Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3571—Microorganisms; Enzymes
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/40—Viruses, e.g. bacteriophages
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
  • A23B4/00—General methods for preserving meat, sausages, fish or fish products
  • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
  • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
  • A23B4/20—Organic compounds; Microorganisms; Enzymes
  • A23B4/22—Microorganisms; Enzymes; Antibiotics
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/34635—Antibiotics
• • 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
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • 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
  • A61P31/04—Antibacterial agents
• • 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
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • 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
  • C12N2795/00—Bacteriophages
  • C12N2795/00011—Details
  • C12N2795/10011—Details dsDNA Bacteriophages
  • C12N2795/10032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• • 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
  • C12N2795/00—Bacteriophages
  • C12N2795/00011—Details
  • C12N2795/10011—Details dsDNA Bacteriophages
  • C12N2795/10111—Myoviridae
  • C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• • 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 present invention relates to a bacteriophage cocktail for eliminating Listeria monocytogenes and Escherichia coli 0157:H7 to be used in food, beverage, food additive, food supplement, feed, antibiotics and disinfectant contents.
• Bacteriophages were defined as viruses eating bacteria in early 1900 for the first time. Bacteriophages that are in great amounts (10 3 °-10 32 ) and diversity were isolated from various sources such as sea water, fresh water, soil, gastrointestinal and Genito-urinary tract of animals and humans, leather and milk. Bacteriophages, of which extracellular form (virion) is nonviable and of which sizes are under 1 gm (that can pass through Millipore filters of 0,22 gm that bacteria cannot pass) comprises DNA or RNA in a protein shell (capsid). Most of them comprise a tail functioning to transmit the genetic material to the bacteria cells.
• bacteriophages Compared to antibacterial agents such as antibiotics and antiseptics, what makes bacteriophages different and functional is that it can be implemented without damaging any living tissues because of the highly selective toxicity.
• Two types of bacteriophages with a very high host capacity (a species or a few species in the same genus) were presented.
• One of those is the type that is known as temperate bacteriophages and stays in the prophage form subsequent to transmitting its genetic material to the bacteria and multiplies as the bacteria multiplies and exhibits lysogenic cycle.
• the latter type is phages that are called as lytic type (virulent to bacteria), transmits its genetic material holding on to the bacteria, multiplies in the bacteria and leaves and lyses the bacteria approximately in 20-60 minutes and infects new bacteria.
• Antibiotics are used in the treatment of diseases caused by bacterial factors. Intense and wrong use of antibiotics both on humans and in veterinaries causes that bacteria develop a rapid and very powerful resistance to antibiotics, and new antibiotics are not developed, resulting in limiting treatment opportunity. Furthermore, this is interpreted nowadays that the post-antibiotic period has started.
• Another disadvantage of antibiotics are allergic reactions developing depending on the increase in the number of sensitive individuals. Since antibiotics are not host-specific like bacteriophages subject of the present invention, they destroy also microbiota for a certain extent along with the target bacteria in livings, on which it is to be applied, in other words, they may kill beneficial and critical microorganisms for livings along with harmful ones. Separate phage preparations for L. monocytogenes and E.
• coli 0157:H7 developed in this area may be found in some countries, excluding Turkey.
• said preparation are not combined as mentioned above, and they only affect on a single factor.
• their effects on local strains in Turkey may be limited.
• phages used around the world can be used for a single microorganism.
• a phage preparation that affects on two different pathogen bacteria simultaneously has not been used so far.
• cocktails-wise mixtures, in which more than one phage are used in different matrices such as disinfectant, feed and food, are not used commercially.
• the inventive product is a cocktail product in which phages against both factors exist together, wherein there is no similar preparation around the world.
• the present product is the first domestic and national phage.
• every bacteriophage is characteristic, wherein every novel bacteriophage is a novel patent subject.
• the bacteriophages used in this cocktail are new phages isolated within the scope of the present invention.
• lytic phages were included by FDA (Food and Drug Administration) in the class GRAS (Generally Recognize as Safe). Therefore, there is use of the bacteriophages with its specified purposes around the world. As mentioned above, the present phage preparations few in number are specific to a single type of bacteria. Further, various chemical additives are used in eliminating bacteria from foods. Antibiotics are used in the treatment of those diseases caused by the bacteria in humans and animals. Disinfectants are used in eliminating bacteria from surfaces. Problems pertaining to the specified current technique is listed below;
• HUS hemolytic uremic syndrome
• the inventive phage preparation is an absolutely natural product, wherein it comprises no chemical additives.
