WO1989005849A1 - Lactic acid bacteria for use in fermented milk products and veterinary compositions - Google Patents

Lactic acid bacteria for use in fermented milk products and veterinary compositions Download PDF

Info

Publication number
WO1989005849A1
WO1989005849A1 PCT/DK1988/000222 DK8800222W WO8905849A1 WO 1989005849 A1 WO1989005849 A1 WO 1989005849A1 DK 8800222 W DK8800222 W DK 8800222W WO 8905849 A1 WO8905849 A1 WO 8905849A1
Authority
WO
WIPO (PCT)
Prior art keywords
according
lactic acid
pigs
bacteria
composition
Prior art date
Application number
PCT/DK1988/000222
Other languages
French (fr)
Inventor
Gerd Trine Suhr-Jessen
Susan Anne Fogh
Henry SØGAARD
Original Assignee
Chr. Hansen's Laboratorium A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to DK6861/87 priority Critical
Priority to DK686187A priority patent/DK686187D0/en
Application filed by Chr. Hansen's Laboratorium A/S filed Critical Chr. Hansen's Laboratorium A/S
Publication of WO1989005849A1 publication Critical patent/WO1989005849A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RPROCESSES USING MICROORGANISMS
    • C12R1/00Processes using microorganisms
    • C12R1/01Processes using microorganisms using bacteria or actinomycetales
    • C12R1/225Lactobacillus
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23CDAIRY PRODUCTS, e.g. MILK, BUTTER, CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00Milk preparations; Milk powder or milk powder preparations
    • A23C9/12Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K10/00Animal feeding-stuffs
    • A23K10/10Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K50/00Feeding-stuffs specially adapted for particular animals
    • A23K50/30Feeding-stuffs specially adapted for particular animals for swines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • A61K35/741Probiotics
    • A61K35/744Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747Lactobacilli, e.g. L. acidophilus or L. brevis
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23YINDEXING SCHEME RELATING TO LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23Y2220/00Lactobacillus
    • A23Y2220/77Sakei

Abstract

Lactic acid bacteria isolated from the gastrointestinal tract of pigs are selected for their ability to survive in a gastrointestinal environment, i.e., for their bile and acid tolerance, and for their ability to adhere to pig gastrointestinal epithelium. The thus selected bacteria may be included in a fermented milk product intended for human consumption or in a veterinary composition for preventing or treating pig gastrointestinal diseases.

Description

LACTIC ACID BACTERIA FOR USE IN FERMENTED MILK PRODUCTS AND VETERINARY COMPOSITIONS

FIELD OF INVENTION

The present invention relates to lactic acid bacteria isolated from the gastrointestinal tract of pigs and a fermented milk product comprising such bacteria, as well as to a powder and a veterinary Composition comprising the bacterium and the use thereof in the. prophylaxis or treatment of pig gastrointestinal diseases.

TECHNICAL BACKGROUND

Fermented milk products have long been known to be a beneficial component of human diet as they combine the nutritive value of milk in terms of protein content, including a favourable amino acid composition, content of fat and overall energy content (slightly less than in unfermented milk due to the conversion of lactose to lactic acid through the action of lactic acid bacteria) and the increased digestibility established for fermented milk products (Livia Aim, "The Effect of Fermentation on Nutrient in Milk and some Properties of Fermented Liquid Milk Products", Naringsforskning 27 , 1983, pp. 2-8). The improved digestibility is obtained by precipitation of the milk protein (casein) into fine curd particles which are more easily digested by the enzymes present in the digestive tract than the large casein particles resulting from the action of gastric juice on unfermented milk. Furthermore, the fermentation process causes a significant hydrolysis of the proteins present in the milk into smaller peptides and free amino acids, and this so-called pre-digestion of the proteins makes it easier to degrade them by the digestive enzymes. Lactic acid fermentation also tends to favourably affect the utilization of calcium in the body in the form of calcium lactate which is more easily absorbed in the body.

Apart from the improved utilization of the nutrient components in milk which in itself would speak in favour of the consumption of fer mented milk products, the lactic acid bacteria present In the fermented milk may inhibit the growth of pathogenic or potentially pathogenic microorganisms in the intestinal tract by producing various organic acids (primarily lactic acid and, to a smaller extent, acetic, propionic and formic acid) which lower the intestinal pH so as to inhibit acid-sensitive organisms. Some lactic acid bacteria also produce antimicrobial substances known as biocines which contribute to controlling the growth of pathogens in the intestines. A high concentration of lactic acid bacteria in the intestines may also be employed prophylactically to protect against attacks from pathogenic bacteria.

Ingestion of fermented milk products may therefore be advantageous in order to maintain a good biological balance of the intestinal flora of a healthy person and possibly also to redress the imbalance in the intestinal flora occasioned by various conditions such as intestinal diseases and disorders (e.g. diarrhoea), functional disorders of the digestive tract, or the after-effects of antibiotic treatment and radiation therapy. By ingesting a fermented milk product, the normal Intestinal flora may be regenerated.

Fermented milk products comprising lactic acid bacteria and alleged to exert a beneficial effect by maintaining the normal balance of the intestinal flora are commercially available and are described by, for instance, Frank V. Kosikowski in Cheese and Fermented Milk Foods , 2nd Ed., 1978, pp. 37-48. To the present inventors' best knowledge, however, the ability of the bacteria used in these products to survive in a gastrointestinal environment has not been adequately documented.

One object of the present invention Is therefore to provide lactic acid bacteria which show advantageous properties with respect to survival in the gastrointestinal tract when included in a fermented milk product intended for human consumption.

Another object of the present invention is to provide lactic acid bacteria with useful properties for the prophylaxis or treatment of pig gastrointestinal diseases. Gastrointestinal infections frequently occur in pigs, especially young pigs, resulting in retardation of the growth rate and even death of some of the infected pigs. Enteric diseases in pigs may be of bacterial, viral or protozoal origin and are, in the case of bacteria, initiated by a colonization of the gastrointestinal epithelium with pathogenic organisms which compete with the normal intestinal flora and, if successfully established, produce (depending on the species) exotoxins (enterotoxins) which in sufficient quantities, that is when the organism producing them has ousted the normal bacterial flora from the intestines, produce a variety of disease symptoms in the host, notably diarrhoea leading to an occasionally fatal loss of fluid from tissues and in a decreased feed consumption as well as utilization of the feed. Enteropathogenic organisms are easily transmitted from one pig to another through their presence in the faeces of infected animals present in their environment, and therefore the disease may very quickly be transmitted from the initially infected animal to the rest of the herd. Hence, gastrointestinal infections present a major source of economic loss to pig breeders.

Gastrointestinal diseases in pigs have usually been combated with antibiotics. Thus, antibiotics have been administered prophylactically to neonatal pigs or to gestating sows before farrowing. However, this procedure suffers from the disadvantage that it eliminates the natural intestinal flora, making the animals more vulnerable to invasion by opportunistic pathogens which colonize the intestines more rapidly than the natural microflora. This may, in turn, give rise to bacterial infection after the antibiotic treatment has ceased. Another frequent result of antibiotic treatment is the development of antibiotic-resistant strains of the pathogens. Furthermore, especially when therapeutic doses of antibiotics are administered to older pigs or porkers, residual amounts of the antibiotics may be present in the meat of slaughtered animals; this is undesirable since it may lead to allergic reactions in humans consuming the meat and to the development of antibiotic-resistant strains of human pathogens after exposure of these to low dosages of the antibiotics in question. For these reasons, health authorities in most countries have prohibited the presence of residual antibiotics in meat intended for human consumption. Alternative procedures involving the use of non-pathogenic bacteria such as lactic acid bacteria have therefore been suggested as a replacement of or supplement to antibiotic treatment in case of animal, Including human, gastrointestinal infections. Thus, US 3,320,130 discloses a process for preparing a medicament comprising separately culturing L. acidophilus and a non-pathogenic strain of E. coli on milk containing gastro-pyloric mucin and lysine, arginlne and histidine. The separate cultures are lyophilized and mixed. Both organisms were Isolated from the faeces of a healthy baby, and the medicament is, presumably, intended for human use. It Is suggested that these bacteria will compete successfully with the other intestinal flora.

US 3,953,609 discloses a method of changing the digestive system bacteria in animal by orally feeding the animals live Lactobacillus lactis NRRL B-5628 in an amount of 1×1010 cells/kg body weight/day to reduce the number of other gastrointestinal tract bacteria. The purpose is to provide prophylaxis or treatment of colibacillosis in animals such as swine, cattle and poultry. The bacterial strain was isolated from human faeces.

US 4,314,995 discloses a method of treating infections by local administration of the Lactobacillus strains FRI 1946, 2779, 2780, 2781 or 2782, optionally together with an antibiotic. Among the diseases suggested for treatment in the patent by means of these bacteria are gastritis and enteritis for which applications the bacteria are preferably administered together with antibiotics. The bacteria are stated to be bile resistant, but their origin is not indicated. The bacteria appear to be intended for use in humans only.

