wherein 1 % pump rate corresponds with a flow rate of approximately 0.3 ml/min.
- a pre-conditioning step was performed with distilled water using the set process parameters until the outlet temperature was stabilized (approximately 15-20 min).
- the feed tube was changed to the feed suspension which was stirred continuously using a magnetic stirrer to obtain a homogeneous sample throughout the process.
- the feed suspension was spray-dried completely, the feed tube was changed back towards the recipient with distilled water.
- the cyclone is cleaned with water to avoid any contamination of the previous spray-dried sample.
- the Buchi spray-dryer was cleaned with distilled water and soap.
- the probiotic suspension is sprayed into a warm airstream after which the drying droplets form powder particles in fraction of seconds. At the end of the dryer, the powder particles are collected via a cyclone or bag filter.
- the powder obtained from these aliquots was delivered in vials containing 100 mg and 200 mg for the spray-dried L. rhamnosus AMB-GG as such and the spray-dried powders with saccharides added, respectively.
- the powder was then resuscitated for 30 minutes in MRS broth at 37°C. All ratios were 1 :1 .
- the spray-drying parameters used to test the saccharide protectants were as described above.
- the saccharides to be tested were lactose monohydrate, trehalose dihydrate, mannose, glucose, mannitol and dextran.
- Each saccharide was added in a 1 :1 , 1 :2 or 1 :5 proportion (L rhamnosus AMB-GG: saccharide) based on the solid content of the feed suspension.
- the saccharides were added to an aliquot of the bacterial suspension. After the addition of the saccharide, the sample was first stirred with a magnetic stirrer to obtain a homogeneous feed suspension for spray- drying and a homogenous powder.
- Lactobacilli appear often in chains of varying length.
- the spray-drying process was found to affect the chain length of L. rhamnosus AMB-GG.
- Spray-dried powder as such and with addition of trehalose was evaluated for their viability using the commercial available LIVE/DEAD BacLight Viability Kit (Molecular Probes, Paisley, UK).
- PBS phosphate buffer
- the coloring agents are added to the samples, which are then incubated for 15 minutes at room temperature. These samples can then be seen through a epifluorescence microscope. Cells colored red equals dead bacteria and cells colored green equals viable bacterial cells. Step 3: Test viability bacteria
- Incubator Memmert INB200 Spray-dried powder is stored at a certain temperature depending on the test. 10 mL MRS or PBS is added to the samples. The samples are incubated for 5 min, 30 min or 3h at 37°C, to resuscitate the bacteria. After resuscitation, the culture is mixed using the vortex to make it as homogeneous as possible. Then, dilutions are made by pipetting 100 ⁇ _ of the culture in 900 ⁇ _ PBS. Then, again 100 ⁇ _ of this sample is transferred to 900 ⁇ _ PBS after pipetting up and down for at least 10 times to make the culture as homogeneous as possible.
- 100 ⁇ _ of the preferred dilution is inoculated on an MRS-plates in duplo or triple
- the bacteria are spread on the plate using glass beads.
- the plates are incubated at 37°C for 72h and the colonies are counted for each plate. Then, the CFU (colony forming units) and the survival are calculated.
- Step 4 Test recommended resuscitation time
- Spray-dried powder is stored at 4°C. 10 mL 0,85% NaCI is added to the samples. The samples are incubated for 5, 15 and 30 minutes at room temperature, to resuscitate the bacteria. After resuscitation, different dilutions of the suspension are plated as explained in step 3. After 3 nights incubation at 37°C, the CFU is calculated and compared to different resuscitation times.
- Step 5 Test water activity of the spray-dried powder
- the water activity is defined as the volume of free, unbound water in the product that is available for metabolic activities of the bacteria. Waste metabolites from these activities may lead to spoilage of the product and thus shorter shelf-life. Water activity measurements were carried out with a water-activity meter (Lab-Swift-aw, Novasina). Approximately 2g of the spray-dried powder is placed in a measurement chamber and the mode is set to S (slow), which allows a more accurate water activity measurement.
- Step 6 Test stability of the spray-dried powder
- the spray-dried powder- is stored at 4°C and at room temperature. After certain periods of time, the viability of the powder is measured as explained in step 3. After 3 nights of incubation at 37°C, the CFU is calculated and compared to different periods of storage.
- the 1 :5 proportion of trehalose increases the viability of L. rhamnosus AMB-GG 14 times.
- the 1 :5 proportion of lactose and dextran enhances the viability 8 and 4 times respectively .
- Figure 5A-D gives an overview of the results of addition of the saccharides to the bacterial (feed) suspensions just before spray-drying. As stated before addition of 5 parts trehalose to the bacterial suspension just before spray-drying gives the best viability results.
- Figure 6B show the results of the addition of lactose and mannitol to the growth medium of L. rhamnosus. After an overnight incubation at 37°C a PBS-feed suspension was made, which was spray-dried. Addition of mannitol to the growth medium gives the highest survivability of the bacteria after spray-drying, compared to addition of lactose or no addition of a saccharide at all.
- Figure 7 represents the cumulative effect of the addition of mannitol -the saccharide with the best result for the growth medium, and trehalose-the saccharide with the best result for the bacterial (feed) suspension just before spray-drying.
