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WO2017105267A1 - Nouvelle culture starter de probiotique pour usage humain et animal - Google Patents

Nouvelle culture starter de probiotique pour usage humain et animal Download PDF

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Publication number
WO2017105267A1
WO2017105267A1 PCT/RS2015/000027 RS2015000027W WO2017105267A1 WO 2017105267 A1 WO2017105267 A1 WO 2017105267A1 RS 2015000027 W RS2015000027 W RS 2015000027W WO 2017105267 A1 WO2017105267 A1 WO 2017105267A1
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WIPO (PCT)
Prior art keywords
lmg
probiotic
starter culture
lactobacillus
bacteria
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PCT/RS2015/000027
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English (en)
Inventor
Nataša GOLIĆ
Amarela TERZIĆ-VIDOJEVIĆ
Katarina VELJOVIĆ
Igor MRVALJEVIĆ
Original Assignee
Institute Of Molecular Genetics And Genetic Engineering, University Of Belgrade
Veterinarska Stanica "Koker" D.O.O.
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Application filed by Institute Of Molecular Genetics And Genetic Engineering, University Of Belgrade, Veterinarska Stanica "Koker" D.O.O. filed Critical Institute Of Molecular Genetics And Genetic Engineering, University Of Belgrade
Priority to TR2018/08116T priority Critical patent/TR201808116T1/tr
Publication of WO2017105267A1 publication Critical patent/WO2017105267A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the subject invention belongs to the field of biotechnology and is referring to the new probiotic starter culture with unique combination of three bacterial strains within genus Streptococcus thermophilus, Lactobacillus fermentum and Lactobacillus helveticus. Also, the use thereof in prevention and/or treatment of intestinal infections in mammals, namely humans and animals, and use thereof as an immunomodulator is described. Background of the invention
  • the technical problem that is solved by the subject invention can be defined as the provision of a new improved probiotic culture as an alternative to use of antibiotics in treatment of intestinal infections in humans and animals, whereby the improvement of overall health of the subject is achieved.
  • the probiotic starter culture of the subject invention is based on the optimal ratio of three unique natural isolates Streptococcus thermophilus LMG P-28585 (BGVL1-44), Lactobacillus fermentum LMG P-28583 (BGHI14) and Lactobacillus helveticus LMG P-24226 (BGRA43) characterized on the strain level by 16S rDNA sequencing and pulsefield gel electrophoresis, for which it has been proven to express proteolytic and antimicrobial activity, as well as an immunomodulatory effect.
  • BGVL1-44 Streptococcus thermophilus LMG P-28585
  • BGHI14 Lactobacillus fermentum LMG P-28583
  • BGRA43 Lactobacillus helveticus LMG P-24226
  • this starter culture for producing the probiotics is described and compositions intended for the treatment and/or prophylaxis of disorders associated with the colonization of pathogens in mammalian gastrointestinal tract, as well as for obtaining the probiotics and compositions for colonization of mammalian gastrointestinal tract after antibiotic therapy.
  • the subject invention is referring to the probiotics and the compositions based on this probiotic starter culture and to use thereof in prevention and/or treatment of intestinal infections in humans and animals, and use thereof as an immunomodulator in maintaining the native balance of the immune system and in the regulation of immune responses in humans and animals, including infants and cubs.
  • the subject invention is also referring to the combination of probiotics based on this probiotic starter culture and the use thereof as an immunomodulator in prevention and/or treatment of inflammatory bowel disease in humans and animals.
  • the subject invention is reffering to a new probiotic starter culture comprising three thermophylic natural isolates of lactic acid bacteria (LAB): Streptococcus thermophilus LMG P- 28585 (BGVL1-44), Lactobacillus fermentum LMG P-28583 (BGHI14) and Lactobacillus helveticus LMG P-24226 (BGRA43), in specific ratios, use thereof in prevention and/or treatment od intestinal infections in mammals and use thereof as an immunomodulator.
  • LAB thermophylic natural isolates of lactic acid bacteria
  • BGVL1-44 Streptococcus thermophilus LMG P- 28585
  • BGHI14 Lactobacillus fermentum LMG P-28583
  • BGRA43 Lactobacillus helveticus LMG P-24226
  • probiotic preparations based on the use of various LAB strains, Bacillus sp. and/or yeasts are well know in the prior art.