EP 203 586 discloses the use of Lactobacillus fermentum ATCC 53113 or mutants thereof for treating gastrointestinal diseases in domestic animals such as pigs, cows, sheep, goats and horses. The strain is indicated to have been isolated from the gut of a healthy newborn Pig. SUMMARY OF THE INVENTION he present inventors have now isolated probiotic lactic acid bacteia from the digestive tract of pigs which bacteria have been careully selected for properties which are useful when the organisms re to be employed in fermented milk products for the prophylaxis or reatment of pig gastrointestinal infections. ccordingly, the present invention relates to a lactic acid bacterium hich is viable in the gastrointestinal tract of human beings and igs, the bacterium being isolated from the gastrointestinal tract of pig and selected according to the following criteria:

adhesion to pig gastrointestinal epithelial tissue after incubaion of bacteria with pig epithelial cells in a ratio of 100:1 inhosphate buffered saline at 37°C for 30 minutes,

production of lactic acid as determined by

i) growth in MRS (Oxoid CM 359) medium, pH 6.2, for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction to at least 4.0, or

ii) growth in 9.5% reconstituted skimmilk, pH 6.3-6.4, for up to 48 hours at 37ºC in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction to at least 5.2, or

iii) growth in modified MRS medium without glucose, pH 6.2, containing equal amounts of maltose and soluble starch for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction to at least 4.5, or

iv) defined as the production of at least 10 g/l of lactic acid (D- and L-forms) when grown in MRS medium, pH 6.2, for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in

H2, c) bile tolerance as determined by growth in MRS medium, pH 6.2, supplemented with 0.9% bile for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction comparable to the one defined in b) (i)-(iii),

d) acid tolerance as determined by a reduction in the number of colony-forming units/ml after incubation for 1 hour at 40°C and a pH of 2.5 not exceeding 3.2 log units, preferably less than 1 log unit,

e) a generation time of about 65 minutes, preferably 30-50 minutes, when grown in MRS medium at 40°C under anaerobic conditions,

or a functionally equivalent mutant thereof.

In the present context, the term "probiotic" is used to indicate cultures of non-pathogenic gastrointestinal bacteria or compositions containing such cultures which, after the ingestlon of effective doses, may survive or even become established in the gastrointestinal tract and thereby preserve or enforce the function of the microflora as a barrier against the colonization of the epithelium by pathogenic organisms.

In order to exert the above-described beneficial effects both with respect to maintaining or restoring a healthy balance of the gastrointestinal microflora and with respect to inhibiting the growth of pathogenic or potentially pathogenic organisms, It is essential that the bacteria of the invention are capable of surviving the conditions prevailing in the gastrointestinal tract. The criteria according to which the bacteria of the invention have been selected have primarily been employed to achieve this overall effect. Thus, contrary to known fermented milk products allegedly producing these effects on Ingestion, the lactic acid bacteria of the invention have been systematically screened for their viability under gastrointestinal conditions in pigs. Due to the well-known similarities between the human and porcine digestive systems, the fact that the bacteria of the invention have been shown to establish themselves in the intestines of pigs renders probable their ability to survive in the human gastrointestinal tract as well. When the bacteria of the invention are inge sted primarily to maintain a favourable balance of the intestinal microflora, survival of the bacteria in the digestive tract is the most important selection criterion as regular ingestion of a fermented milk product containing the bacteria will usually be sufficient to ensure a sufficient number of the bacteria in the intestines to provide a favourable regulation of the intestinal flora.

In connection with research leading to the present invention, it has been found that lactic acid bacteria isolated from the intestinal flora of the same animal species as the one to which it will eventually be administered for therapeutic purposes show superior properties with respect to therapeutic effect. This may, presumably, be ascribed to the fact that the bacteria have already adapted to the conditions prevailing in the intestines of the animal species in question so that they will be able to colonize and compete successfully with the other intestinal flora and establish itself in the intestines. To the best of the present inventors' knowledge, this finding has not previously been reported, and in the prior literature nobody appears to have speculated that it might be important for obtaining the desired effect, cf. EP 203 586 which although it discloses an organism isolated from a pig which is used to treat diarrhoea in pigs , also indicates that the same organism may be used for treating animals of other species .

In particular when the bacteria of the invention are intended to be used therapeutically, however, it has been found to be advantageous that they show good adhesion properties since this improves their ability to colonize the gastrointestinal mucosa and compete successfully with the pathogens or potential pathogens present in the intestines, thus obtaining a favourable balance of the gastrointestinal microflora. The adhering bacteria form a layer on the epithelium and prevent access of the pathogens to the receptors present on the epithelial cells. The bacteria preferably adhere to pig gastrointestinal tissue in a number of at least 4, preferably at least 16, most preferably at least 50, per epithelial cell. The production of lactic acid is another important selection criterion since most pathogens show a significantly decreased growth rate in the presence of acid. Bile and acid tolerance are also essential for a probiotic bacterium to be employed successfully in the intestines, in particular the upper small intestines in order to be able to survive passage of the stomach and Intestines, and possibly to colonize the epithelium In the vicinity of the biliary tract. Since most pathogens tend to multlply very quickly, It is also essential that the lactic acid bacteria employed as the probiotic organisms have a brief generation time so that they may compete with the pathogens in the intestines.

Certain lactic acid bacteria have been found to produce one or more antimicrobial agents such as hydrogen peroxide. Such substances are known to possess an inhibitory effect on a variety of microorganisms and therefore supplement the effect of pH decrease ascribable to the production of lactic acid.

Mutants of the bacteria may either occur spontaneously (as is often the case in nature) or be produced deliberately such as by treatment with a chemical mutagen such as mitomycin C, 5-bromouracil, methylmethane sulphonate, nitrogen mustard and the nitrofurans, ionizing radiation, ultraviolet radiation, or by applying recombinant DNA techniques. The term "functionally equivalent" should be taken to mean a mutant which shows similar properties to the parent strain with respect to establishing itself and competing with the pathogens present in the intestines at the time the bacteria are administered or entering the Intestines at some later point.

Ingestion of a fermented milk product containing lactic acid bacteria of the invention for the purpose of regulating digestive functions or prophylactic or therapeutic administration of a lactic acid bacterium of the invention promotes the establishment of the normal, healthy microflora in the Intestines thereby creating or recreating a balanced intestinal environment where enteropathogens have difficulties in establishing themselves. As a result of this, it may, in case of actual gastrointestinal infections, furthermore be advantageous to administer the lactic acid bacteria concomitantly with or subsequently to antibiotic treatment in order to restore a healthy balance of the intestinal flora. It is, however, preferred that antibiotic treatment of pig gastrointestinal tract infections be dispensed with altogether and replaced by administration of the lactic acid bacteria of the invention.

DETAILED DISCLOSURE OF THE INVENTION

Probiotic bacteria useful for the present purpose may, in principle, be any lactic acid bacteria isolated from porcine gastrointestinal epithelium, preferably the epithelium of healthy pigs. Suitable strains may be selected after isolation by screening procedures comprising testing for adhesive properties, production of lactic acid, acid and bile tolerance and generation time as described in further detail in Examples 1-5 and 8 below. For treating pig gastrointestinal infections in particular, the bacteria may be isolated from pigs of varying ages so as to ensure the availability of strains which are able to colonize the epithelium at different stages of the pigs' lives. Specific examples of lactic acid bacterial strains which may be employed in the present invention include strains selected from the genus Lactobacillus , e.g. L. acidophilus/L . gasseri isolated from the duodenum of a suckling pig; L. salivarius subsp. salivarius isolated from the jejunum of a weaning pig; L . acidophilus/L . gasseri isolated from the ileum of a weaning pig; L . crispatus isolated from the jejunum of a suckling pig; and L. salivarius subsp. salivarius isolated from the pars oesophagea of a suckling pig.

Samples of thus isolated strains of these Lactobacillus species were deposited in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on 22 December, 1987 in the Deutsche Sammlung von Mikroorganismen, Mascheroder Weg IB, 3300 Braunschweig, Federal Republic of Germany, with the following accession numbers:

L. acidophilus/L . gasseri SS28 DSM 4324

L. salivarius subsp. salivarius SS129 DSM 4325 L. acidophilus/L. gasseri SS131 DSM 4326

L . crispatus SS151 DSM 4327

L. salivarius subsp. salivarius SS258 DSM 4328 It will, however, be evident to persons skilled In the art that other lactic acid bacterial strains may be isolated in a similar fashion, using the selection criteria indicated above and the procedures described in the examples, without departing from the spirit and scope of the present invention.

For use in a fermented milk product, the two Lactobacillus salivarius strains indicated above have been found to be particularly suitable as they not only provide the beneficial effects described above, but also result in an agreeable taste of the product.

Once Isolated, the bacteria may be propagated by growing the selected strain or strains in a suitable culture medium under anaerobic conditions for a period of time sufficient to provide at least 106 viable bacteria/ml of medium and harvesting the resulting bacteria from the medium. The cultivation temperature will typically be about 37°C, and the pH in the range of about 5.5-6.5.

In another aspect, the present invention relates to a liquid or frozen concentrate comprising lactic acid bacteria of the invention. Although it is theoretically possible to use the culture medium containing the bacteria directly, it is generally preferred to produce a bacterial concentrate in order to obtain a higher count of bacteria for administration or inoculation. The concentrate may be prepared by, for instance, centrifugation or filtration. The concentrate may suitably comprise 1×104-1×1012 viable colony-forming units/ml. The concentrate may contain a mixture of two or more of the Lactobacillus strains indicated above.

In a further aspect, the present invention relates to a freeze-dried or spray-dried powder comprising a lactic acid bacterium as described above. In this case, too, it is in theory possible to dry the bacterial cultures as grown, but it is preferred to concentrate the bacteria before drying for the reason indicated above. To ensure a sufficient viability of the bacteria, it is preferred to add a stabilising or cryoprotective agent to the concentrate before drying as for instance disclosed in US 3,897,307. The resulting dried powder preferably comprises 104-1012 viable colony-forming units/g. It may be an advantage that the powder comprises a mixture of two or more lactic acid strains such as those listed above, in order to secure a broad-spectrum activity of the powder when used as or in a preparation to be administered to pigs of varying ages.

In a still further aspect, the invention relates to a fermented milk product which comprises a lactic acid bacterium of the present invention. The product Is primarily intended for human consumption, and although it is primarily ingested for its nutritive value, it may be particularly useful for dietary purposes, i.e. for its favourable influence on digestive properties ascribable to the ability of the lactic acid bacteria to inhibit the growth of pathogenic or potentially pathogenic microorganisms. The fermented milk product of the invention may contain a mixture of two or more of the Lactobacillus strains listed above, and preferably also contains a Bifidobacterium sp. such as B . bifidum or B . longum, as well as L . acidophilus .

