WO2002076223A1 - A probiotic, a food product containing a probiotic, a method for preparation of said food product, a pharmaceutical composition and a use of the probiotic strain - Google Patents
A probiotic, a food product containing a probiotic, a method for preparation of said food product, a pharmaceutical composition and a use of the probiotic strain Download PDFInfo
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- WO2002076223A1 WO2002076223A1 PCT/NL2002/000191 NL0200191W WO02076223A1 WO 2002076223 A1 WO2002076223 A1 WO 2002076223A1 NL 0200191 W NL0200191 W NL 0200191W WO 02076223 A1 WO02076223 A1 WO 02076223A1
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- probiotic
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Links
- 239000006041 probiotic Substances 0.000 title claims abstract description 53
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 52
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 50
- 235000013305 food Nutrition 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 7
- 235000014897 Streptococcus lactis Nutrition 0.000 claims abstract description 10
- 241000194035 Lactococcus lactis Species 0.000 claims abstract 4
- 244000005700 microbiome Species 0.000 claims description 25
- 208000015181 infectious disease Diseases 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 244000144977 poultry Species 0.000 claims description 6
- 235000013365 dairy product Nutrition 0.000 claims description 5
- 235000013351 cheese Nutrition 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 206010016952 Food poisoning Diseases 0.000 claims description 2
- 208000019331 Foodborne disease Diseases 0.000 claims description 2
- 240000002129 Malva sylvestris Species 0.000 claims description 2
- 235000006770 Malva sylvestris Nutrition 0.000 claims description 2
- 235000015155 buttermilk Nutrition 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims description 2
- 230000002147 killing effect Effects 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 235000008983 soft cheese Nutrition 0.000 claims description 2
- 235000013618 yogurt Nutrition 0.000 claims description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 2
- 230000001681 protective effect Effects 0.000 abstract description 2
- 241001465754 Metazoa Species 0.000 description 12
- 241000607142 Salmonella Species 0.000 description 6
- 244000057717 Streptococcus lactis Species 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 230000002354 daily effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 230000036642 wellbeing Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 206010017964 Gastrointestinal infection Diseases 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 241000917009 Lactobacillus rhamnosus GG Species 0.000 description 2
- 241000194036 Lactococcus Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 229940059406 lactobacillus rhamnosus gg Drugs 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000006872 mrs medium Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108091092566 Extrachromosomal DNA Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- HXCILVUBKWANLN-UHFFFAOYSA-N brilliant green cation Chemical class C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 HXCILVUBKWANLN-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- OKTJSMMVPCPJKN-YPZZEJLDSA-N carbon-10 atom Chemical compound [10C] OKTJSMMVPCPJKN-YPZZEJLDSA-N 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1236—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using Leuconostoc, Pediococcus or Streptococcus sp. other than Streptococcus Thermophilus; Artificial sour buttermilk in general
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
- A23C19/061—Addition of, or treatment with, microorganisms
- A23C19/062—Addition of, or treatment with, microorganisms using only lactic acid bacteria, e.g. pediococcus, leconostoc or bifidus sp., or propionic acid bacteria; Treatment with non-specified acidifying bacterial cultures
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
-
- 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
Definitions
- a probiotic a food product containing a probiotic, a method for preparation of said food product, a pharmaceutical composition and a use of the probiotic strain
- the present invention relates to a probiotic.
- Probiotic-comprising food products are known for many years.
- a probiotic is a probiotic micro-organism is a micro-organism and in the context of the present invention it also encompasses a product excreted by a probiotic-organism, said probiotic contributing to the health and/or the well- being of a bird, such as poultry, or a mammal, such as a human.
- probiotic-comprising food products are known having a beneficial effect when combating gastro- intestinal infections, such as infections with Rotavirus.
- the European patent application 0,904,784 discloses a food product comprising a mixture of probiotic strains, amongst which optionally a Lactococcus strain, for which Lactococcus lactis L -P53 is mentioned as an example.
- probiotics are neither always effective with gastro-intestinal infections, nor capable of being included in any food product, there is a continuous need for new probiotics.
- a mixture of several probiotic strains is necessary for achieving an effect.