  • probiotics such as mono- or mixed cultures of live microorganisms, which when administered in adequate amounts confer a health benefit on the host (humans or animals), improving thereby the existing features of gut microbiota, are well known.
  • the probiotic bacteria have a number of beneficial effects, among others, in prevention and treatment of diarrhea, the beneficial effect on the immune system (probiotic microorganisms form an intestinal ecosystem that inhibits the growth of pathogenic and opportunistic bacteria (E. coli, Salmonella Typhimurium, Clostridium perfrigens, Clostridium difficile, Pseudonomas, Listeria, Staphylococcus aureus%) in prevention of candidiasis, etc.
  • pathogenic and opportunistic bacteria E. coli, Salmonella Typhimurium, Clostridium perfrigens, Clostridium difficile, P
  • compositions for treatment of intestinal infections in domestic animals are designed for feeding the new born animals (Method for correction of immunobiological indices with piglets in period of weaning - RU2471359 (C1)— 2013-01-10), prepared in the form of dry feed with addition of various active substances such as zeolite (Complex probiotic preparation for beef strains - RU2011153643 (A)— 2013-07-10), medicinal plants (Chinese herbal medicine composite probiotic feed additive for pigs and preparation method of additive - CN103549141 (A) — 2014-02-05), prebiotics (Feed additive for veterinary drug solid fermentation and preparation method of feed additive - CN103039700 (A) — 2013-04-17), vitamins (Pig feed capable of preventing piglets from diarrhoea and traditional Chinese medicine composition - CN 102835609 (A)— 2012-12-26) etc.
  • active substances such as zeolite (Complex probiotic preparation for beef strains - RU20111536
  • US patent US 5,656,268 describes the process for producing of biological product in liquid form or as cream, comprising pure milk or milk ingredients and the ferments or bacterial cultures of Lactobacillus fermentum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei and Streptococcus thermophilus species.
  • This invention can be considered as the closest prior art document since the biological product comprises milk and at least two of the bacteria as previously stated.
  • the product of defined acidity is obtained, which can further comprise an oil (olive oil and aromatic oil) as well as fruits, vegetables, plants or honey, and based on milk or whey that can be used as an additive for improving users' health, especially when in dry or sublimed form.
  • the product can be used as a cosmetic cream, cream and liquid for body and massage, as cream for gum treatment and in spa for taking baths.
  • the probiotic starter culture of the subject invention substantially differs from the stated patent, among other things, in that it is based entirely on optimal ratios of three clearly defined bacterial strains within the genus of Streptococcus thermophilus, Lactobacillus fermentum and
  • Lactobacillus helveticus stated in the subject invention namely: Streptococcus thermophilus LMG P-28585 (BGVLJ1 -44), Lactobacillus fermentum LMG P-28583 (BGHI14) and
  • Lactobacillus helveticus LMG P-24226 (BGRA43), that is prerequisite for use thereof in humans and animals.
  • the ratio of components in the biological product is not defined, only the process for obtaining the product, which is substantially different from the process for obtaining the probiotic starter culture of the subject invention.
  • probiotic starter culture expresses proteolytic and antimicrobial activity and immunomodulatory effect, compared to the prior art document wherein such activity is not shown in any way.
  • the probiotic starter culture of the subject invention does not contain any oils, nor other ingredients as in stated prior art document, and differs by the forms of use. It's well known that the probiotic characteristics are strain-dependent, not genus-dependent. Therefore, the combination of strains expresses its probiotic characteristics, and is supported by the description and as such can be used, compared to the product of the prior art document that refers to a species comprising broader (all strains), but biologically is absolutely inapplicable.
  • European patent EP1359816 relates to a combination of probiotics comprising lactobacilli, propionic acid bacteria and bifidobacteria.
  • the probiotics are preferably combined with a suitable prebiotic to produce a symbiotic.
  • the combination of the invention may be consumed as such or combined with a suitable foodstuff, such as a dairy product or a drink, and it is therapeutically useful for example for stimulating the immune system and for general health improvement.
  • the given combination is prepared in unit dosage form, precisely in the form of a capsule.
  • European patent EP1541673 relates to novel lactic acid bacteria and particularly microorganisms of the genera Lactobacillus, Bifidobacterium and Streptococcus (Enterococcus) that have been isolated and used thereof for preparing the composition for use in the preparation of pet food compositions intended to improve the health of pets (mainly canine and feline).