The fermented milk product may be prepared by inoculating milk with a culture of the bacterium of the invention in a manner known per se , e.g. as described by A.Y. Tamine and R.K. Robinson in Yoghurt Science and Technology, 1985, preferably in the form of the liquid or frozen concentrate or freeze-dried or spray-dried powder described above. Consequently, the present invention also relates to the use of the lactic acid bacterium or a liquid or frozen concentrate or freeze- dried or spray-dried powder containing it for the production of a fermented milk product. The inoculation level is typically in the range of 1×105-1×108 bacteria/ml of milk, such as 5×106 bacteria/ml.

The lactic acid bacteria of the invention have been found capable of reducing the occurrence and/or severity of gastrointestinal tract infections in pigs caused by enteropathogenic microorganisms. The term "reducing the occurrence" is understood to mean that there are fewer cases of gastrointestinal infections both among individual pigs within each herd and among herds on the same farm, relative to pigs which have not been treated with the lactic acid bacteria of the invention. The gastrointestinal tract infections which may be prevented or treated by administering the bacteria of the invention may be any infections resulting from the presence and multiplication of enteropathogenie microorganisms in the Intestines, e.g. diarrhoeas, scours, etc. Examples of enteropathogens are E. coli and rotavirus.

In a still further aspect, the present invention relates to a veterinary composition for the prophylaxis or treatment of gastrointestinal tract infections in pigs caused by enteropathogenie microorganisms, the composition comprising a lactic acid bacterium as described above and an excipient or carrier. The composition should preferably comprise 103-1012 viable colony-forming units/g since in this range a sufficient number of bacteria will normally survive the passage of the pigs stomach to colonize the intestinal epithelium in sufficient numbers to compete successfully with the pathogens or other microorganisms present.

The composition may be formulated according to conventional veterinary practice as a powder, granulate, tablet, capsule, paste, gel, drench, mixture or suspension. Solid formulations, i.e. powders, granulates, tablets and capsules, may contains fillers, e.g. sugars, sorbitol, mannitol and silicic acid; binders, e.g. cellulose derivatives such as carboxymethyl cellulose and polyvinylpyrrolidone; disintegrants, e.g. starch, sodium bicarbonate and calcium carbonate; lubricants, e.g. magnesium stearate, talc and calcium stearate.

Semi-solid formulations, i.e. pastes or gels, may comprise a gelling agent such as an alginate, gelatin, carrageenan, tragacanth gum and pectin, a mineral oil such as liquid paraffin, a vegetable oil such as corn oil, sunflower oil, rape oil and grape kernel oil, as well as a thickener such as a starch, gum, gelatin, etc. Liquid formulations, i.e. drenches, mixtures and suspensions, may comprise a liquid or oily vehicle, e.g. water (favourably the drinking water of the pigs to which the composition is administered), an electrolyte solution (often given to pigs to compensate for the electrolyte loss caused by the loss of fluids from tissue which is characteristic of diarrhoea) or reconstituted sowmilk replacer. Oily vehicles comprise a mineral oil such as liquid paraffin, a vegetable oil such as corn oil, sunflower oil, rape oil, grape kernel oil, etc. The freeze-dried or spray-dried powder of the invention may be suspended in the liquid vehicle in accordance with usual practice. The powder formulation may advantageously be the freeze-dried or spray-dried powder described above, either in itself or formulated with one or more further excipients.

The solid formulations may be provided with a coating to protect the bacteria from gastric conditions so that they will more easily survive the passage of the stomach. The coating should be one which is degradable in a gastrointestinal environment. The coating is preferably one which is soluble in the stomach (though only gradually so that the protective effect will be maintained) or in the upper part of the small intestines. Examples of suitable coatings are alginates or lipids.

The composition of the invention may also comprise a mixture of two or more lactic acid bacterial strains such as the strains listed above. Apart from the lactic acid bacteria, the composition may further comprise other active agents such as antibiotics, chemical growth promoters, or microorganisms or enzymes which have a growth promoting effect.

In a still further aspect, the present invention relates to the use of a lactic acid bacterium as defined above for preparing a veterinary composition for the prophylaxis or treatment of gastrointestinal tract infections in pigs caused by enteropathogenic microorganisms.

It is known that pigs are most susceptible to gastrointestinal infections at three points in their lives: when they are newborn, when they are being weaned and when they have just been weaned, since their digestive system is under stress, being forced to adapt to changed feeding conditions. The composition of the invention is therefore advantageously administered to suckling or weaning pigs. However, older pigs .(porkers) may also succumb to gastrointestinal diseases which may spread very quickly to an entire herd of the pigs thereby causing substantial economic loss due to death or considerable loss of weight.

The composition may be formulated in any of the ways described above, but is preferably adapted to be sprayed or sprinkled in the environ ment of the pigs, or Is in the form of a suspension in an oily or aqueous medium such as the drinking water, an electrolyte solution or a reconstituted sowmilk replacer. When the composition is sprayed or sprinkled in the environment of the pigs, the pigs will take up the bacteria when they root about in the sty, the bacteria colonizing the intestinal epithelium and competing with enteropathogens taken up in a similar way. For weaning pigs or porkers, the composition may also advantageously be mixed with the feed. The composition is preferably the freeze-dried or spray-dried powder described above.

When the composition is intended for oral use, the amount of lactic acid bacteria per dosage of the composition is in the range of 104-1012, preferably 106-1010, colony-forming units. At this dosage level, the composition is suitably administered 1-3 times a day for a period of up to 14 days, the period being to some extent determined by the level of contamination in the sty or by the condition of the pigs, i.e. whether the composition is to be used therapeutically or prophylactically.

The lactic acid bacteria of the invention may optionally be administered concomitantly with or subsequent to antibiotic therapy in order to counteract the unfavourable effect of antibiotic treatment that the healthy intestinal flora is killed, thus giving pathogens ample opportunity to colonize the intestinal epithelium without being held in check by other organisms in the intestinal environment.

Alternatively, the composition of the invention may be administered to a gestating or lactating sow in an amount sufficient to provide a therapeutically effective amount of the bacteria in the environment of the sow. This form of administration therefore has the same effect as spraying or sprinkling the bacteria directly in the environment of the pigs. The amount of bacteria administered is in the range of 105-1012, preferably 107-1011, viable colony-forming units per dosage. At this dosage level, the bacteria are suitably administered 1-3 times a day for a period of up to 28 days, the period being to some extent determined by the level of contamination by pathogens in the sty. The invention is described in further detail in the following examples which should not be construed as limiting in any way to the scope of the invention.

EXAMPLE 1

Isolation and characterization of Lactobacillus strains

Description of strains

A large number of bacteria were isolated from a number of healthy piglets and weaning pigs from several different swine herds, and after comprehensive screening as described below in Examples 1-5 and 8, the following strains were selected:

SS28 L. acidophilus/L. gasseri isolated from duodenum of a suckling pig.

SS129 L . salivarius subsp. salivarius isolated from jejunum of a weaning pig.

SS131 L . acidophilus/L. gasseri isolated from ileum of a weaning pig.

SS151 L. crispatus isolated from jejunum of a suckling pig.

SS258 L. salivarius subsp. salivarius isolated from pars oesophagea of a suckling pig.

Fermentation pattern

The fermentation profile of the bacteria is shown in Tables 2, 2a and 3. The profiles were performed using a commercially available test system (API-50CH), Tables 2 and 2a, and using a modified MRS (Oxoid CM 359) medium and incubation method according to Bergey's Manual of Systematic Bacteriology, Volume 2, 1986, Table 3. For identification, the fermentation profiles of both tests were compared to the fermentation patterns described in Bergey's Manual 1986. In addition to the fermentation patterns of the 22 carbohydrates from the API-50CH used for comparison with Bergey's Manual , eight more criteria shown in Table 2a were also taken into account in the identification, resulting in 30 criteria according to Bergey's Manual .

Table 2

Fermentation patterns as recorded by API-50CH

Medium Reaction

SS28 SS129 SS131 SS151 SS258

Glycerol - - - - - Erythritol - - - - -

) D-Arabinose - - - - -

X) L-Arabinose - - - - - x) Ribose - - - - -

) D-Xylose - - - - - x) L-Xylose - - - - -

Adonitol - - - - - β Methyl-Xyloside - - - - - x) Galactose + + + + + x) D-Glucose + + + + + x) D-Fructose + + + + + x) D-Mannose + + + + +

L-Sorbose - - - - - x) Rhamnose - + - - +

Dulcitol - - - - -

Inositol - - - - - x) Mannitol - + - - + x) Sorbitol - + - - - α Methyl-D-Mannoside - - - - - α Methyl-D-Glucoside - - - - -

N Acetyl Glucosamine + + + + + x) Amygdaline + - - + -

Arbutine + - - + - x) Esculine + - + + - x) Salicine + - - + - x) Cellobiose + - + + - x) Maltose + + + + + x) Lactose + + + + + x) Melibiose + + - - + x) Saccharose + + + + + x) Trehalose + - + - +

Inuline - - - - - x) Melezitose - - - - - x) D-Raffinose + + + - +

Amidon + - + + -

Glycogene - - - - -

Xylitol - + - - + β Gentibiose + - + + -

D-Turanose - - - - -

D-Lyxose - - - - -

D-Tagatose - - - - -

D-Fucose - - - - -

L-Fucose - - - - -

D-Arabitol - + - - +

L-Arabitol - - . - .

Figure imgf000020_0001

* The Boehringer Mannheim Kit (Cat. No. 139084) used to determine the concentration of L- and D-lactic acid in the growth medium consists of a) glycylglycine. buffer, L-glutamic acid and stabilizers; b) β-nicotlnamide-adenine dinucleotide (NAD) ; c) 1100 U of glutamatepyruvate transaminase (GPT); and d) 3800 U of L-lactate dehydrogenase (for L-lactic acid) or D-lactate dehydrogenase (for D-lactic acid) (LDH) , in separate containers.