• Lytic bacteriophages are included by FDA in the list of GRAS, wherein it is considered as safe in terms of human, animal and environment life. Further, it was observed no negative findings in terms of health in in vivo acute toxicity analysis carried out in this invention.
• Bacteriophages are host-specific, wherein it exhibits a lytic effect on only target microorganism species. It constitutes no positive or negative effect on other bacteria in the medium.
• the present phage preparation is developed as an alternative treatment opportunity to the antibiotics in L. monocytogenes and E. coli 0157:H7 infections.
• the inventive phage preparation is an absolutely natural product, wherein it comprises no chemical additives. It is safe in terms of human, animal and environment life.
• the inventive bacteriophages cause no corrosion on surfaces.
• the present invention relates to joint use of lytic bacteriophages specific to L monocytogenes and E. coli 0157:H7 that constitute a crucial importance for public health. Thanks to the product obtained, it is ensured to eliminate said bacteria on food, feed, beverage and different surfaces. It can be used for the treatment by means of killing causes of disease in living organism in case of diseases on humans and animals caused by said bacteria.
• the present invention relates to a cocktail (mixture) comprised of 4 bacteriophages intended for the destruction of L. monocytogenes and E. coli 0157:H7 to be used in the specified contents.
• L. monocytogenes strains house e isolate code characteristics
• monocytogenes in total including reference strains of E. coli 0157:H7, E. faecium, E. faecalis, S. Typhimurium, A. hydrophila, Y. enterocolitica, R. equi, C. perfringens and S. aureus and 9 units of non-Z. monocytogenes were used so as to determine the host specifities and lytic effect profiles of the bacteriophages isolated in the study. None of the bacteriophages obtained showed any lytic effect on the reference strains of non-Z. monocytogenes. It was detected that the bacteriophages had lytic effect profiles on Z. monocytogeneses used in the study in a different range. In the light of those data, it was detected that LMF-M61, M83, Ml 17, Ml 19 and Ml 35 had the lytic effect on Z. monocytogeneses used in the study in the widest spectrum.
• L. monocytogenes ATCC 19111 strain to give nalidixic acid resistance that increases as a selective feature (so as to give a characteristic feature to the strain used in food models) was given resistance to nalidixic acid of 50 pg/ml with passages carried out on the LB (Tryptone [pancreatic digest of casein] 10 g/1, yeast extract 5 g/1, NaCl 5 g/1, agar 15 g/1) agar comprising nalidixic acid in the increasing concentration ((10, 20, 30, 40 and 50 pg/ml).
• the in-vitro lytic activities of the candidate phages specified for the cocktail to the host NA-LM19111 strain was evaluated with performing countings of phage and bacteria from samples taken in different periods (0., 30., 60. and 90. min. and 3., 6., 24. and 48. hours) with adding (Mol, Multiplicity of Infection, phage number/bacteria) with different multiplicities of infection to the L. monocytogenes strain in the log-reproduction phase, enriched for a night at 37°C in TSB.
• the spot cultivations were performed on the double-layer agars in the result of determination analyses of bacterio-sensitivity profiles, and the number of the phage-insensitive mutant L. monocytogenes strains that developed resistance to bacteriophage with which it was cultivated was detected, and then it was observed that there was no difference between their colony morphologies, when comparing their colony morphologies with L. monocytogenes strains that did not develop the resistance.
• Lysate (>10 9 pob/ml) of the phages determined so as to form the cocktail was centrifuged in 21.000 x g in ammonium acetate of 0,1 mol/L for two hours and the phages suspended in SM buffer were dyed with aqueous uranyl acetate solution of 2 % in carbonized copper grids.
• the dyed bacteriophages were observed at 800 kY with Transmission Electron Microcopy (TEM) and then they were photographed. It was detected that the bacteriophages were comprised of head, tail and holding plaque and they did not comprise pili. In the imaging performed, it was detected the bacteriophage in two different morphologies.
• head diameter of the phages in the first morphology was of 67,96 and 93,32 nm and the tail length was of 190,08 - 304,15 nm and the tail width was of 19,95-26,42. It was also determined that the phages characterizing the latter morphology were similar with the first phage in terms of structure, that its head diameter was almost the same with the first type, however its tail length was approximately half of the other type (150,88 nm) and its tail width was thicker (30,17 nm). It was detected in the performed imaging that the order Caudovirales was from the family Myoviridae according to the morphological characteristics exhibited by the bacteriophages.