- the cumulative effect of both added saccharides enhances the viability in comparison to no addition of saccharides at all or to addition of mannitol to the growth medium.
- addition of solely trehalose to the bacterial suspension just before spray-drying increases the viability of L. rhamnosus AMB-GG the most.
- FIG. 8A-C represents the samples without addition of trehalose and those with addition of 2 parts trehalose regarding their viability after tablet compression. The samples without trehalose showed no or retarded growth (grey lines on graph). Samples spray-dried with addition of 2 parts trehalose still showed good viability (black lines on graph).
- control sample (bacterial suspension before spray-drying) shows more living, bacterial cells than the spray-dried samples (data not shown).
- the chain length of the control sample is also clearly longer than these of the spray-dried ones, with an average of 10 cells per chain to an average of 3-4 cell per chain for the spray-dried samples.
- the spray-dried samples with addition of trehalose shows more viable cells, than the as such spray-died sample, which is in conclusion with previous findings (data not shown).
- L. rhamnosus AMB-GG was stored as such and with addition of 2 parts trehalose at two storage conditions, 4°C and room temperature. After 1 year the viability of the stored samples was calculated, using the plate-count method as explained before. The samples, as such as well as 1 :2 trehalose, stored at room temperature for 1 year showed a viability less than 10 7 CFU/100mg. The samples stored at 4°C had a viability of more than 10 8 CFU/100mg. The sample as such stored at 4°C showed a 1 log reduction, the sample stored at 4°C with addition of 2 parts trehalose showed no viability loss after 1 year of storage, as can be seen in figure 13. 5. Maintenance of the anti-pathogenic activity
- Negative control no bacteria With reference to figure 14 and table 10, spray-dried bacteria do not lose their antipathogenic activity.
- the inhibition zones against Moraxella observed for L. rhamnosus AMB-GG spray-dried as such are comparable with the positive control.
- L. rhamnosus AMB-GG spray-dried after adding trehalose shows even higher inhibition zones compared to the positive control.
- Even bacteria that were spray-dried with 7x trehalose 1 ,5 years ago and stored at room temperature still showed antipathogenic activity against M.
- MC Moraxella catarrhalis
- SA Staphylococcus aureus
- MRSA methicillin resistant Staphylococcus aureus
- MSSA methicillin sensitive Staphylococcus aureus
- CT Corynebacterium tuberculostearicum
- SPN Streptococcus pneumoniae
- SPY Streptococcus pyogenes.
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Abstract
La présente invention concerne une nouvelle formule probiotique naso-oro-pharyngée de bactéries séchées par atomisation comprenant des saccharides en tant qu'agents protecteurs, pour administration dans la cavité naso-oro-pharyngée. La reconstitution de la formule selon la présente invention permet d'obtenir des micro-organismes rapidement déployables et viables conservant une activité antimicrobienne, et il a été découvert qu'elle était particulièrement utile pour l'administration naso-pharyngée dans le traitement des états pathologiques respiratoires chez l'humain et l'animal. Dans un aspect supplémentaire, la présente invention concerne les micro-organismes probiotiques utilisés dans la formule susmentionnée. Comme mis en évidence dans les exemples ci-après, les compositions séchées par atomisation comprenant des souches de Lactobacillus, en particulier comprenant Lactobacillus rhamnosus, se sont avérées optimales dans les applications naso-oro-pharyngées de la présente invention. La demande concerne également l'utilisation de souches de Lactobacillus en tant qu'agents antipathogènes et antibiofilm, en particulier contre les agents pathogènes naso-pharyngés communs (les acides produits tels que l'acide lactique étant dans ce contexte d'importants facteurs antimicrobiens). La présente invention concerne également les procédés associés à la fabrication des formules probiotiques séchées par atomisation selon la présente invention, ainsi que l'utilisation de celles-ci dans le traitement prophylactique ou thérapeutique d'infections de la cavité naso-oro-pharyngée.
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| WO2018158306A1 (fr) * | 2017-02-28 | 2018-09-07 | Alimentary Health Limited | Bifidobacterium longum pouvant moduler avantageusement une réponse immunitaire à une infection à virus respiratoire |
| WO2018158309A1 (fr) * | 2017-02-28 | 2018-09-07 | Alimentary Health Limited | Bifidobacterium longum apte à moduler de manière bénéfique une réponse immunitaire à l'encontre d'une infection virale respiratoire |
| CN110121353A (zh) * | 2016-06-21 | 2019-08-13 | Yun股份有限公司 | 用于维持和/或恢复健康皮肤微生物群的皮肤科制剂 |
| EP3470075A4 (fr) * | 2016-06-13 | 2020-04-15 | Murata Manufacturing Co., Ltd. | Médicament antimicrobien et antiviral, élément antimicrobien et antiviral, et procédé de production de médicament antimicrobien et antiviral |
| US20220143104A1 (en) * | 2018-12-21 | 2022-05-12 | Universiteit Antwerpen | Novel dolosigranulum pigrum strains and uses thereof |
| CN116024120A (zh) * | 2022-09-09 | 2023-04-28 | 青岛蔚蓝生物股份有限公司 | 一株具有抑制呼吸道感染致病菌功效的鼠李糖乳酪杆菌及其应用 |
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