  • Given compositions are selected for the regulation of animal's immune response primarily for its ability to colonize the gastrointestinal tract of a pet and to exert a beneficial probiotic activity on pet health.
  • European patent EP2219658 relates to a composition based on probiotic bacteria and a substance having probiotic properties and the use thereof to prevent and/or treat respiratory pathologies and/or infections and simultaneously improve intestinal functionality.
  • a symbiotic composition comprising the following bacterial strains Lactobacillus plantarum (LMG P-21020), Lactobacillus plantarum (LMG P-21021 ), Lactobacillus rhamnosus (DSM 16605), Lactobacillus rhamnosus (DSM 19739), Bifidobacterium lactis (LMG P-21384) and prebiotic component comprising i.e. fructo-oligosaccharides or galacto-oligosaccharides.
  • European patent EP2101796 discloses the strain of microorganism Bacillus smithii TBMI12 MSCL P737, and the compositions comprising the said strain, compositions for use, as a probiotic for antibacterial purposes, for colonizing the gastrointestinal tract and for stimulating the immune system, as well as use of said strain as a food supplement and as a component in therapeutic and non-therapeutic probiotic composition.
  • European patent EP0856259 describes the composition, comprising lyophilized live active lactic acid bacteria in combination with oligosaccharides for use as food supplement, intended for balancing intestinal flora. Stated lyophilizate comprises the strains of genus Bifidobacterium, Lactobacillus, Streptococcus.
  • the invention is disclosing a kit comprising two containers, wherein one is holding a liquid, creamy or pasty foodstuff, and the other one is holding the stated composition for the purpose of supplementing said foodstuff with the composition held therein, at the moment of consuming the foodstuff.
  • the foodstuff in this case is a milk, a yoghurt, or another type of fermented milk, a milk-based dessert, a milk-based beverage or a beverage based on milk serum or milk permeate enriched with fruit, a fruit juice, a tomato juice, a carrot juice, a tea, or a beverage based on vegetable extracts.
  • Microorganisms ingested with food have a positive effect on human and animal health. While the use of probiotics for humans is extensive, the use of probiotics in livestock is negligible. The latest studies are intensively engaged in the development of probiotic preparations for animals, based on the use of lactic acid bacteria (LAB), Bacillus sp. or yeasts. Increased interest in use of probiotics in livestock has arisen from tight European regulations related to use of the antibiotics in livestock production. Therefore, probiotics are considered as an appropriate alternative especially in terms of intestinal infections in animals, especially cubs. Establishing good microflora by colonisation with the LAB in the intestines of newborn animals immediately after birth results in healthier animals.
  • LAB lactic acid bacteria
  • Fermented dairy products with probiotic bacteria are products obtained by fermentation of the milk by probiotic starter culture activity or by combining the starter cultures with other lactic acid bacteria. Detailed description of the invention
  • the subject invention is specifically related to the new probiotic starter culture for human and animal use, the technological process for preparation thereof and use of this probiotic culture in prevention and/or treatment of intestinal infections of various ethyology in humans and animals as well as use thereof as an immunomodulator.
  • the basic unit of product is the probiotic culture, which represents a unique combination of bacteria with specific probiotic potential, for prevention and/or treatment of intestinal infections and other gut immunological diseases in humans and animals.
  • Probiotic culture is particularly effective in therapy of intestinal infections of various ethyology and reduces the number of pathogenic bacteria with unique wide range of antimicrobial activity.
  • Probiotic culture exhibits exceptional probiotic potential, has the ability of immunomodulation as well as specific antimicrobial effect against large number of bacterial strains.
  • the new probiotic culture represents an alternative to use of antibiotics in treatment of intestinal infections, based on wide range of antimicrobial activity, especially for severe hardly curable infections e.g. infections caused by Clostridium perfrigens and Clostridium difficille, and thereby the improvement of overall health of humans and animals is achieved.
  • the subject invention is related to a new probiotic starter culture comprising three thermophilic LAB natural isolates: Streptococcus thermophilus LMG P-28585, Lactobacillus fermentum LMG P-28583 and Lactobacillus helveticus LMG P-24226 in a specific ratio that provides desired effects, use thereof for producing probiotics and compositions intended for prevention and/or treatment of disorders related to colonisation of mammalian gastrointestinal tract by pathogenic microorganisms and have an immunomodulator effect.