- the present invention relates to a new probiotic which is characterized in that it is a probiotic microorganism or a composition derived from such a micro-organism chosen from the group consisting of
- Lactococcus lactis strain B970 which was deposited on 27 June, 2000 with the Centraalbureau voor Schimmelcultures,
- a probiotic micro-organism is understood to be a living micro-organism which may be in the form of spores, lyophilized form or otherwise non-lethally inacti- vated.
- an inactivated probiotic micro-organism will be in an active form again before, during or after ingestion by a mammal or a bird in the gastro-intestinal tract, in which active state it may contribute to the health and/or the well- being of the animal, in particular a human being.
- a mutant is understood to be any probiotic strain derived from the claimed strains, undergone a change in the nucleotide sequence in its chromosomal or extra- chromosomal DNA while having retained its probiotic quality.
- the change may be the result of a natural process, or an in- duction by various agents or by genetic engineering techniques. See for a detailed description and examples for instance J.H. Miller "A short course in bacterial genetics" Cold 1992, page 83 112.
- a derivative is understood to be any probiotic strain derived from the claimed strain that may be provided with extra DNA while having retained its probiotic quality.
- Mutants and derivatives may be produced using any technique for genetic engineering known in the art, for example as disclosed by Sambrook et al in Molecular Cloning - a laboratory manual, Cold Spring Harbor, New York, 1989.
- the invention also relates to a food product containing the probiotic according to the invention.
- a food product is understood to be a food product for mammals, in particular for a human being, or animal feed, such as feed for poultry such as chicken.
- the food product is a dairy product, preferably a dairy product chosen from buttermilk, yoghurt, cheeses and, in particular, soft cheeses, cottage cheese, curd and quark.
- the probiotic according to the invention seems to be particularly suited for solid or semi-solid food products.
- a particular embodiment of the food product is a feed containing a probiotic according to the invention, in particular poultry feed.
- the present invention also relates to a method for the preparation of a food product containing the probiotic according to the invention.
- the method is characterized in that the probiotic micro-organism is grown and after an optional isolation in part or whole the micro-organism or an optionally purified probiotically active composition derived from said microorganism is added to the food product, wherein after the ad- dition the food product is not subjected to any probiotic micro-organism-killing or pro-biotic activity of the composition eliminating processes.
- a food product can be obtained having beneficial effects to a animal.
- the process or processes to which the food product is subjected after addition of the probiotic may comprise inactivation of the probiotic micro-organism according to the invention as long the micro-organism is not killed.
- the food product according to the invention comprises more than 10 6 probiotic micro-organism/g food prod- uct, such as at least 10 7 /g.
- the probiotic micro-organism may be grown on the food product, a half- inished product or starting material, or may be added at any stage during the preparation of the food product. Addition occurs, depending on what is desired, in the presence of substrate on which the probiotic micro-organism was grown or after separation from the probiotic micro-organism from the substrate.
- the present invention also relates to a pharmaceutical composition containing the probiotic according to the invention, together with a pharmaceutically acceptable carrier or excipient.
- Such a composition may be used for curative or prophylactic purposes in order to improve or maintain the health and/or the well-being of a mammal, in particular a human being. Similarly, it may be used to reduce the occurrence of Salmonella spp. in poultry. That is, it may reduce the number of Salmonella bacteria per chicken, as well reduce the number of chicken carrying Salmonella.
- the invention relates to the use of a probi- otic according to the invention for the preparation of pharmaceutical composition, suitable for the oral application against infections and/or food poisoning caused by Gram- negative micro-organisms.
- the importance is not the least the fact that more and more micro-organisms become resistant against antibiotics.
- the claimed use for poultry and mammals, in particular a human being, may eliminate (prevent) , replace or supplement treatment with antibiotics.
- the present invention will be elucidated with reference to the following examples and the drawing in which the only figure graphically depicts the intestinal colonization of Salmonella as measured by the faecal excretion of that pathogen in time. 1. Animal experiments
- Specific pathogen-free mail istar rats ( u, Harlan, Horst, the Netherlands) , age 8 weeks having a mean body weight of 263 g, were individually housed in cages in a room with controlled temperature (22-24°C), relative humidity (50- 60%) and a light/dark cycle (light from 6 a.m. to 6 p.m.).
- the animals were randomly divided in 4 groups of 8 animals each. For the entire period of the experiment, the animals had free excess to demineralized drinking water and food. Every 2 days the food consumption was measured and the body weight was measured every day.