The sample. (0.1-10 ml) is pipetted into a cuvette (1 cm light path) containing a)+b)+c). The absorbance is read at 340 nm after which d) is added initiating the following reaction:

LDH 1) L-lactate + NAD ↔ pyruvate + NADH + H+.

The amount of NADH formed in the above reaction is stoichiometric with the concentration of lactic acid. The absorbance is measured at 340 nm after completion of the reaction (about 10 minutes). The difference in absorbance before and after reaction is calculated and compared to that of a blank (a cuvette not containing any sample). The amount of lactic acid in the sample is then calculated according to the following formulae:

Figure imgf000021_0001
where ΔA - absorbance difference of blank subtracted from that of sample, V = final volume (ml), v = sample volume (ml)

MW = molecular weight of the substance to be assayed, d = light path (cm) , e = absorption coefficient of NADH at 340 nm = 6.3 [1 × mmol-1 x cm-1]

It follows for L-lactic acid

Figure imgf000021_0002

2.018 × ΔA/E [g L-lactic acid/1 sample solution]

Figure imgf000021_0003
2.045 × ΔA/E [g D-lactic acid/1 sample solution] ** Catalogue No. 148261. Table 3

Fermentation patterns recorded in Bergey's Modified Medium No. SS28 SS129 SS131 SS151 SS258

1 Gram + + + + +

2 Catalase - - - - -

3 Oxydase - - - -

4 Motility - - - - -

5 Spores - - - - -

6 Strictly aerobic - - - - -

7 Microaerophilic + + + + +

8 Pigment - - - - -

9 Growth, 15°C - - - - -

10 Growth, 45°C + + + + +

11 Starch hydrolysis + - (-) + -

12 Arginine hydrolysis - - - - -

13 Nitrate reduced - - - - -

14 Gelatin liquefaction - - - - -

15 Casein hydrolysis - - - - -

16 Indole - - - - -

17 H2S - - - - -

18 D-Lactic acid + + + + +

19 L-Lactic acid + - + + -

20 Amygdalin + - + + -

21 L(+) Arabinose - - - - -

22 Cellobiose + - + + -

23 Aesculin hydrolysis + - + + -

24 D(-) Fructose + + + + +

25 D(+) Galactose + + + + +

26 D(+) Glucose, acid + + + + +

27 Glucose, gas - - - - -

28 Gluconate - - - - -

29 Lactose + + + + +

30 Maltose + + + + +

31 D(-) Mannitol - + - - +

32 D(+) Mannose + + + + +

33 D(+) Melezitose - - - - -

34 D(+) Melibiose + + + - +

35 D(+) Raffinose + + + - +

36 L(+) Rhamnose - + - - +

37 Ribose - - - - -

38 Salicin + - + + -

39 D(-) Sorbitol - + - - -

40 Sucrose + + + + +

41 Trehalose + - + - +

42 D(+) Xylose - - - - - Identification

SS28 identified as L. acidophilus/L. gasseri . 100% accordance with Sergey's Manual . No atypical reactions.

SS129 identified as L . salivarius subsp. salivarius . 87% accordance with Bergey 's Manual . Atypical reactions: mannose, trehalose, maltose, sorbitol.

SS131 identified as L. acidophilus/L . gasseri . 97% accordance with Bergey's Manual . Atypical reactions: amygdalin.

SS151 identified as L. crispatus .

100% accordance with Bergey 's Manual . No atypical reactions.

SS258 identified as L. salivarius subsp. salivarius . 97% accordance with Bergey 's Manual .

Atypical reactions: mannose.

EXAMPLE 2

Adhesion to squamous epithelial cells from pars oesophagea

A characteristic considered important for a probiotic strain is the ability to attach to the cells of the gastrointestinal tract . This attachment will enable the bacteria to multiply and colonize the stomach and the intestinal tract, thus contributing to maintaining a well-balanced microflora in the digestive tract.

It has been generally observed that the ability to attach to and colonize the surface of gastric epithelial cells is connected with host specificity (Lin, J.H.-C. and D.C. Savage, "Host specificity of the colonization of murine gastric epithelium by Lactobacilli", FEMS Microbiol . Letters 24 , 1984, pp. 67-71; Tannock, G.W. , O. Szylit, Y. Duval and P. Raibau, "Colonization of tissue surfaces in the gastrointestinal tract of gnotobiotic animals by Lactobacillus strains", Can. J. Microbiol . 28 , 1982, pp. 1196-1198; Kotarski, S.F., Savage, D.C, "Models for study of the specificity by which indigenous Lactobacilli adhere to murine gastric epithelia" , Inf. and Immun . 26 ,

1979, pp. 966-975; Fuller, R., "Ecological Studies of the Lactobacillus Flora Associated with the Crop Epithelium of the Fowl", J. Appl . Bacteriol . 36, 1973, pp. 131-139).

The five strains in question have been selected for their ability to attach to squamous epithelial cells of pars oesophagea from pig stomachs (Table 4).

Materials and Methods

The test is a slight modification of a test described by Fuller et al. (Appl. and Environ. Microbiol. 35 (3), 1978).

Isolation of epithelial cells from pigs

Newborn piglets which had not yet been fed were killed by an intracardiac overdose of pentobarbitone. The stomach was opened by a slit along the greater curvature, and the oesophageal and gastric walls were gently washed with phosphate buffered saline (PBS), pH 7.3. Squamous epithelial cells from the pars oesophagea were brushed off into 2.5 ml of PBS and homogenized gently. This preparation can be stored frozen at -20°C.

Test for adhesion of bacteria to epithelial cells

The bacteria (all Lactobacilli) were grown overnight at 37°C in MRS medium (Oxoid CM 359) . The test culture was diluted or centrifuged and resuspended in PBS, and the total count was adjusted by using a counting chamber to about 5x10' to 1x10° cells per ml. 0.2 ml of the epithelial cell suspension was mixed with 0.05 ml of bacterial suspension to give a ratio of approximately 100 bacteria to I epithelial cell. The mixture was rotated at 37°C for 30 minutes and examined for adhesion by phase-contrast microscopy. At least 5 cells were exam ined. Evaluation of the adhesion ability is described as : + = 4-15 bacteria, attached to the surface of 1 epithelial cell; ++ = 16-50 bacteria, attached to the surface of 1 epithelial cell, +++- = ≥ 50 bacteria, attached to the surface of 1 epithelial cell.

TABLE 4

Adhesion of bacteria to epithelial cells

Strain Adhesion

SS28 +++

SS129 +++ SS131 +++

SS151 +

SS258 ++

EXAMPLE 3

Acidification activity

In order to multiply in the intestinal tract, it is important that the bacteria can ferment the carbohydrates available in the partially decomposed feed, i.e. lactose from sowmilk and glucose, maltose and, to some extent, starch from the weaning feed.

One of the characteristics which is also considered important for a probiotic organism is the ability to produce lactic acid, thus reducing the pH, both in the stomach and in the upper part of the small intestine.

The five strains in question have been selected for acidification activity in media resembling partially decomposed feed.

The time it takes to reach the final pH is dependent on the growth conditions of the bacteria before the acidification test. Materials and methods

Acidification activity in glucose

From an overnight MRS culture of the strain in question, 0.10 ml was inoculated into 10 ml of MRS broth (Oxoid CM 359) of pH 6.2. After incubation at 37°C for up to 48 hours in an atmosphere containing 10% CO2 in H2, the pH was measured.

Acidification activity in lactose

From an overnight MRS culture of the strain in question, 0.10 ml was inoculated Into 10 ml of 9.5% reconstituted skim milk (SKM) which had been UHT treated and pasteurised. After incubation at 37°C for up to 48 hours in an atmosphere containing 10% CO2 in H2, the pH was measured.

Acidification activity in maltose/starch-containing medium

The medium (called MRS-P) was a modified MRS medium in which the glucose had been replaced by a mixture of maltose and soluble starch in equal amounts. The composition of the medium was as follows:

Bacteriological peptone 10.0 g

Bacto soytone 8.0 g

Yeast extract 4.0 g Soluble starch 10.0 g

Maltose 10.0 g

Tween 80 1.0 ml

K2HPO4 2.0 g

Sodium acetate, 3H2O 5.0 g Triammonium citrate 2.0 g

MgSO4, 7H2O 0.2 g

MnSO4, 4H2O 0.05 g

Demineralised water ad 1000 ml

pH = 6.2 From an overnight MRS culture of the strain in question, 0.10 ml was inoculated into 10 ml of MRS-P. After incubation at 37°C for up to 48 hours in an atmosphere containing 10% CO2 in H2, the pH was measured.

Typical final pH values are shown in Table 5.

EXAMPLE 4

Bile tolerance

It is considered important for a probiotic organism to be resistant to the presence of bile in the digestive tract.

The amount of bile produced during 24 hours varies with the feeding time and the content of fat in the feed. In pigs the bile concentration is unlikely to exceed 0.9% in the contents of the small intestine. The strains in question have been selected for their tolerance to bile.

The bile tolerance was measured as the acidification activity in MRS (Oxoid CM 359) containing 0.9% of bile, added as 0.9 g of Bactooxgall (Difco B 128) per 1000 ml of broth.

Method

From an overnight-grown MRS broth of the strain in question, 0.10 ml was inoculated into 10 ml of MRS containing 0.9% of bile.

After incubation at 37°C for up to 48 hours in an atmosphere containing 10% CO2 in H2, the pH was measured.

Typical final values are shown in Table 5. EXAMPLE 5

Acid tolerance

An important criterion for a probiotic strain is the ability to survive the passage of the stomach. The HCl production in a pig's stomach increases during the first period of the piglet's life until it is fully developed after approximately two months. The pH of the stomach varies greatly, depending on the nature and amount of the stomach contents. The pH Is rarely less than 4.5 In a stomach containing feed, but pH in an empty stomach of a pig can be as low as 1.0-2.0.