  • probiotic starter culture of the subject invention comprises three natural thermophilic LAB isolates: Streptococcus thermophilus LMG P-28585, Lactobacillus fermentum LMG P-28583 and Lactobacillus helveticus LMG P-24226 in optimal ratio. Specific ratio thereof is the key factor of effect of this starter culture in prevention and/or treatment of intestinal infections in mammals, both in humans and animals, and will be described in details below.
  • thermophilus LMG P-28585 from 10% to 40%
  • Lactobacillus fermentum LMG P-28583 from 10% to 50%
  • Lactobacillus helveticus LMG P-24226 from 40% to 80%. Results showed that in all combinations tested, strains expressed probiotic potential.
  • the preffered ratio od strains Streptococcus thermophilus LMG P-28585, Lactobacillus fermentum LMG P-28583 and Lactobacillus helveticus LMG P-24226 is 10%: 10%: 80% (1 :1 :8) or 10%: 20%: 70% (1 :2:7). This ratio provides larger number of
  • Ratio of Streptococcus thermophilus LMG P-28585, Lactobacillus fermentum LMG P-28583 and Lactobacillus helveticus LMG P-24226 of 40%:20%:40% (4:2:4) has a potential use in the immunocompromised group of patients, since in this combination the strain present in least amount is Lactobacillus fermentum LMG P-28583, which expresses mild proinflammatory activity.
  • probiotic starter culture comprises Streptococcus thermophilus LMG P- 28585, Lactobacillus fermentum LMG P-28583 and Lactobacillus helveticus LMG P-24226 in the ratio of 30%: 40%: 30% (3:4:3).
  • the most present strain is Lactobacillus fermentum LMG P-28583 that by its characteristics bursts immune response.
  • the subject starter culture shows exceptional outcome in the treatment of infections caused by bacteria Clostridium perfringens and Clostridium difficile, as well as by families of Enterobacteriaceae, when the numbers of live bacteria are in the following ranges: Streptococcus thermophilus LMG P-28585 from 6x10 7 to 1.25x10 11 , Lactobacillus fermentum LMG P-28583 from 1 .1 x10 8 to 1 .25x10 11 and Lactobacillus helveticus LMG P-24226 from 4x10 6 to 2.5x10 9 .
  • probiotics from the prior art are intended for feeding of the cubs, wherein this probiotic culture (due to safe status of lactic acid bacteria) in one embodiment is designated for gravid animals wherein cubs receive the passive immunity from their mothers and are therefore already immunised during their first days of life. Also, during the last 15 days of gravidity, the immunity in animals lowers and the number of enterobacterias in mother's intestines increases, and immediately after the birth they are transmitted to offspring that becomes infected.
  • probiotic starter culture comprising Streptococcus thermophilus LMG P- 28585, Lactobacillus fermentum LMG P-28583 and Lactobacillus helveticus LMG P-24226 wherein the range of number of live bacteria for Streptococcus thermophilus LMG P-28585 is from 4x10 9 to 8x10 10 , for Lactobacillus fermentum LMG P-28583 is from 3x10 9 to 1 x10 10 and for Lactobacillus helveticus LMG P-24226 is from 4x10 6 to 9x10 8 CFU/ml.
  • the new probiotic culture of the subject invention was tested in farm trials and was proven to be extremely effective in the treatment of intestinal infections of various ethyology, and specifically successfully reduced (below detection level) the number of Clostridium perfringens, as well as Enterobacteriaceae. All previously described combinations can be prepared in the form of a fermented dairy product, suspension, lyofilisate, premix, spray-dry, tablets, capsules and other forms that are know to a person skilled in the art and as such are for human and animal use. All these forms comprise probiotic starter culture of the subject invention obtained by known methods.
  • Method for preparation of probiotic starter culture comprises: 1 ) the cultivation of natural isolates included in the composition of a probiotic starter culture under optimal conditions for growth of these bacteria; 2) combination of natural isolates that form a part of a probiotic starter culture in an optimal ratio. Due to the presence of strain Streptococcus thermophilus LMG P-28585 in the probiotic culture, the one can be also used for the preparation of a fermented dairy product that can be used for prevention of intestinal infections in mammals including humans and animals (pigs, canine, feline, etc.).