• LMF-M61, M83, Ml 17, Ml 19 and M135 phages applied separately on the animals as a result of the analysis did not exhibit any acute toxic effects. It was further reported in the pathological examination that it was encountered no pathological results on esophagus, stomach, duodenum, jejunum, ileum, cecum and colon of animals.
• Bacteriophag log [pob/cuf]/g) Number of ature (log cuf/g) e bacteria (°C) l h 3 h 6 h 24 h
• Detection limit log 1,00 cuf/ml Table 10. Amount of the reduction seen in L. monocytogenes in the meatball food model.
• Bacterioph log [pob/cuf]/g) Number ature (log cuf/g) age of bacteria (°C) 1 h 3 h 6 h 24 h
• E. coli 0157:H7 It was performed a lytic bacteriophage isolation specific to E. coli 0157:H7 from the samples of slaughterhouse waste from. To this end, it was used the reference strains E. coli 0157:H7 ATCC 43895, E. coli 0157:H7 ATCC 43888, E. coli 0157:H7 ATCC 35150, E. coli 0157:H7 NCTC 12900 and E. coli 0157:H7 ECl. In the invention, thirty-one bacteriophages virulent (lytic) to E. coli 0157:H7 in total were isolated from 9 (37,5%) of 24 slaughterhouse waste waters in total.
• the bacteriophages M 12A-BEC 16 showed a more lytic effect on the two isolate E. coli 0157:H7 along with the isolate E. coli 0157:H7- coded 210KB.
• the other 23 bacteriophages obtained in the study demonstrated a more lytic effect on one isolate E. coli 0157:H7, four of those demonstrated a more lytic effect on the isolate 1 E. coli 0157:H7- in addition to the isolate 1 E. coli 0157:H7.
• the bacteriophages M1AEC00, M8AEC16, M9AEC95 and M12AEC50 were used in preparing the bacteriophage cocktail demonstrating the lytic effect in the widest spectrum against the isolates E. coli 0157:H7.
• the cocktail comprised of 4 bacteriophages obtained demonstrated the lytic effect against all isolates E. coli 0157:H7+/H7-. It was detected that the bacteriophages were of the family Myoviridae and the order Caudovirales in the TEM (Transmission Electron Microscope) imagings.
• the strain E. coli 0157:H7 ATCC 43895 (EC95)-effective bacteriophage numbered M8AEC were used so as to be employed in the experimental studies.
• the activity of the bacteriophage to the strain E. coli 0157:H7 ATCC 43895 were evaluated in-vitro, and to this end, NAEC95 cultures diluted at the level of 10 4 and 10 6 cuf/ml with TSB in line with OD600 values were infected such that their final titre was 10 8 pob /ml (plaque forming unit/ml) and were incubated for 48 hours at 37°C.
• the antibiotic therapy may increase the risk of HUS formation in human due to the fact that cells are lysed, the gene expression is increased because of stress and Shiga toxin release is increased in intestine channel. Therefore, it is crucial to develop an alternative therapy to antibiotics and protection precautions in infections E. coli 0157:H7.
• This mixture can be used directly and/or as additives in food and beverage industry, feed and animal feeding industry, slaughterhouses, every sort of hand and facial cleansing materials and even in pharmaceutical industry by means of eliminating the above-mentioned drawbacks originated from chemical additive substances, disinfectants and antibiotics.
• the reduction of both Gram-negative ( E . coli 0157:H7) and Gram-positive (L. monocytogenes ) microorganism to 3 logarithms in very various mediums- matrices can be achieved.
• the present invention has a high host-target specification. It can show its effect without damaging any living tissue. It constitutes no toxic effect on human, animals and environment. There is no risk of residue, mainly including chemical substances. It can be used repeatedly. There is no time limitation on its use. It can be used repeatedly. Its effect may be observed in 30 minutes after its application. There is no dosing limitation. It can be used in every stage in the related areas. It can be used on different products. Its range of use is pretty wide. It is absolutely natural and comprises no chemical substance.

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• 2019
  • 2019-11-12 WO PCT/TR2019/000078 patent/WO2021080520A1/en unknown
  • 2019-11-12 EP EP19949570.6A patent/EP3886593A4/de active Pending

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