The strains in question have been selected for their acid tolerance by testing the survival in a PBS medium of pH 2.5.

Materials and Methods

From a fully outgrown MRS culture, 0.10 ml was inoculated into 10 ml of phosphate buffered saline (PBS), pH 2.50 (adjusted with 0.1 N HCl).

Dilution series and plating on MRS was carried out at time 0 and 1 hour, and the log reduction was determined.

Typical reduction values are shown in Table 6.

TABLE 5

Acidification activities and bile tolerance (incubation at 37°C for up to 48 hours)

Typical final pH values

Medium

MRS MRS + 0.9% MRS-P SKM

Strain of bile

SS28 3.86 4.21 4.02 4.93

SS129 3.72 4.55 4.11 4.43

SS131 3.89 3.92 4.01 5.07

SS151 3.74 3.88 3.75 3.82

SS258 3.69 4.57 4.10 4.36

TABLE 6

Acid tolerance (log CFU/ml reduction in PBS, pH 2.5, 1 hour, 40°C)

Typical reduction values

Strain Log CFU/ml reduction

SS28 3.23

SS129 0.67

SS131 1.92

SS151 0.63

SS258 0.59 EXAMPLE 6

Susceptibility to feed additives

The five selected strains have been tested for susceptibility to a number of growth promoters and antibiotics frequently used as feed additives. The susceptibility was measured as the minimum inhibitory concentration (MIC) values by inoculating the organisms into two-fold dilution series of the individual additives in an MRS broth. The bacterial concentration was approximately 5×105/ml. The results are shown in Table 7.

TABLE 7

MIC values (ppm)

Feed additive SS28 SS129 SS131 SS151 SS258

Avoparcin <1.5 >100 25 100 >100

Olaqulndox >100 >100 >100 (>)100 >100

Carbadox 100 100 100 25 50

Flavofosfolipol 5 0.6 10 10 2.5

Nitrovin 31 >248 248 248 >248

Spiramycin >200 >200 3.1 3.1 6.3

Tylosln >62.5 >62.5 3.9 3.9 3.9

Virginiamycin 7.8 3.9 31 2.0 2.0

Zn-bacitracin 12.5 12.5 <1.6 12.5 12.5

ZnCl2 250 1000 250 1000 1000

EXAMPLE 7

Susceptibility to feed additives present in pig feed

The five selected strains which were all rifr were plated on solid MRS medium containing 50 μg/ml rifampicin and Incubated anaerobically for 48 hours at 37°C. One colony of each strain was grown anaerobically in liquid MRS medium containing 50 μg/ml rifampicin for 18 hours at 37°C after which the strains were mixed in a ratio of 1:1:1:1:1 CFU/ml to a total of 4-10×108 CFU/ml.

25 g of a pig starter feed (Pri-mor from A/S Korn- og Foderstof Kompagniet, Denmark) were crushed and mixed with 75 ml of water. The feed additives were added immediately prior to the addition of 2.5 ml (1.6×107 CFU/ml) of the mixed bacterial cultures. The mixture was incubated with stirring for 4 hours at 37°C after which the pH was measured and dilution series were prepared. The incubated mixture was plated on solid MRS medium containing 50 μg/ml rifampicillin.

The results are shown in Table 8 in which the figures are the average of three experiments.

TABLE 8

Feed additive Amount* CFU/ml pH

Avotan 40 ppm 7.4×107 5.68

Olaquindox 20 ppm 6.4×107 5.66

Carbadox 20 ppm 2.5×107 5.66

Flavomycin 25 ppm 5.3×107 5.69

Nitravin 30 ppm 2 ×107 5.79

Spiramycin 80 ppm 1.8×107 5.84

Tylosin 40 ppm 3.6×107 5.82

Virginiamycin 50 ppm 1.3×107 5.83

Zn-bacitracin 80 ppm 6.5×106 5.79

ZnCl2 100 mM 3.2x107 5.24

Control 0 7.6×107 5.61

* Maximum allowable dosage

It appears from Table 8 that the mixed bacterial cultures survive 4 hours of incubation in the feed containing the ten feed additives although bacterial growth is inhibited to a greater or lesser extent relative to the control. EXAMPLE 8

H2O2 production

It is characteristic of some Lactobacilli to be able to produce H2O2 which may show an inhibitory effect against other microorganisms.

The five selected strains were tested for the ability to produce H2O2 by a method described by Marshall, V.M. (J. Appl . Bacteriol . 47, 1979, pp. 327-328), slightly modified with respect to the basic growth medium (MRS instead of acetate agar).

The reactions are shown in Table 9.

TABLE 9

Strain H2O2 production

SS28 ++

SS129 - SS131 +

SS151 +

SS258 -

++ = strong production + = weak production

- = no production

EXAMPLE 9

Generation time

In order to be able to compete with the intestinal microflora, the probiotic lactic acid bacteria should have a brief generation time. When grown In MRS medium at 40°C under anaerobic conditions, the selected strains showed the generation times indicated in Table 10.

TABLE 10

Strain Generation time

SS28 62 minutes

SS129 40 minutes

SS131 50 minutes

SS151 47 minutes

SS258 34 minutes

EXAMPLE 10

Protective effect against experimentally induced E. coli diarrhoea in weaning pigs

The effect of the strains was investigated in weaning pigs challenged with E. coli 0149/K88.

28 pigs of both sexes from 3 litters, age 33-34 days, were removed from their mothers while still nursing and prior to exposure to solid feed. Each litter was divided into two groups, taking the weight and the sex of each animal into account in order to make the two groups as uniform as possible. All the pigs were ear-tagged, and faecal swab samples were taken from each animal.

One group from each litter was housed together as a treatment group, and the remaining three groups were housed together as the control group.

Both groups were housed in pens heavily contaminated with 0.5 1 each of a fully outgrown hemolytic E. coli 0149/K88 towards which the pigs were known to be sensitive. Each animal from the treatment group received an oral dose (by gavage) of a 10 ml mixture of equal amounts of overnight broths from the five strains = 2×109/dose on arrival (day 1) and twice a day for two weeks. Faecal swab samples were taken from each animal in both groups every morning on days 1-16, 19 and 22.

Results: E. coli 0149/K88 was isolated from all animals at some time during the period, peaking on days 7-11.

From the control group, 7 out of 14 pigs died with the typical symptoms of colienteroxemia. The diagnosis was verified by autopsy, followed by bacteriology and serotyping of E. coli 0149/K88.

From the treated group, only 1 out of 14 pigs died, E. coli 0149/K88 being the primary cause.

χ2 log rank = 5.95, p < 0.015

EXAMPLE 11

Effect on reducing the severity of experimentally induced E. coli diarrhoea In weaning pigs

In the experiment described in Example 10, all surviving pigs in the control group and in the group to which lactic acid bacteria were administered suffered from diarrhoea. In order to assess an effect on the severity of non-fatal cases, the body weights were recorded at the beginning of the experiment and after 14 days. The results are shown in Table 11. TABLE 11

Mean body weight, kg Average daily Day 0 Day 14 weight gain, g

Control group 9.16 10.60 101

Treatment group 13 8.45 11.70 231

The average daily weight gain of the Lactobacillus treated pigs was more than twice as high as that of the untreated group, indicating that the severity of the diarrhoeal outbreaks was reduced by the administration of the Lactobacillus cultures.

EXAMPLE 12

Field trial with a freeze-dried probiotic mixture

The field trial included 1260 newly weaned pigs from four herds, all suffering from weaning diarrhoea induced by E. coli 0149. 562 pigs were treated with a freeze-dried mixture of four probiotic strains (SS129, SS131, SS151 and SS258) which was administered as a "top dressing" in a dosage of 5×109 colony-forming units/pig/day for the first ten days of weaning. 437 pigs were treated conventionally with antibiotics, chemotherapeutic agens and/or a high dosage of zinc (positive control). The remaining 261 pigs did not receive any treatment (negative control). The results are shown in Table 12 below. TABLE 12

Group % days with Mortality diarrhoea (%)

Non-treated 11.2 5.4

Zn-antibiotics 0.8 0

Probiotic bacteria 6.3 1.6

It appears from the Table that the frequence of diarrhoea was reduced by 50% by treatment with the lactic acid bacteria of the invention. Likewise, the mortality rate was considerably reduced. In herds with a low infection pressure, a positive effect of the bacteria in the form of an increased daily weight gain throughout the weaning period was also observed.