  • Method for preparation of a fermented probiotic product for human and animal use which includes the milk fermentation process by use of a defined starter and probiotic bacteria, that includes the following stages: 1 ) Pre-cultivation of natural isolates that form a probiotic starter culture under the optimal conditions for growth of these bacteria; 2) Milk is inoculated with natural isolates prepared in stage 1 , in optimal ratio; 3) Incubation of milk inoculated in stage 2 under optimal conditions for fermentation.
  • the obtained fermented product is stable, the number of live probiotic bacteria is reproducible and the technology of producing it is easily scaled up. Moreover, such product exhibits better probiotic effect probably due to presence of specific bioactive peptides incurred by degradation of milk protein by proteolytic enzyme activity of probiotic bacteria.
  • probiotic culture in other mammals ruminants
  • other animals ruminants
  • probiotic culture prepared without milk as previously described.
  • FIG. Genotyping of the strains included in the probiotic composition by pulse- field gel electrophoresis (PFGE).
  • PFGE pulse- field gel electrophoresis
  • probiotic combination (synergism) on Clostridium difficile. 1. Control C.
  • Figure 4 Survival of strain LMG P-28583 in simulated conditions of the gastrointestinal tract in a saline buffer and in the dairy matrix.
  • Figure 5 Survival of strain LMG P-24226 in simulated conditions of the gastrointestinal tract in a saline buffer and in the dairy matrix.
  • LMG P-28585, LMG P-28583 and LMG P-24226 The values given in the graph represent mean values of 3 measurements and are expressed in percentages. Statistically significant values compared to the control are marked with asterisks ( * p ⁇ 0.5, ** p ⁇ 0.1 ).
  • FIG. 8 Adhesion of Salmonella 654 7E (veterinary isolate) in the presence and the absence of strains LMG P-28585, LMG P-28583 and LMG P-24226.
  • the values given in the graph represent mean values of 3 measurements and are expressed in percentages.
  • Statistically significant values compared to the control are marked with asterisks ( * p ⁇ 0.5, ** p ⁇ 0.1 ).
  • the values given in the graph represent the mean values of 4 measurements and are expressed in percentages. Statistically significant values compared to the control are marked with asterisks ( * p ⁇ 0.5, ** p ⁇ 0.1 ).
  • Enterobacteriaceae family significantly decreases after the probiotic treatment of animals, while the number of same bacteria in
  • perfringens genus is reduced below the detection treshold after the probiotic treatment of animals
  • the probiotic starter culture is composed of three thermophilic LAB natural isolates Streptococcus thermophilus BGVLJ1 -44 (natural isolate from an artisanal yogurt produced from uncooked cow's milk, the strain from laboratory collection of Laboratory of Molecular Microbiology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade - LMM-IMGGE), Lactobacillus fermentum BGHI14 (natural isolate from the newborn feces, the strain from laboratory collection of LMM-IMGGE) and Lactobacillus helveticus BGRA43 (natural isolate from the human gastrointestinal tract, from the laboratory strain collection of LMM- IMGGE).
  • the strains were isolated by classical microbiological methods described previously (Terzic Vidojevic et al., 2007). For the cultivation of BGVLJ 1 -44 liquid and solid M17 medium (Merck GmbH, Darmstadt, Germany), supplemented with 0.5% glucose (GM17) was used, while for the cultivation of strains BGHI14 and BGRA43 liquid and solid MRS medium (Merck, GmbH) was used. The cultures were stored at -80°C in a suitable growth medium (GM17 or MRS supplemented with 15% glycerol) in cryovials.
  • GM17 or MRS supplemented with 15% glycerol 15% glycerol
  • strain BGRA43 was deposited on July 31 , 2007 by Ljubisa Topisirovic, the Head of Laboratory for Molecular Genetics of Industrial Microorganisms (former name of Laboratory for Molecular Microbiology), IMGGE, as the depositor.
  • Strains BGVLJ1 -44 and BGHI14 were deposited on October 28, 2014 by Milan Kojic, the Head of the Laboratory of Molecular Microbiology, IMGGE, as the depositor.
  • the strain Streptococcus thermophilus BGVLJ1 -44 was deposited under LMG P-28585 number, the strain Lactobacillus fermentum BGHI14 under LMG P-28583 number, and the strain Lactobacillus helveticus BGRA43 has a deposit number LMG P-24226.