- Lactococcus lactis B971 according to the invention (isolated from saliva of a cow) .
- the lactic acid bacteria were suspended in 0.5 ml fresh sterile MRS broth (Merck, Darmstadt, Germany) .
- a con- trol group received only 0.5 ml fresh MRS broth.
- the daily doses of lactic acid bacteria was about 10 10 CFU, as determined by growing on agar plates.
- the probiotic suspensions were freshly prepared daily by thawing of a frozen (-80°C) vial containing 1% of a stationary culture (suspended in litmus-containing skimmed milk with 0.5% yeast extract) on a water bath at room temperature.
- the medium was inocculated with 2% of a thawed suspension of bacteria.
- this comprised:
- Lactobacilli were grown under an atmosphere of 80% nitrogen, 10% carbon dioxide and 10% hydrogen at 37°C for 24 h.
- the Lactococci were aerobically grown for 24 h. at 37°C.
- the lactic acid bacteria were harvested by means of centrifu- gation (20 min. at 2,000 g; tabletop centrifuge) and re- suspended in fresh sterile MRS medium and administered to the animals.
- Salmonella entiritidis was grown as described (ref . 2 and 3) for infection of the animals. After 11 days of treatment with probiotics the animals were infected by oral administration of a 0.5 ml saline comprising 3% sodium bicarbonate and 3 x 10 8 SFU S. enteritidis. Before and several days after infection fresh faecal samples were obtained directly from the anus of the animals. The number of Samonella bacteria in the faeces were quantified by plating 10-fold dilu- tions of faeces on modified Brilliant Green Agar (Oxoid) containing sulphamandelate (ref. 4). The experiment was ended 10 days after infection.
- Oxoid modified Brilliant Green Agar
- the food uptake by and growth of the animals were neither affected by the treatment with probiotics nor by the infection.
- the average daily gain in body weight was 3 g and the intake of food was 21 g per day (as dry weight) .
- the Lactococci according to the invention improved the resistance to colonization of the rats against S. enteriditis signifi- cantly, as shown by the reduced faecal excretion of this pathogen in time (Fig. 1. Please note the logarithmic scale).
- Lactobacillus rhamnosus GG did not protect against S. enteritidis infection as the faecal excretion of this pathogen was not significantly different from the sham-treated control group.
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- Polymers & Plastics (AREA)
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Abstract
The invention relates to a probiotic capable of exerting a protective effect in the gastro-intestinal tract. The probiotic is a deposited Lactococcus lactis strain or a mutant or derivative thereof. The invention also relates to a food product and a pharmaceutical composition containing the probiotic, as well as a method for the preparation thereof.
Description
A probiotic, a food product containing a probiotic, a method for preparation of said food product, a pharmaceutical composition and a use of the probiotic strain
The present invention relates to a probiotic. Probiotic-comprising food products are known for many years. A probiotic is a probiotic micro-organism is a micro-organism and in the context of the present invention it also encompasses a product excreted by a probiotic-organism, said probiotic contributing to the health and/or the well- being of a bird, such as poultry, or a mammal, such as a human. For example, probiotic-comprising food products are known having a beneficial effect when combating gastro- intestinal infections, such as infections with Rotavirus. The European patent application 0,904,784 discloses a food product comprising a mixture of probiotic strains, amongst which optionally a Lactococcus strain, for which Lactococcus lactis L -P53 is mentioned as an example. As probiotics are neither always effective with gastro-intestinal infections, nor capable of being included in any food product, there is a continuous need for new probiotics. In addition, in many cases a mixture of several probiotic strains is necessary for achieving an effect. The present invention relates to a new probiotic which is characterized in that it is a probiotic microorganism or a composition derived from such a micro-organism chosen from the group consisting of
Lactococcus lactis strain B970 which was deposited on 27 June, 2000 with the Centraalbureau voor Schimmelcultures,
(CBS, Oosterstraat 1, 3740 AG Baarn, the Netherlands) under accession number CBS 108918, and
- Lactococcus lactis strain B971 which was deposited on 20 November, 2000 with the CBS under accession number CBS 109208
- or a mutant or derivative thereof.