Figure imgf000037_0001

Figure imgf000038_0001

Figure imgf000039_0001

Figure imgf000040_0001

Figure imgf000041_0001

Claims

1. A lactic acid bacterium isolated from the gastrointestinal tract of a pig and selected according to the following criteria:
a) adhesion to pig gastrointestinal epithelial tissue after incubation of bacteria with pig epithelial cells in a ratio of 100:1 in phosphate buffered saline at 37°C for 30 minutes,
b) production of lactic acid as determined by
i) growth in MRS (Oxoid CM 359) medium, pH 6.2, for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction to at least 4.0, or
ii) growth in 9.5% reconstituted skimmilk, pH 6.3-6.4, for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction to at least 5.2, or
iii) growth in modified MRS medium without glucose, pH 6.2, containing equal amounts of maltose and soluble starch for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction to at least 4.5, or
iv) defined as the production of at least 10 g/l of lactic acid (D- and L-forms) when grown in MRS medium, pH 6.2, for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2,
c) bile tolerance as determined by growth in MRS medium, pH 6.2, supplemented with 0.9% bile for up to 48 hours at 37°C in an atmosphere containing 10% CO2 in H2, giving rise to a pH reduction comparable to the one defined in b) (i)-(iii),
d) acid tolerance as determined by a reduction in the number of colony-forming units/ml after incubation for 1 hour at 40°C and a pH of 2.5 not exceeding 3.2 log units, preferably less than 1 log unit, e) a generation time of about 65 minutes, preferably 30-50 minutes, when grown in MRS medium at 40°C under anaerobic conditions,
or a functionally equivalent mutant thereof.
2. A bacterium according to claim 1, which adheres to pig gastrointestinal epithelial tissue in a number of at least 4, preferably at least 16, most preferably at least 50, bacteria per epithelial cell.
3. A bacterium according to claim 1 , which is able to produce an antimicrobial agent such as hydrogen peroxide.
4. A bacterium according to claim 1, wherein the strain is selected from the group consisting of: L. acidophilus/L. gasseri isolated from the duodenum of a suckling pig; L . salivarius subsp. salivarius isolated from the jejunum of a weaning pig; L. acidophilus/L. gasseri isolated from the ileum of a weaning pig; L. crispatus isolated from the jejunum of a suckling pig; and L . salivarius subsp. salivarius isolated from the pars oesophagea of a suckling pig.
5. A bacterium according to claim 4 which is selected from the group consisting of:
L . acidophilus/L. gasseri SS28 DSM 4324
L. salivarius subsp. salivarius SS129 DSM 4325 L. acidophilus/L . gasseri SS131 DSM 4326
L. crispatus SS151 DSM 4327
L . salivarius subsp. salivarius SS258 DSM 4328
6. A bacterium according to claim 5, which is L . salivarius subsp. salivarius SS129, DSM 4325, or L. salivarius subsp. salivarius SS258, DSM 4328.
7. A liquid or frozen concentrate comprising a lactic acid bacterium according to any of claims 1-6.
8. A concentrate according to claim 7, which comprises 1×104-1×1012 viable colony-forming units/ml.
9. A concentrate according to claim 7 , which comprises a mixture of two or more Lactobacillus strains according to any of claims 4-6.
10. A freeze-dried or spray-dried powder comprising a lactic acid bacterium according to any of claims 1-6.
11. A powder according to claim 10 which comprises 104-1012 viable colony-forming units/g.
12. A powder according to claim 10 which comprises a mixture of two or more Lactobacillus strains according to any of claims 4-6.
13. A fermented milk product, which comprises a lactic acid bacterium according to any of claims 1-6.
14. A fermented milk product according to claim 13, which additionally comprises L. acidophilus and/or Bifidobacterium spp.
15. A fermented milk product according to claim 13 or 14, which comprises a mixture of two or more Lactobacillus strains according to any of claims 4-6.
16. A veterinary composition for the prophylaxis or treatment of gastrointestinal tract infections in pigs caused by enteropathogenic microorganisms, the composition comprising a lactic acid bacterium according to any of claims 1-6 and an excipient or carrier.
17. A composition according to claim 16 which comprises 10 -1012 viable colony-forming units/g.
18. A composition according to claim 16 which is formulated as a powder, granulate, tablet, capsule, paste, gel, drench, mixture or suspension.
19. A composition according to claim 18, wherein the powder is the powder according to any of claims 10-12.
20. A composition according to claim 18 or 19, wherein the powder, granulate, tablet or capsule is provided with a coating which is degradable in a gastrointestinal environment.
21. A composition according to claim 20, wherein the coating is one which is soluble in the stomach or in the upper part of the small intestine.
22. A composition according to claim 18, wherein the suspension is a suspension of the powder of any of claims 10-12 in an aqueous or oily medium, such as in the drinking water of the pig, an electrolyte solution or in a reconstituted sowmilk replacer.
23. A composition according to any of claims 16-22 which comprises a mixture of two or more Lactobacillus strains according to any of claims 4-6.
24. A process of producing a lactic acid bacterium according to claim 1, the process comprising isolating and selecting a lactic acid bacterial strain in accordance with the criteria defined in claim 1, growing the so selected bacterium in a suitable medium under anaerobic conditions for a period of time sufficient to provide at least 10° viable lactic acid bacteria/ml of medium, and harvesting the resuiting bacteria from the medium.
25. Use of a lactic acid bacterium according to any of claims 1-6 for the production of a fermented milk product.
26. Use of a liquid or frozen concentrate according to any of claims 7-9 for the production of a fermented milk product.
27. Use of a freeze-dried or spray-dried powder according to .any of claims 10-12 for the production of a fermented milk product.
28. Use of a lactic acid bacterium according to any of claims 1-6 for preparing a veterinary composition for the prophylaxis or treatment of gastrointestinal tract infections in pigs caused by enteropathogenie microorganisms.
29. Use according to claim 28, wherein the pigs for which the composition is intended are suckling or weaning pigs or porkers.
30. Use according to claim 28, wherein the composition is adapted to be sprayed or sprinkled in the environment of the pigs .
31. Use according to claim 30, wherein the composition is in the form of a powder according to any of claims 10-12.
32. Use according to claim 28, wherein the composition is in the form of a suspension in an aqueous or oily medium, such as the drinking water of the pigs, an electrolyte solution or a reconstituted sowmilk replacer.
33. Use according to claim 28, wherein the composition is in a form suited for administration to a sow.
34. Use according to claim 28, wherein the composition is adapted to be mixed with the feed of weaning pigs or porkers.
35. Use according to claim 34, wherein the composition is in the-form of a powder according to any of claims 10-12.
36. Use according to any of claims 32-35, wherein the amount of lactic acid bacteria per dosage of the composition is in the range of 104-1012, preferably 106-10i0, colony-forming units.
37. A method of preventing or treating gastrointestinal infections in pigs caused by enteropathogenic microorganisms, the method comprising administering a therapeutically effective dosage of a lactic acid bacterium according to any of claims 1-6 to pigs.
38. A method according to claim 37, wherein the pigs are suckling or weaning pigs or porkers.
39. A method according to claim 37, wherein the lactic acid bacterium is administered as a powder according to any of claims 10-12, the powder being sprayed or sprinkled in the environment of the pigs.
40. A method according to claim 37, wherein the lactic acid bacteria are administered in the form of a suspension thereof in an aqueous or oily medium, such as the drinking water of the pigs, an electrolyte solution or a reconstituted sowmilk replacer.
41. A method according to claim 40, wherein the amount of lactic acid bacteria is in the range of 104-1012, preferably 106-1010, viable colony-forming units per dosage.
42. A method according to claim 41, wherein the lactic acid bacteria are administered 1-3 times a day, for a period of up to 14 days.
43. A method according to claim 37, wherein the lactic acid bacteria are administered in the form of a powder according to any of claims 10-12, the powder being admixed with the feed of the pigs.
44. A method according to claim 43, wherein the amount of lactic acid bacteria administered is in the range of 104-1012, preferably
106-1010, viable colony-forming units per dosage.
45. A method according to claim 44, wherein the lactic acid bacteria are administered 1-3 times a day, for a period of up to 14 days.
46. A method of preventing or treating gastrointestinal tract infections in pigs caused by enteropathogenic microorganisms, the method comprising administering to a gestating or lactating sow an amount of lactic acid bacteria according to any of claims 1-6 which is sufficient to provide a therapeutically effective amount of the bacteria in the environment of the sow.
47. A method according to claim 46, wherein the amount of lactic acid bacteria administered is in the range of 105-1012, preferably
107-1011, viable colony-forming units per dosage.
48. A method according to claim 47, wherein the lactic acid bacteria are administered 1-3 times a day for a period of up to 28 days.
49. A method according to any of claims 46-48, wherein the lactic acid bacteria are administered in the form of a powder according to any of claims 10-12.
PCT/DK1988/000222 1987-12-23 1988-12-23 Lactic acid bacteria for use in fermented milk products and veterinary compositions WO1989005849A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
DK6861/87 1987-12-23
DK686187A DK686187D0 (en) 1987-12-23 1987-12-23 veterinary medicinal

Publications (1)

Publication Number Publication Date
WO1989005849A1 true WO1989005849A1 (en) 1989-06-29

Family

ID=8149123

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1988/000222 WO1989005849A1 (en) 1987-12-23 1988-12-23 Lactic acid bacteria for use in fermented milk products and veterinary compositions

Country Status (2)

Country Link
DK (1) DK686187D0 (en)
WO (1) WO1989005849A1 (en)