  • the strain Lactobacillus helveticus BGRA43 was deposited on 31/07/2007; the strain assigned number is LMG P-24226.
  • the depositor is Ljubisa Topisirovic, the head of Laboratory for Molecular Genetics of Industrial Microorganisms (former name of Laboratory for Molecular Microbiology), IMGGE, Belgrade, Serbia.
  • the strain Streptococcus thermophilus BGVLJ1 -44 was deposited under the number LMG P- 28585, on 28/10/2014.
  • the depositor is Dr. Milan Kojic, the Head of Laboratory of Molecular Microbiology, IMGGE, Belgrade, Serbia.
  • strain Lactobacillus fermentum BGHI14 was deposited under the number LMG P-28583, on 28/10/2014.
  • the depositor is Dr. Milan Kojic, the Head of Laboratory of Molecular
  • proteolytic activity of strains Streptococcus thermophilus LMG P- 28585, Lactobacillus helveticus LMG P-24226 and Lactobacillus fermentum LMG P-28583 was tested as previously described (Kojic et al., 1991 ). The ability of ⁇ -, ⁇ - and ⁇ -casein hydrolysis was tested.
  • Antimicrobial activity was tested by the deferred antagonism method using overnight cultures of Streptococcus thermophilus LMG P-28585, Lactobacillus helveticus LMG P-24226 and Lactobacillus fermentum LMG P-28583 and various indicator strains. Briefly, soft GM17 and MRS agars (0.7% w/v) containing indicator strains were overlaid onto GM17 and MRS plates, respectively. The plates were incubated overnight at 37°C. A clear zone of inhibition of indicator strain growth around the well was taken as a positive signal for production of antimicrobial compound.
  • anaerobic bacteria Clostridium sp.
  • antimicrobial activity was tested as follows: the glass tubes were filled with overnight culture of C. difficile or C. sporogenes (1 ml), followed by a soft agar of growth medium for C. difficile, and on top of it with the soft GM17 containing 200 ⁇ of overnight culture of Streptococcus thermophilus LMG P-28585, or MRS with 200 ⁇ of overnight cultures of Lactobacillus helveticus LMG P-24226 or Lactobacillus fermentum LMG P- 28583 or 200 ⁇ of mixed probiotic culture.
  • Production of gas by Clostridium sporogenes and Clostridium difficile manifested in the form of cracking agar, represents the signal for the growth of Clostridium sporogenes and Clostridium difficile and absence of antimicrobial activity.
  • the antimicrobial activity of probiotic strains on Clostridium sporogenes and Clostridium difficile is scored as the absence of gas production, i.e. agar remains intact.
  • L. helveticus LMG P-24226 exhibits antimicrobial activity at various strains of the genera Lactococcus sp. and Lactobacillus sp., Bacillus subtilis ATCC8, Bacillus cereus ATCC1 1778, Bacillus mycoides, as well as the pathogenic bacteria, Staphylococcus aureus ATCC25923, Staphylococcus sp.
  • the strains of Streptococcus thermophilus LMG P-28585 and Lactobacillus fermentum LMG P-28583 exhibited weak antimicrobial activity on Clostridium difficile, while probiotic combination shows a synergistic effect and exhibits the strongest antimicrobial effect on Clostridium sporogenes and Clostridium difficile ( Figure 2). Antibiotic resistance
  • strains LMG P-24226, LMG P-28583 and LMG P-28585 are sensitive to the recommended levels of antibiotics on the basis of the recommendations of EFSA (2012).
  • Adhesion to epithelial intestinal cell (EIC) lines is an essential prerequisite for the probiotic strain showing positive effects on the host's health. A higher percentage of adhesion enables better antagonistic activity against enteropatogens by competing for the same binding sites at EIC (Coconniere et al., 1993). On the other hand, the strain that shows a greater degree of adhesion to EIC retains longer in the gastrointestinal tract and makes better contact with the EIC stimulating the immune system of the host (Schiffrin et al., 1995).
  • GALT Gut Associated Lymphoid Tissue
  • the effect of the probiotic combination (including strains LMG P-28585, LMG P-28583 and LMG P-24226) on the health status of farm animals was monitored in vivo in farm trial.