These Lactococcus strains are, surprisingly, capable to exert a protective effect, as shown by experiment, without the presence of further probiotic strains, something that has
been disclosed up to now only for some Lactobacillus strains. In the present invention a probiotic micro-organism is understood to be a living micro-organism which may be in the form of spores, lyophilized form or otherwise non-lethally inacti- vated. Here, an inactivated probiotic micro-organism will be in an active form again before, during or after ingestion by a mammal or a bird in the gastro-intestinal tract, in which active state it may contribute to the health and/or the well- being of the animal, in particular a human being. In the pre- sent invention a mutant is understood to be any probiotic strain derived from the claimed strains, undergone a change in the nucleotide sequence in its chromosomal or extra- chromosomal DNA while having retained its probiotic quality. The change may be the result of a natural process, or an in- duction by various agents or by genetic engineering techniques. See for a detailed description and examples for instance J.H. Miller "A short course in bacterial genetics" Cold 1992, page 83 112. A derivative is understood to be any probiotic strain derived from the claimed strain that may be provided with extra DNA while having retained its probiotic quality. Mutants and derivatives may be produced using any technique for genetic engineering known in the art, for example as disclosed by Sambrook et al in Molecular Cloning - a laboratory manual, Cold Spring Harbor, New York, 1989. The invention also relates to a food product containing the probiotic according to the invention.
In the present invention a food product is understood to be a food product for mammals, in particular for a human being, or animal feed, such as feed for poultry such as chicken.
According to a preferred embodiment the food product is a dairy product, preferably a dairy product chosen from buttermilk, yoghurt, cheeses and, in particular, soft cheeses, cottage cheese, curd and quark. The probiotic according to the invention seems to be particularly suited for solid or semi-solid food products. For non-human animals, a particular embodiment of the food product is a feed containing a probiotic according
to the invention, in particular poultry feed.
The present invention also relates to a method for the preparation of a food product containing the probiotic according to the invention. The method is characterized in that the probiotic micro-organism is grown and after an optional isolation in part or whole the micro-organism or an optionally purified probiotically active composition derived from said microorganism is added to the food product, wherein after the ad- dition the food product is not subjected to any probiotic micro-organism-killing or pro-biotic activity of the composition eliminating processes.
Thus a food product can be obtained having beneficial effects to a animal. The process or processes to which the food product is subjected after addition of the probiotic may comprise inactivation of the probiotic micro-organism according to the invention as long the micro-organism is not killed. Typically the food product according to the invention comprises more than 106 probiotic micro-organism/g food prod- uct, such as at least 107/g. The probiotic micro-organism may be grown on the food product, a half- inished product or starting material, or may be added at any stage during the preparation of the food product. Addition occurs, depending on what is desired, in the presence of substrate on which the probiotic micro-organism was grown or after separation from the probiotic micro-organism from the substrate.
The present invention also relates to a pharmaceutical composition containing the probiotic according to the invention, together with a pharmaceutically acceptable carrier or excipient.
Such a composition may be used for curative or prophylactic purposes in order to improve or maintain the health and/or the well-being of a mammal, in particular a human being. Similarly, it may be used to reduce the occurrence of Salmonella spp. in poultry. That is, it may reduce the number of Salmonella bacteria per chicken, as well reduce the number of chicken carrying Salmonella.
Finally the invention relates to the use of a probi-
otic according to the invention for the preparation of pharmaceutical composition, suitable for the oral application against infections and/or food poisoning caused by Gram- negative micro-organisms. The importance is not the least the fact that more and more micro-organisms become resistant against antibiotics. The claimed use for poultry and mammals, in particular a human being, may eliminate (prevent) , replace or supplement treatment with antibiotics. The present invention will be elucidated with reference to the following examples and the drawing in which the only figure graphically depicts the intestinal colonization of Salmonella as measured by the faecal excretion of that pathogen in time. 1. Animal experiments
Specific pathogen-free mail istar rats ( u, Harlan, Horst, the Netherlands) , age 8 weeks having a mean body weight of 263 g, were individually housed in cages in a room with controlled temperature (22-24°C), relative humidity (50- 60%) and a light/dark cycle (light from 6 a.m. to 6 p.m.). The animals were randomly divided in 4 groups of 8 animals each. For the entire period of the experiment, the animals had free excess to demineralized drinking water and food. Every 2 days the food consumption was measured and the body weight was measured every day.