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0577903A1 (en) * 1992-07-06 1994-01-12 Societe Des Produits Nestle S.A. Agent antigastrite contenant lactobacillus acidophilus
WO1995007090A1 (en) * 1993-09-06 1995-03-16 Zeneca Limited Probiotics
US5474932A (en) * 1991-07-25 1995-12-12 Probi Ab Intestine colonizing strains of lactobacilli
WO1997016198A1 (en) * 1995-11-02 1997-05-09 Chr. Hansen Biosystems A/S Probiotically acting formulation
WO1998035014A2 (en) * 1997-02-11 1998-08-13 Enterprise Ireland Trading As Bioresearch Ireland Probiotic strains from lactobacillus salivarius and antimicrobial agents obtained therefrom
EP0949330A2 (en) * 1998-04-10 1999-10-13 Proge Farm S.r.l. Lactobacilli strains having inhibitory and/or microbicidal activity against pathogenic microorganisms and a method for inducing and keeping said activity in lactobacilli cultures
WO2000010582A2 (en) * 1998-08-24 2000-03-02 Ganeden Biotech, Inc. Probiotic, lactic acid-producing bacteria and uses thereof
JP2001002578A (en) * 1999-06-24 2001-01-09 Yasuhiro Koga Helicobacter pylori-removing medicament
DE19945746C1 (en) * 1999-09-24 2001-04-19 Theodor Backs Gmbh New Lactobacillus salivarius DSM 12000, for preventing and treating digestive tract infections in ornamental birds and for use as a feed supplement
FR2826020A1 (en) * 2001-06-14 2002-12-20 Ceva Sante Animale Selecting strains of lactic acid bacteria, useful for controlling infections by pathogenic bacteria, based on adhesion to tissue, each other and determinants of the pathogen
WO2002103034A1 (en) * 2001-06-14 2002-12-27 Ceva Sante Animale Method of selecting non-pathogenic gram-positive lactic acid bacteria strains
FR2831555A1 (en) * 2001-10-29 2003-05-02 Ceva Sante Animale Selecting strains of lactic acid bacteria, useful for controlling infections by pathogenic bacteria, based on adhesion to tissue, each other and determinants of the pathogen
DE19943488B4 (en) * 1999-09-10 2004-10-07 Schäfer, Ulrich, Dipl.Agr.Ing. Agents for the treatment of pigeons
US7195906B2 (en) 1999-01-15 2007-03-27 Enterprise Ireland (Trading As Bioresearch Ireland) Bifidobacterium in the treatment of inflammatory disease
US7323166B2 (en) 2002-06-19 2008-01-29 Board Of Regents University Of Nebraska Lactic acid bacteria cultures that inhibit food-borne pathogens
US7785635B1 (en) 2003-12-19 2010-08-31 The Procter & Gamble Company Methods of use of probiotic lactobacilli for companion animals
GB2469059A (en) * 2009-03-31 2010-10-06 Univ Plymouth Fermented feed comprising lactic acid producing bacteria
US7906112B2 (en) 2003-12-19 2011-03-15 The Procter & Gamble Company Canine probiotic Lactobacilli
WO2011050426A2 (en) 2009-10-27 2011-05-05 "Selur Vk Holding" Eood New strains of lactic acid bacteria and their combinations producing probiotic preparations
US7998473B2 (en) 2003-12-19 2011-08-16 The Procter & Gamble Company Methods of treatment or prevention of gastrointestinal disorders using canine probiotic bifidobacterium
US8034601B2 (en) 2005-05-31 2011-10-11 The Procter & Gamble Company Feline probiotic bifidobacteria
GB2484126A (en) * 2010-09-30 2012-04-04 Univ Plymouth Foodstuff fermented with lactic acid producing bacteria
US8551473B2 (en) 2004-09-27 2013-10-08 Multigerm Uk Enterprises Ltd. Metabolically active micro organisms and methods for their production
US8563522B2 (en) 1997-07-08 2013-10-22 The Iams Company Method of maintaining and/or attenuating a decline in quality of life
GB2501999A (en) * 2009-03-31 2013-11-13 Univ Plymouth Fermented feed for pigs comprising Lactobacillus plantarum
US8691303B2 (en) 2009-07-31 2014-04-08 The Iams Company Dusted animal food
US8809035B2 (en) 2003-12-19 2014-08-19 The Iams Company Canine probiotic Bifidobacterium
US8846029B2 (en) 2005-09-28 2014-09-30 Nordisk Rebalance A/S Treatment of IBD and IBS using both probiotic bacteria and fermented cereal as treatment effectors
US8877178B2 (en) 2003-12-19 2014-11-04 The Iams Company Methods of use of probiotic bifidobacteria for companion animals
US9173423B2 (en) 2009-07-31 2015-11-03 The Iams Company Animal food kibble with electrostatically adhered dusting
US9192177B2 (en) 2005-05-31 2015-11-24 The Iams Company Feline probiotic Lactobacilli
US9210945B2 (en) 2009-07-31 2015-12-15 The Iams Company Animal food having low water activity
US9415083B2 (en) 2004-05-10 2016-08-16 Mars, Incorporated Method for decreasing inflammation and stress in a mammal
US9446111B2 (en) 2009-04-29 2016-09-20 Ganeden Biotech, Inc. Inactivated bacterial cell formulation
US9622502B2 (en) 2008-10-16 2017-04-18 Ganeden Biotech, Inc. Probiotic Bacillus pasta compositions
EP3168292A1 (en) 2015-11-06 2017-05-17 Probis Sp. z o.o. New lactobacillus plantarum strain amt14 and composition containing the strain of lactobacillus plantarum amt14
US9771199B2 (en) 2008-07-07 2017-09-26 Mars, Incorporated Probiotic supplement, process for making, and packaging
US10104903B2 (en) 2009-07-31 2018-10-23 Mars, Incorporated Animal food and its appearance
US10111916B2 (en) 2003-12-05 2018-10-30 Ganeden Biotech, Inc. Compositions comprising Bacillus coagulans spores and whey
US10166262B2 (en) 2017-04-20 2019-01-01 Ewelina Sosnowska-Turek Strain of bacteria and composition comprising the same

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0033584A2 (en) * 1980-01-11 1981-08-12 Farmos-Yhtyma Oy Process for the production of a bacterial preparation for the prophylaxis of intestinal disturbances in poultry
EP0199535A2 (en) * 1985-04-17 1986-10-29 Sherwood L. Gorbach Lactobacillus acidophilus strains of bacteria and compositions thereof
EP0203586A2 (en) * 1985-05-29 1986-12-03 Pioneer Hi-Bred International A composition for treating gastrointestinal disease in animals
EP0271364A2 (en) * 1986-12-12 1988-06-15 Biorem C.C. Preparation suitable for treating enteric disorders

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0033584A2 (en) * 1980-01-11 1981-08-12 Farmos-Yhtyma Oy Process for the production of a bacterial preparation for the prophylaxis of intestinal disturbances in poultry
EP0199535A2 (en) * 1985-04-17 1986-10-29 Sherwood L. Gorbach Lactobacillus acidophilus strains of bacteria and compositions thereof
EP0203586A2 (en) * 1985-05-29 1986-12-03 Pioneer Hi-Bred International A composition for treating gastrointestinal disease in animals
EP0271364A2 (en) * 1986-12-12 1988-06-15 Biorem C.C. Preparation suitable for treating enteric disorders

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF APPLIED BACTERIOLOGY, 1-39, 1980, Vol. 48, BARROW et al., "The Attachment of Bacteria to the Gastric Epithelium of the Pig and its Importance in the Microecology of the Intestine", p. 147-154, see p. 150, line 33-40. *