  • 25 were treated with fermented dairy probiotic product prepared as given above for 7-15 days before farrowing (at a time when the natural immunity of pregnant sows usually declines).
  • Pregnant sows have been received the probiotic every day during the first morning feeding.
  • 25 control sows, not treated with the probiotic were followed.
  • Probiotic starter culture of the subject invention will, according to ratios thereof of bacterial isolates in probiotic culture, exert its proteolytic activity, antimicrobial and/or imunomodulatory activity. Therefore, according to needs and intended purposes this probiotic starter culture find its human and animal use in prophylaxis and/or treatment of intestinal pathologies of various ethyologies and as an immunomodulator.
  • This probiotic starter culture can be prepared as such and in forms suitable for target group of mammals, namely humans or animals. Special use of the probiotic starter culture is as food supplement for humans and animals.
  • the probiotic starter culture can be prepared as standard pharmaceutical forms that find the veterinary use, but not limited to, fermented probiotic dairy product, lyofilisate, premix, granules, spray-dry (sprinkled over the food), powders that can be mixed with water or food, suspensions, tablets, capsules and other forms known to the person skilled in art.
  • the probiotic starter culture can be used preferably as forage additive, and especially preferable as spray dry.
  • the probiotic starter culture/probiotic/composition of the subject invention comprising three thermophylic natural bacterial isolates, as previously described, can be prepared in the form of fermented probiotic product, lyophilisate, powder, oral suspension, pastilles and chewable tablets, tablets, capsules and other forms known to the person skilled in art.

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Abstract

La présente invention concerne une nouvelle culture de probiotique starter qui est composé de trois isolats naturels de bactéries lactiques thermophiles (LAB) : Streptococcus thermophilus LMG P- 28585 (BGVLJ1-44), Lactobacillus fermentum LMG P-28583 (BGHI14) et Lactobacillus helveticus LMG P-24226 (BGRA43), dans des rapports spécifiques. La culture starter de la présente invention présente une activité protéolytique et antimicrobienne, ainsi qu'un effet immunomodulateur. L'invention concerne en outre l'utilisation de celle-ci dans la prévention et/ou le traitement d'infections intestinales chez des mammifères, à savoir des humains et des animaux et en tant qu'immunomodulateur.
PCT/RS2015/000027 2015-12-17 2015-12-22 Nouvelle culture starter de probiotique pour usage humain et animal WO2017105267A1 (fr)

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WO2019118843A1 (fr) * 2017-12-14 2019-06-20 Pure Cultures, Inc. Probiotiques et métabolites de fermentation pour la prévention et le traitement d'états pathologiques chez des animaux
WO2021133877A1 (fr) * 2019-12-24 2021-07-01 Iowa State University Research Foundation, Inc. Régulation de l'histamine pour favoriser la santé et contrôler l'entérocolite à l'aide de compositions probiotiques et/ou d'enzymes dégradant l'histamine
CN113197312A (zh) * 2020-07-14 2021-08-03 内蒙古蒙牛乳业（集团）股份有限公司 嗜热链球菌mn002在免疫调节产品中的用途及膳食补充剂

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019118843A1 (fr) * 2017-12-14 2019-06-20 Pure Cultures, Inc. Probiotiques et métabolites de fermentation pour la prévention et le traitement d'états pathologiques chez des animaux
US10758577B2 (en) 2017-12-14 2020-09-01 Pure Cultures 2020, Inc. Probiotics and fermentation metabolites for the prevention and treatment of disease conditions in animals
CN112020308A (zh) * 2017-12-14 2020-12-01 纯文化2020股份有限公司 用于预防和治疗动物病症的益生菌和发酵代谢物
US12257275B2 (en) 2017-12-14 2025-03-25 Pure Cultures, Inc. Probiotics and fermentation metabolites for the prevention and treatment of disease conditions in animals
WO2021133877A1 (fr) * 2019-12-24 2021-07-01 Iowa State University Research Foundation, Inc. Régulation de l'histamine pour favoriser la santé et contrôler l'entérocolite à l'aide de compositions probiotiques et/ou d'enzymes dégradant l'histamine
CN113197312A (zh) * 2020-07-14 2021-08-03 内蒙古蒙牛乳业（集团）股份有限公司 嗜热链球菌mn002在免疫调节产品中的用途及膳食补充剂

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