After 4 days the following probiotics were orally administered daily:
- Lactobacillus rhamnosus GG, a probiotic known from a dairy product currently on the market, or - Lactococcus lactis B970 according to the invention, or
- Lactococcus lactis B971 according to the invention (isolated from saliva of a cow) .
The lactic acid bacteria were suspended in 0.5 ml fresh sterile MRS broth (Merck, Darmstadt, Germany) . A con- trol group received only 0.5 ml fresh MRS broth. The daily doses of lactic acid bacteria was about 1010 CFU, as determined by growing on agar plates. The probiotic suspensions were freshly prepared daily by thawing of a frozen (-80°C)
vial containing 1% of a stationary culture (suspended in litmus-containing skimmed milk with 0.5% yeast extract) on a water bath at room temperature. The medium was inocculated with 2% of a thawed suspension of bacteria. In particular this comprised:
- MRS medium supplemented with 0.05% cystein for Lb. Rhamno- sus GG; and
- M17 medium (Oxoid, Basingstoke, England) supplemented with 0.5% lactose for Lactococcus lactis B970 and B971. Lactobacilli were grown under an atmosphere of 80% nitrogen, 10% carbon dioxide and 10% hydrogen at 37°C for 24 h. The Lactococci were aerobically grown for 24 h. at 37°C. The lactic acid bacteria were harvested by means of centrifu- gation (20 min. at 2,000 g; tabletop centrifuge) and re- suspended in fresh sterile MRS medium and administered to the animals.
Salmonella entiritidis was grown as described (ref . 2 and 3) for infection of the animals. After 11 days of treatment with probiotics the animals were infected by oral administration of a 0.5 ml saline comprising 3% sodium bicarbonate and 3 x 108 SFU S. enteritidis. Before and several days after infection fresh faecal samples were obtained directly from the anus of the animals. The number of Samonella bacteria in the faeces were quantified by plating 10-fold dilu- tions of faeces on modified Brilliant Green Agar (Oxoid) containing sulphamandelate (ref. 4). The experiment was ended 10 days after infection. Results: The food uptake by and growth of the animals were neither affected by the treatment with probiotics nor by the infection. The average daily gain in body weight was 3 g and the intake of food was 21 g per day (as dry weight) . The Lactococci according to the invention improved the resistance to colonization of the rats against S. enteriditis signifi- cantly, as shown by the reduced faecal excretion of this pathogen in time (Fig. 1. Please note the logarithmic scale). There was a significantly reduced colonization on the days 4- 6 after infection of the rats. In contrast, Lactobacillus
rhamnosus GG did not protect against S. enteritidis infection as the faecal excretion of this pathogen was not significantly different from the sham-treated control group.
REFERENCES
1. Reeves et al. J. Nutr. 123, pp. 1939-1951 (1993).
2. Bovee-Oudenhoven, I.M.J. et al. Gut 40, pp. 497-504 (1997).
3. Oudenhoven, I.M.J. et al. Gastroenterol. 107, pp. 47-53 (1994) .
4. Bartram, H.P. et al. Am. J. Clin. Nutr. 59: pp. 428-432 (1994) .
BP/A/II/12 page 14
BUDAPEST TREATY ON THE INTERNATIONAL ECOGNITION OP HE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
NIZO Food Research RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Postb'js 20 issued pursuant to Rule 7 . 1 by the 6710 BA EDE INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page Ne eiiand
name and address of depositor
Form BP/4 (sole page) CBS/9107
Claims
1. Probiotic characterized in that it is a probiotic micro-organism or a composition derived from such a micro-organism chosen from the group consisting of
- Lactococcus lactis strain B970 which was deposited on 27 June, 2000 with the Centraalbureau voor Schimmelcultures,
(CBS, Oosterstraat 1, 3740 AG Baarn, the Netherlands) under accession number CBS 108918, and
- Lactococcus lactis strain B971 which was deposited on 20 November, 2000 with the CBS under accession number CBS 109208
- or a mutant or derivative thereof.
2. Food product containing a probiotic, characterized, in that the food product contains Lactococcus lactis strain with accession number CBS 108918 and/or number CBS 109208 as a probiotic.
3. Food product according to claim 2, characterized, in that the food product is a dairy product.
4. Food product according to claim 3, characterized, in that the dairy product is chosen from buttermilk, yoghurt, cheeses, and in particular soft cheeses, cottage cheese, curd and quark.
5. Food product according to claim 2, characterized in that the food product is poultry feed.
6. Method for the preparation of a food product containing a probiotic, characterized, in that the probiotic micro-organism is grown and after an optional isolation in part or whole the micro-organism or an optionally purified probiotically active composition derived from said microorganism is added to the food product, wherein after the ad- dition the food product is not subjected to any probiotic micro-organism-killing or pro-biotic activity of the composition eliminating processes.
7. Pharmaceutical composition containing the strain according to claim 1, together with a pharmaceutically ac- ceptable carrier or excipient.
8. Use of a probiotic according to claim 1, for the preparation of a pharmaceutical composition, suitable for oral administration against infections and/or food poisoning by Gram-negative micro-organisms.
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NL01201086.4 | 2001-03-23 | ||
NL1201086 | 2001-03-23 |
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WO2002076223A1 true WO2002076223A1 (en) | 2002-10-03 |
Family
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PCT/NL2002/000191 WO2002076223A1 (en) | 2001-03-23 | 2002-03-25 | A probiotic, a food product containing a probiotic, a method for preparation of said food product, a pharmaceutical composition and a use of the probiotic strain |
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WO (1) | WO2002076223A1 (en) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2616390B1 (en) * | 1976-04-14 | 1977-10-06 | Inst Przemyslu Mleczarskiego | Production of biologically nisinated milk powder |
US5527505A (en) * | 1991-03-18 | 1996-06-18 | Snow Brand Milk Products Co., Ltd. | Process for the manufacture of fermented milk |
RU2063755C1 (en) * | 1993-07-14 | 1996-07-20 | Товарищество с ограниченной ответственностью "Алэф" | Dry probiotic for ruminant animals |
EP0904784A1 (en) * | 1997-09-22 | 1999-03-31 | N.V. Nutricia | Probiotic nutritional preparation |
WO1999062348A1 (en) * | 1998-05-29 | 1999-12-09 | Enterprise Ireland (Trading As Bioresearch Ireland) | Process for the manufacture of probiotic cheese |
-
2002
- 2002-03-25 WO PCT/NL2002/000191 patent/WO2002076223A1/en not_active Application Discontinuation
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2616390B1 (en) * | 1976-04-14 | 1977-10-06 | Inst Przemyslu Mleczarskiego | Production of biologically nisinated milk powder |
US5527505A (en) * | 1991-03-18 | 1996-06-18 | Snow Brand Milk Products Co., Ltd. | Process for the manufacture of fermented milk |
RU2063755C1 (en) * | 1993-07-14 | 1996-07-20 | Товарищество с ограниченной ответственностью "Алэф" | Dry probiotic for ruminant animals |
EP0904784A1 (en) * | 1997-09-22 | 1999-03-31 | N.V. Nutricia | Probiotic nutritional preparation |
WO1999062348A1 (en) * | 1998-05-29 | 1999-12-09 | Enterprise Ireland (Trading As Bioresearch Ireland) | Process for the manufacture of probiotic cheese |
Non-Patent Citations (4)
Title |
---|
DATABASE FSTA [online] INTERNATIONAL FOOD INFORMATION SERVICE (IFIS), FRANFURT/MAIN, DE; KIMOTO H ET AL: "Lactococci as probiotic strains: adhesion to human enterocyte-like Caco-2 cells and tolerance to low pH and bile.", XP002174436, Database accession no. 2000-00-p0536 * |
DATABASE WPI Section Ch Week 199713, Derwent World Patents Index; Class B04, AN 1997-143791, XP002174437 * |
KIMOTO, OHMOMO, NOMURA, KOBAYASHI, OKAMOTO: "In vitro studies on probiotic properties of lactococci", MILCHWISSENSCHAFT, vol. 55, no. 5, 2000, pages 245 - 249, XP002174435 * |
LETTERS IN APPLIED MICROBIOLOGY 29 (5) 313-316 1999 NAT. INST. OF ANIMAL IND., TSUKUBA NORIN-DANCHI, PO BOX 5, IBARAKI 305-0901, JAPAN. E-MAIL ANNE(A)NIAI.AFFRC.GO.JP * |
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