Cited By (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474932A (en) * 1991-07-25 1995-12-12 Probi Ab Intestine colonizing strains of lactobacilli
US5578302A (en) * 1992-07-06 1996-11-26 Nestec S.A. Treatment of stomach ulcers
EP0577903A1 (en) * 1992-07-06 1994-01-12 Societe Des Produits Nestle S.A. Agent antigastrite contenant lactobacillus acidophilus
WO1995007090A1 (en) * 1993-09-06 1995-03-16 Zeneca Limited Probiotics
WO1997016198A1 (en) * 1995-11-02 1997-05-09 Chr. Hansen Biosystems A/S Probiotically acting formulation
JP2002514068A (en) * 1997-02-11 2002-05-14 エンタープライズ アイルランド トレーディング アズ バイオリサーチ アイルランド Useful strains bio-derived Lactobacillus salivarius and it from the resulting antimicrobial agent
WO1998035014A2 (en) * 1997-02-11 1998-08-13 Enterprise Ireland Trading As Bioresearch Ireland Probiotic strains from lactobacillus salivarius and antimicrobial agents obtained therefrom
WO1998035014A3 (en) * 1997-02-11 1998-11-19 Forbairt Trading As Bioresearc Probiotic strains from lactobacillus salivarius and antimicrobial agents obtained therefrom
JP4743925B2 (en) * 1997-02-11 2011-08-10 エンタープライズ アイルランド トレーディング アズ バイオリサーチ アイルランド Useful strains bio-derived Lactobacillus salivarius and it from the resulting antimicrobial agent
US7186545B2 (en) 1997-02-11 2007-03-06 Enterprise Ireland Probiotic strains from Lactobacillus salivarius and antimicrobial agents obtained therefrom
AU748744B2 (en) * 1997-02-11 2002-06-13 Enterprise Ireland Trading As Bioresearch Ireland Probiotic strains from lactobacillus salivarius and antimicrobial agents obtained therefrom
US8563522B2 (en) 1997-07-08 2013-10-22 The Iams Company Method of maintaining and/or attenuating a decline in quality of life
EP0949330A2 (en) * 1998-04-10 1999-10-13 Proge Farm S.r.l. Lactobacilli strains having inhibitory and/or microbicidal activity against pathogenic microorganisms and a method for inducing and keeping said activity in lactobacilli cultures
EP1894996A1 (en) * 1998-04-10 2008-03-05 Proge Farm S.r.l. Lactobacilli strains having inhibitory and/or microbicidal activity against pathogenic microorganisms and a method for inducing and keeping said activity in lactobacilli cultures
EP0949330A3 (en) * 1998-04-10 2003-05-28 Proge Farm S.r.l. Lactobacilli strains having inhibitory and/or microbicidal activity against pathogenic microorganisms and a method for inducing and keeping said activity in lactobacilli cultures
WO2000010582A3 (en) * 1998-08-24 2000-07-13 Ganeden Biotech Inc Probiotic, lactic acid-producing bacteria and uses thereof
US6461607B1 (en) 1998-08-24 2002-10-08 Ganeden Biotech, Inc. Probiotic, lactic acid-producing bacteria and uses thereof
US8187590B2 (en) 1998-08-24 2012-05-29 Ganeden Biotech, Inc. Probiotic, lactic acid-producing bacteria and uses thereof
US7807151B2 (en) 1998-08-24 2010-10-05 Ganeden Biotech, Incorporated Probiotic, lactic acid-producing bacteria and uses thereof
JP2002523372A (en) * 1998-08-24 2002-07-30 ガネデン バイオテック, インコーポレイテッド Symbiotic lactic acid-producing bacteria and their use
US8097247B2 (en) 1998-08-24 2012-01-17 Ganeden Biotech, Inc. Probiotic, lactic acid-producing bacteria and uses thereof
WO2000010582A2 (en) * 1998-08-24 2000-03-02 Ganeden Biotech, Inc. Probiotic, lactic acid-producing bacteria and uses thereof
US7708988B2 (en) 1998-08-24 2010-05-04 Ganeden Biotech, Incorporated Probiotic, lactic acid-producing bacteria and uses thereof
US8697055B2 (en) 1998-08-24 2014-04-15 Ganeden Biotech, Inc. Probiotic, lactic acid-producing bacteria
US7195906B2 (en) 1999-01-15 2007-03-27 Enterprise Ireland (Trading As Bioresearch Ireland) Bifidobacterium in the treatment of inflammatory disease
JP4509250B2 (en) * 1999-06-24 2010-07-21 明治乳業株式会社 Helicobacterpylori disinfectant drugs
JP2001002578A (en) * 1999-06-24 2001-01-09 Yasuhiro Koga Helicobacter pylori-removing medicament
DE19943488B4 (en) * 1999-09-10 2004-10-07 Schäfer, Ulrich, Dipl.Agr.Ing. Agents for the treatment of pigeons
DE19945746C1 (en) * 1999-09-24 2001-04-19 Theodor Backs Gmbh New Lactobacillus salivarius DSM 12000, for preventing and treating digestive tract infections in ornamental birds and for use as a feed supplement
WO2002103034A1 (en) * 2001-06-14 2002-12-27 Ceva Sante Animale Method of selecting non-pathogenic gram-positive lactic acid bacteria strains
FR2826020A1 (en) * 2001-06-14 2002-12-20 Ceva Sante Animale Selecting strains of lactic acid bacteria, useful for controlling infections by pathogenic bacteria, based on adhesion to tissue, each other and determinants of the pathogen
FR2831555A1 (en) * 2001-10-29 2003-05-02 Ceva Sante Animale Selecting strains of lactic acid bacteria, useful for controlling infections by pathogenic bacteria, based on adhesion to tissue, each other and determinants of the pathogen
US7323166B2 (en) 2002-06-19 2008-01-29 Board Of Regents University Of Nebraska Lactic acid bacteria cultures that inhibit food-borne pathogens
US10111916B2 (en) 2003-12-05 2018-10-30 Ganeden Biotech, Inc. Compositions comprising Bacillus coagulans spores and whey
US8802158B2 (en) 2003-12-19 2014-08-12 The Iams Company Methods of use of probiotic Lactobacilli for companion animals
US8900569B2 (en) 2003-12-19 2014-12-02 The Iams Company Method of treating diarrhea in a canine
US7906112B2 (en) 2003-12-19 2011-03-15 The Procter & Gamble Company Canine probiotic Lactobacilli
US9580680B2 (en) 2003-12-19 2017-02-28 Mars, Incorporated Canine probiotic bifidobacterium pseudolongum
US9821015B2 (en) 2003-12-19 2017-11-21 Mars, Incorporated Methods of use of probiotic bifidobacteria for companion animals
US8900568B2 (en) 2003-12-19 2014-12-02 The Iams Company Method of treating diarrhea in a canine
US8894991B2 (en) 2003-12-19 2014-11-25 The Iams Company Canine probiotic Lactobacilli
US8877178B2 (en) 2003-12-19 2014-11-04 The Iams Company Methods of use of probiotic bifidobacteria for companion animals
US8840880B2 (en) 2003-12-19 2014-09-23 The Iams Company Canine probiotic bifidobacteria globosum
US7785635B1 (en) 2003-12-19 2010-08-31 The Procter & Gamble Company Methods of use of probiotic lactobacilli for companion animals
US8809035B2 (en) 2003-12-19 2014-08-19 The Iams Company Canine probiotic Bifidobacterium
US7998473B2 (en) 2003-12-19 2011-08-16 The Procter & Gamble Company Methods of treatment or prevention of gastrointestinal disorders using canine probiotic bifidobacterium
US9415083B2 (en) 2004-05-10 2016-08-16 Mars, Incorporated Method for decreasing inflammation and stress in a mammal
US8551473B2 (en) 2004-09-27 2013-10-08 Multigerm Uk Enterprises Ltd. Metabolically active micro organisms and methods for their production
US9404162B2 (en) 2005-05-31 2016-08-02 Mars, Incorporated Feline probiotic bifidobacteria and methods
US8034601B2 (en) 2005-05-31 2011-10-11 The Procter & Gamble Company Feline probiotic bifidobacteria
US9192177B2 (en) 2005-05-31 2015-11-24 The Iams Company Feline probiotic Lactobacilli
US9427000B2 (en) 2005-05-31 2016-08-30 Mars, Incorporated Feline probiotic lactobacilli composition and methods
US8846029B2 (en) 2005-09-28 2014-09-30 Nordisk Rebalance A/S Treatment of IBD and IBS using both probiotic bacteria and fermented cereal as treatment effectors
US9205114B2 (en) 2005-09-28 2015-12-08 Nordisk Rebalance A/S Probiotic fermented cereal compositions and methods for treatment of gastrointestinal diseases caused by pro-inflammatory bacteria
US8900570B2 (en) 2005-09-28 2014-12-02 Nordisk Rebalance A/S Treatment of IBD and IBS using both probiotic bacteria and fermented cereal as treatment effectors
US9771199B2 (en) 2008-07-07 2017-09-26 Mars, Incorporated Probiotic supplement, process for making, and packaging
US9622502B2 (en) 2008-10-16 2017-04-18 Ganeden Biotech, Inc. Probiotic Bacillus pasta compositions
GB2501999A (en) * 2009-03-31 2013-11-13 Univ Plymouth Fermented feed for pigs comprising Lactobacillus plantarum
GB2469059A (en) * 2009-03-31 2010-10-06 Univ Plymouth Fermented feed comprising lactic acid producing bacteria
WO2010112861A1 (en) * 2009-03-31 2010-10-07 University Of Plymouth Components for animal feed and use thereof
GB2469059B (en) * 2009-03-31 2014-01-08 Univ Plymouth Components for animal feed and use thereof
CN102480995A (en) * 2009-03-31 2012-05-30 普利茅斯大学 Components for animal feed and use thereof
CN102480995B (en) 2009-03-31 2014-06-04 普利茅斯大学 Components for animal feed and use thereof
US9757442B2 (en) 2009-04-29 2017-09-12 Ganeden Biotech, Inc. Inactivated bacterial cell formulation
US9446111B2 (en) 2009-04-29 2016-09-20 Ganeden Biotech, Inc. Inactivated bacterial cell formulation
US9210945B2 (en) 2009-07-31 2015-12-15 The Iams Company Animal food having low water activity
US9173423B2 (en) 2009-07-31 2015-11-03 The Iams Company Animal food kibble with electrostatically adhered dusting
US8691303B2 (en) 2009-07-31 2014-04-08 The Iams Company Dusted animal food
US10104903B2 (en) 2009-07-31 2018-10-23 Mars, Incorporated Animal food and its appearance
WO2011050426A2 (en) 2009-10-27 2011-05-05 "Selur Vk Holding" Eood New strains of lactic acid bacteria and their combinations producing probiotic preparations
EP2634269A1 (en) 2009-10-27 2013-09-04 "Selur Vk Holding" Eood New strain of lactic acid bacteria and its combinations producing probiotic preparations
GB2484126A (en) * 2010-09-30 2012-04-04 Univ Plymouth Foodstuff fermented with lactic acid producing bacteria
WO2012042223A2 (en) 2010-09-30 2012-04-05 University Of Plymouth Fermented foodstuff
EP3168292A1 (en) 2015-11-06 2017-05-17 Probis Sp. z o.o. New lactobacillus plantarum strain amt14 and composition containing the strain of lactobacillus plantarum amt14
US10166262B2 (en) 2017-04-20 2019-01-01 Ewelina Sosnowska-Turek Strain of bacteria and composition comprising the same

Also Published As

Publication number Publication date
DK686187D0 (en) 1987-12-23

Similar Documents

Publication Publication Date Title
Kabir The role of probiotics in the poultry industry
Fuller Probiotics in human medicine.
RU2308483C2 (en) Strain bifidobacterium longum infantis for probiotic production, anti-microbial agent and preparation based on strain bifidobacterium longum infantis with immunomodulating properties
Naidu et al. Probiotic spectra of lactic acid bacteria (LAB)
EP1541673B1 (en) Probiotics for pet food applications
Pollmann et al. Effects of microbial feed additives on performance of starter and growing-finishing pigs
Oyetayo et al. Safety and protective effect of Lactobacillus acidophilus and Lactobacillus casei used as probiotic agent in vivo
FERNANDES et al. Anticarcinogenic and immunological properties of dietary lactobacilli
EP0577904B1 (en) Lactic bacteria
US5709857A (en) Lactobacillus strains of human origin, their compositons and uses thereof
EP1036160B1 (en) Strain of bacteria of the species lactobacillus paracasei subsp. paracasei, composition thereof for use in food and product containing said strain
Vanbelle et al. Probiotics in animal nutrition: a review
RU2313572C2 (en) Bifidobacterium bifidum strain possessing galactosidase activity, galactooligosaccharide composition for stimulation of bifidobacterium growth, synbiotic composition for improving bowel state, their using (variants) for preparing medicinal preparation and method for preparing bifidobacterium growth stimulating agent
AU750136B2 (en) Probiotic lactic acid bacterium to treat bacterial infections associated with SIDS
Edens et al. Principles of ex ovo competitive exclusion and in ovo administration of Lactobacillus reuteri
CN100378214C (en) Probiotic bacterium: lactobacillus fermentum
US20020192202A1 (en) Probiotic compounds derived from lactobacillus casei strain KE01
AU734194B2 (en) Treatment of diarrhea
US8021654B2 (en) Methods of treating pigs with Bacillus strains
JP4176715B2 (en) Probiotic strain, selection methods, compositions thereof, and use thereof
US7150986B2 (en) Lactic acid bacteria-containing probiotics products
EP1185618B1 (en) A bacterial strain, processed plant extracts and probiotic compositions for human and veterinary use
Seo et al. Direct-fed microbials for ruminant animals
JP3186202B2 (en) Parent biological
Abe et al. Effect of administration of bifidobacteria and lactic acid bacteria to newborn calves and piglets

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): DK FI JP NO US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE