ES2367518T3 - LACTOBACILLI FELIN PROBIOTICS. - Google Patents

Abstract

A strain of lactic acid bacteria of the genus Lactobacilli obtainable by isolation from feline gastrointestinal tract removed and washed, which has probiotic activity, in which said strain is selected from the group consisting of Lactobacillus salivarius ss salicinius NCIMB 41287 (AHF 122A) , Lactobacillus animalis NCIMB 41288 (AHF 223C), Lactobacillus reuteri NCIMB 41289 (AHF 5119), mutants thereof and mixtures thereof.

Description

FIELD OF THE INVENTION

The present invention relates to the field of probiotic microorganisms, more specifically to feline probiotic lactic acid bacteria and methods of use.

BACKGROUND OF THE INVENTION

The defense mechanisms to protect the gastrointestinal (GI) tract of mammals from colonization by bacteria are extremely complex. The GI tract of most mammals is colonized by natural microflora and by invasive pathogenic microorganisms. In a state of health, this competing microflora is in a state of equilibrium. Modifying the balance of the intestinal microflora can lead to many GI disorders, or prevent them, both in humans and in other species of mammals, such as pets, including cats, dogs and rabbits. The welfare of companion animals is closely related to their feeding and GI health, and maintaining the balance of the intestinal microflora can improve the health of these companion animals.

The amount and composition of the intestinal microflora tends to be stable, although age and diet can change it. It is considered that gastric acidity, bile, intestinal peristaltic movement and local immunity are important factors in the regulation of bacterial flora in the small intestine of humans and other diverse mammals. Frequently, IC disorders of companion animals, including those observed in felines, are linked to bacterial overpopulation and the production of enterotoxins by pathogenic bacteria. These factors affect the balance of the intestinal microflora and can cause inflammation and autoimmune diseases.

In recent years, research has begun to discover some valuable strains of bacteria and their potential use as probiotic agents. Probiotics are prepared from bacteria, viable or dead, with constituents such as proteins or carbohydrates, or purified fractions of bacterial ferments that stimulate the health of mammals by preserving the natural microflora of the GI tract and strengthening the controls of autoimmune diseases. There are those who believe that probiotic bacteria are more effective if they are obtained from the species that are intended to be treated, or from species closely related to them. Therefore, for use in companion animals, probiotic strains obtained from companion animals, different from those obtained from humans, are necessary.

WO 01/90311 describes probiotic microorganisms isolated from faecal samples obtained from cats with probiotic activity. However, these bacteria were obtained from faecal samples and may not be part of the natural intestinal microflora present in the upper part of the GI tract.

The American Journal of Veterinary Research; vol. 32, dated September 9, 1971, pages 1399-1405, also describes microflora in the cat's alimentary tract. Some additional information can be found in The Journal of Nutrition, Ag. 2004, vol. 38, 8 Suppl .; pages 2022S-2026S. In The Veterinary Clinics of North America; Small Animal Practice, Mar 1999, vol. 29; Pages 523-550 published a study on bacterial overpopulation of the small intestine. In WO 03/010298 describes certain probiotic bacteria originating from the human gastrointestinal tract.

In US-2003/0190309 it refers to new probiotics for use in pet food.

G.W. Osbaldiston and others in Am. J. Vet. Res., Vol 32, No. 9, September 1971, pages 1399-1405, refers to a study of the microflora of the alimentary tract of cats.

The article by R. A. Rastall in Am. Soc. Nut. Sci., Vol 134, No. 8, August 2004, pages 2022-2026 refers to a study of bacteria in the intestine.

The article by Johnston K.L. in Veterinary clinics of North-America: Small animal practice, vol. 29, No. 2, March 1999, pages 523-550, refers to bacterial overpopulation in the small intestine.

In W003 / 010298 it refers to the strains of a probiotic Lactobacillus Salivarus.

Consequently, there is a need for strains of bacteria that can be obtained by isolating the natural intestinal microflora present in the upper part of the GI tract that are specially adapted to cats, and that have been selected for their probiotic properties and their ability to survive the processing, and incorporate said strains into compositions suitable for use.

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SUMMARY OF THE INVENTION The present invention relates to a strain of lactic acid bacteria of the genus Lactobacilli obtainable by isolation of the gastrointestinal tract of felines removed and washed, with probiotic activity, wherein said strain is selected from the group formed by Lactobacillus salivarius ss. Salicinius NCIMB 41287 (AHF 122A),

Lactobacillus animalis NCIMB 41288 (AHF 223C), Lactobacillus reuteri NCIMB 41289 (AHF 5119); mutants of the same and mixtures thereof. In addition, the present invention is directed to provide uses of lactic acid bacteria obtainable by isolation

of gastrointestinal tract of felines removed and washed, to maintain and improve the health of pets, and

Compositions comprising lactic acid bacteria. These features and other features, aspects and advantages of the present invention will be apparent to Those skilled in the art after reading the present example.

Sequences

I KNOW THAT. No. 1 - 16s-23s nucleotide sequence of the intergenic spacer of Lactobacillus salivarius subsp. Salicinius, NCIMB 41287 I KNOW THAT. No. 2 - 16s-23s nucleotide sequence of the intergenic spacer of Lactobacillus animalis, NCIMB 41288 I KNOW THAT. No. 3 - 16s-23s nucleotide sequence of the intergenic spacer of Lactobacillus reuteri, NCIMB 41289 I KNOW THAT. # 4 - Left 16s-23s PCR primer sequence for sequencing. I KNOW THAT. # 5 - Right 16s-23s PCR primer sequence for sequencing. Bacterial Deposit Numbers The following table indicates the Lactobacillus species and numbers of strains of the strains that are examples of the

present invention Bacterial strains are deposited in the body "National Collections of Industrial Food and Marine Bacteria (NCIMB) ”, Aberdeen, United Kingdom.

Strain

Deposit number 16s-23s sequence

Lactobacillus salivarius subsp. salicinius

NCIMB 41287 I KNOW THAT. # 1

Lactobacillus animalis

NCIMB 41288 I KNOW THAT. # 2

Lactobacillus reuteri

NCIMB 41289 I KNOW THAT. # 3

DETAILED DESCRIPTION OF THE INVENTION

All weights, measurements and concentrations herein are measured at 25 ° C in the composition as a whole, unless otherwise indicated.

Unless otherwise indicated, all percentages of compositions referred to herein are weight percentages and all ratios are weight ratios.

Unless otherwise indicated, all molecular weights are average molecular weights by weight.

Except in cases where the actual measurement values of specific examples are detailed, the numerical values referred to herein should be considered accompanied by the word "approximately".

In the following description the abbreviation UFC ("colony forming units") designates the number of bacterial cells obtained by microbial count on agar plates, in the manner generally understood in the art.

As used herein, the term "mutants thereof" includes derived bacterial strains that comprise DNA mutations in other DNA sequences of the bacterial genome, excluding the 16s-23s intergenic sequence.

As used herein, the term "DNA mutations" includes natural or induced mutations that comprise, at a minimum, single base alterations including deletions, insertions, transversions, and other DNA modifications known to those skilled in the art. , including genetic modifications introduced into a precursor nucleotide or amino acid sequence while maintaining at least 50% homology with respect to the precursor sequence. Preferably, the sequence comprising the mutation or DNA mutations has, with respect to the parental sequence, a homology of at least 60%, more preferably, at least 75%, even more preferably 85%. As used herein, "homology" of sequences can be determined using standard techniques known to those skilled in the art. For example, homology can be determined using the "BLAST" online homology algorithm program publicly available at http://www.ncbi.nlm.nih.gov/BLAST/.

As used herein, "genetic modification" includes the introduction of exogenous and / or endogenous DNA sequences into the genome of an organism, either by insertion into the genome of that organism, either by vectors including plasmid or bacteriophage DNA. as those known to those skilled in the art, said DNA sequence having a length of at least two bases of deoxyribonucleic acid.

Here, "companion animal" means a pet. Preferably, "companion animal" means a feline (cat), a can (dog), rabbit, ferret, horse, cow, or similar, domestic animal. More preferably, "companion animal" means a domestic feline.

Lactobacilli strains of lactic acid

The first aspect of the present invention comprises a strain of lactobacilli lactic acid bacteria obtainable by isolation from the gastrointestinal tract of feline, removed and washed, which has probiotic activity in animals. Probiotics are viable or dead microorganisms, processed compositions of microorganisms, their constituents such as proteins or carbohydrates, or purified fractions of bacterial ferments that beneficially influence a host. Probiotic bacteria are generally used in the form of viable cells. However, this use can be extended to non-viable cells such as dead cultures or compositions that contain the beneficial factors expressed by probiotic bacteria. This may include thermally killed microorganisms, or killed microorganisms by exposure to an altered pH or under pressure. For the purposes of the present invention, it is envisioned that the term "probiotics" also includes the metabolites generated by the microorganisms of the present invention during fermentation, in case they are not cited separately. These metabolites can be released into the fermentation medium, or they can be stored inside the microorganism. As used herein, "probiotics" also includes bacteria, homogenized bacteria, bacterial proteins, bacterial extracts, bacterial ferment supernatants, and mixtures thereof, which perform beneficial functions in the animal that receives them when it is received. administered in therapeutic doses.

It has been found that lactobacilli lactic acid bacteria obtainable directly by isolation from the mammalian GI tract, removed and washed, adhere to the GI tract after feeding viable bacterial cells, and also have a considerable immunomodulatory effect when they administer to animals in a viable, non-viable or fractional way. Without intending to impose any theory, it is believed that Lactobacilli obtainable by isolation from the GI tract removed and washed are closely associated with intestinal mucosa tissues. Likewise without intending to impose any theory, it is believed that this leads to the probiotic Lactobacilli of the present invention generating alternative host responses that cause their probiotic action. It has been found that probiotic bacteria obtainable by isolation from the removed and washed GI tract can modulate the host's immune system by direct interaction with the mucosal epithelium and the host's immune cells. This immunomodulation, together with the traditional mechanism of action associated with probiotic bacteria, that is, the inhibition of pathogenic adhesion to the intestine by occlusion and competition for nutrients, makes the Lactobacilli of the present invention very effective as probiotic organisms. .

The Lactobacilli the present invention, obtainable by isolation from GI tract of feline removed and washed, possess in vitro antimicrobial activity against a series of pathogenic bacterial strains / species. & Without intending to impose any theory, it is believed that this in vitro antimicrobial activity It is indicative of a potential probiotic activity in vivo in animals, preferably companion animals such as felines. The lactic acid bacteria of the present invention preferably have an in vitro antimicrobial activity against Salmonella typhimurium, Listeria monocytogenes, Listeria innocua or Eschericia coli, more preferably a mixture of these strains and, even more preferably, all these strains.

Without intending to impose any theory, it is believed that the antimicrobial activity of the lactic acid bacteria of the present invention may be the result of a series of different actions of the lactic acid bacteria herein. It has previously been suggested in the art that several strains of bacteria isolated from stool samples exert a probiotic effect on the GI tract after oral use, inhibiting the adhesion of pathogenic organisms to the intestinal mucosa by occlusion. This requires oral consumption of "live" or viable bacterial cells, so that a colony of bacteria is established in the intestine. However, it is believed that the Lactobacilli of the present invention, obtainable by isolation from the GI tract of felines removed and washed, although exerting a certain probiotic effect due to occlusion if administered in a viable manner, can have a considerable effect. probiotic, both in a viable and non-viable way, because during the in vitro fermentation a substance or substances that inhibit the growth of pathogenic microorganisms are produced or killed, and / or alter the immune competence of the host animal. This form of probiotic activity is desirable, since the bacteria of the present invention can be administered in the form of cultures.

or purified fermentation products both viable and non-viable, always providing a beneficial therapeutic effect to the host animal.

Preferably, the lactic acid bacteria of the present invention are capable of maintaining their viability during transit through the GI tract. This is desirable so that the live cultures of the bacteria can be administered orally, and so that colonization occurs in the intestines and in the large intestine after transit through the esophagus and stomach. Colonization of the intestines and large intestine by the lactic acid bacteria of the present invention is desirable in order to provide the host with long-lasting probiotic advantages. Oral dosing of non-viable cells or purified material isolated from them produces temporary advantages but, since the bacteria are not viable, they cannot multiply and continuously produce an in situ probiotic effect. As a consequence, this may require the host to receive a periodic dose, to maintain the health benefits. On the contrary, viable cells capable of surviving gastric transit in a viable way and then forming colonies adhering to the intestinal mucosa and can proliferate in it, can continuously provide probiotic effects in situ.

Therefore, it is preferable that the lactic acid bacteria of the present invention retain their viability after being suspended in a pH medium of 2.5 for 1 hour. As used herein, "conserving viability" means that at least 25% of the bacteria initially suspended in the assay medium are viable using the plate counting method known to those skilled in the art. Preferably, "conserving viability" means that at least 50% of the initially suspended bacteria are viable. It is desirable that the lactic acid bacteria of the present invention retain their viability after being exposed to a low pH, since this mimics the exposure to gastric juices of the stomach and upper intestine in vivo after oral intake by animals.

Furthermore, it is preferable that the lactic acid bacteria of the present invention have a growth of at least 33% when they are in the presence of at least 0.5% of bile salts of felines. The growth, as used herein, is described in more detail in Example 3. More preferably, the bacteria of the present invention have at least 33% growth in the presence of at least 1% of bile salts of felines Even more preferably, the bacteria of the present invention have a 100% growth in the presence of 0.5% feline bile salts. Without intending to impose any theory, it is believed that the lactic acid bacteria of the present invention, capable of preserving the viability in the presence of at least 0.5% of bile salts of felines, can survive in the conditions existing in the intestine. This is considered to be the result of the addition of feline bile to the culture medium, mimicking the conditions of the intestine.

Additionally, it is preferable that the lactic acid bacteria of the present invention have significant adhesion to intestinal epithelial cells in vitro. As used herein, "significant adhesion" means that at least 1% of the total number of lactic acid bacteria incubated together with in vitro epithelial cells adhere to epithelial cells. More preferably, at least 5% of the co-incubated bacterial cells adhere to the epithelial cells in vitro. Without intending to impose any theory, it is believed that adhesion to intestinal epithelial cells in vitro is an indication of the ability of lactic acid bacteria to colonize an animal's GI tract in vivo.

Preferably, the strain of lactic acid bacteria according to the present invention is of a species selected from the group comprising Lactobacillus salivarius, Lactobacillus animalis, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus johnsonni, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus , Lactobacillus curvatus, Lactobacillus delbruekii, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, or Lactobacillus plantarum. Preferably, the lactic acid bacteria strain is selected from the group comprising Lactobacillus salivarius, Lactobacillus animalis, Lactobacillus reuteri, Lactobacillus acidophilus,

or Lactobacillus johnsonni.

The 16s-23s sequence of intergenic polynucleotides is known to those skilled in the art as the DNA sequence of the bacterial genome that can be used to identify different species and strains of a bacterium. This intergenic polynucleotide sequence can be determined with the method detailed below in Example 4.

In a preferred embodiment of the present invention, the lactic acid bacteria strain, according to the present invention, is selected from the group comprising the Lactobacilli with a 16s-23s sequence of polynucleotides selected from SEQ. No. 1, SEQ. No. 2, or SEQ. No. 3. More preferably, the lactic acid bacteria strain, according to the present invention, is selected from the group comprising Lactobacillus salivarius ss salicinius NCIMB 41287 (AHF 122A), Lactobacillus animalis NCIMB 41288 (AHF 223C), Lactobacillus reuteri NCIMB 41289 (AHF 5119) or a mutant thereof.

The strain of lactic acid bacteria of the genus Lactobacilli obtainable by isolation from feline gastrointestinal tract, removed and washed, can be used to provide probiotic advantages after oral consumption by animals, preferably pets, or humans. This probiotic advantage generally maintains and improves the general health status of the animal. Non-limiting elements of animal health and physiology that benefit, whether by therapeutically relieving symptoms or preventing diseases by prophylaxis, include inflammatory disorders, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancers (especially those of the gastrointestinal systems and immune), diarrhea, antibiotic-associated diarrhea, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, amyloidosis, rheumatoid arthritis, arthritis, joint mobility, diabetes mellitus, insulin resistance, bacterial infections, viral infections, infections by fungi, periodontosis, urogenital diseases, trauma associated with surgery, postsurgical metastases, sepsis, weight loss, weight gain, excessive accumulation of adipose tissue, anorexia, fever control, cachexia, wound healing, ulcers, barrier infection of the intestine, allergy, asthma, respiratory disorders, circulatory disorders, coronary heart disease, anemia, blood clotting system disorders, kidney disease, central nervous system disorders, liver disease, ischemia, nutritional disorders, osteoporosis, endocrine disorders, and dermatological disorders. The treatment of the gastrointestinal tract, including the treatment or prevention of diarrhea, is preferred; the regulation of the immune system, preferably the treatment or prevention of inflammation or autoimmune disease; maintain and improve the health of the skin and / or fur system, preferably treat or prevent atopic dermatitis; improve or reduce the effects of aging, including activity levels and mental awareness; prevent disorders associated with the hypothalamus-pituitary-adrenal axis, and improve joint health to improve mobility.

The treatment of the disorders described above can be measured using techniques known to those skilled in the art. For example, inflammatory disorders including autoimmune diseases and inflammation can be detected and monitored using in vivo immune function tests such as lymphocyte blastogenesis, natural killer cell activity, antibody response to vaccines, hypersensitivity delayed and mixtures thereof. Such methods are briefly described herein, but are well known to those skilled in the art.

one.

Lymphocyte blastogenesis: This assay measures the in vitro proliferation response of lymphocytes isolated from fresh whole blood from test and control animals against various mitogens, and is a measure of the overall function of B and T cells. Concisely explained, Peripheral blood mononucleocytes (PBMC) are isolated from whole blood by Ficoll-Hypaque density centrifugation methods known to those skilled in the art. The isolated PBMCs are washed twice in a RPMI 1640 cellular medium supplemented with HEPES, L-glutamine and penicillin / streptomycin. The washed cells are resuspended in RPMI 1640, counted and cell density adjusted appropriately. The 2x105 cells are exposed to a range of concentrations (0.1 µg / ml to 100 µg / ml) of various mitogens, including examples of the mitogen of Phytolacca americana (Gibco), phytohemagglutinin (Gibco) and conconavalin A (Sigma ), in triplicate, for 72 hours at 37 ° C, and 5% CO2 with 10% fetal bovine serum (Sigma). At 54 hours, the cells are pulsed with 1 µCi 3H-thymidine, and the cells are collected and scintillation counts are read in a TopCount NXT at 72 hours.

2.

Natural killer cell activity: As described in US 6,310,090, this assay measures the in vitro effector activity of natural killer cells isolated from fresh whole blood from test and control animals. Natural killer cells are a component of the immune function of a mammal. Feline thyroid adenocarcinoma cells were used as target cells to evaluate the cytotoxic activity of NK (natural killer) cells. It had previously been shown that this cell line was susceptible to being killed by feline natural killer cells. The target cells were cultured in a T75 flask with 20 ml of minimum essential medium (MEM; Sigma Chem. Co., St. Louis, Mo.) supplemented with 10% fetal calf serum (FCS), 100 U / ml of penicillin and 100 µg / ml streptomycin. Once the confluence was reached, the target cells were treated with trypsin, washed 3 times and resuspended with 5x105 cells / ml in a complete medium (RPMI-1640 + 10% FCS + 100 U / ml penicillin + 100 µg / ml of streptomycin). Triplicate 100 µL aliquots of the white cells were pipetted into 96-well plates and U-shaped bottom (Costar, Cambridge, Mass.) And incubated for 8 hours to allow cell adhesion. Lymphocytes (effector cells; 100 µl) isolated by a Ficoll-Hypaque separation (as described above) were then added to the target cells to obtain a ratio of effector / target cells (E: B) of 10: 1. After incubation for 10 hours at 37 ° C., 20 µl of a substrate containing 5 µg of 3- (4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium bromide (MTT) was added. The mixture was incubated for 4 hours at 37 ° C, after which the non-metabolized MTT was extracted by aspiration. Formazan crystals were dissolved by adding 200 µl of 95% ethanol. The optical density was measured at 570 nm using a microplate reader. The percentage of specific lysis of NK cells was calculated as follows:

Specific cytotoxicity (%) = 100 x {1 - [(OD of target cells and effector cells

- OD of effector cells) / (OD of target cells)]}

3. Antibody response to vaccines: subjects who perform the test are given a series (of up to 5) vaccines after at least 12 weeks of control or probiotic feeding. Vaccines can be a mixture of new and redundant vaccines. Non-limiting examples of vaccine series that can be used include vaccine mixtures prepared by Fort Dodge Animal Health. Non-limiting examples of vaccines suitable for use in the present invention include the vaccine for canine distemper, adenovirus, coronavirus, parainfluenza and parvovirus. The vaccination history of the test subject will determine the vaccines to be used. Antibodies specific to the administered vaccines are measured in the blood for 3 weeks and the duration and resistance of the response in the probiotic and control feeding groups is compared.

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4. Delayed type hypersensitivity: in vivo, non-invasive method to assess the state of the immune system. This test includes an intradermal injection of the phytohemagglutinin polyclonal mitogen (PHA) together with sheep red blood cells as a multivalent vaccine, histamine (100 µl of 0.0275 g / l histamine phosphate; Greer, Lenoir, NC), or PBS (100 µl of Phosphate Buffered Saline, 8.5 g / l; Sigma). The immune response to the antigen is recorded as the thickness of the skinfold using calipers at time intervals of 0, 24, 48 and 72 hours after administering the injection. An increase in the thickness of the skin fold is indicative of a greater response to hypersensitivity that should be decreased by treatment with the bacteria of the present invention.

Additional methods for determining the effect of the Lactobacilli bacteria of the present invention are described in US 6,133,323 and US 6,310,090.

In addition, the improvement of the effects of age can be determined using double X-ray absorptiometry or a CT scan to measure body composition, including body fat mass, fat-free mass and content bone mineral. Similarly, this method can be used to determine changes in anatomy such as weight loss or bone density in the subjects after infection.

The Lactobacilli of the present invention can also be used in a method to reduce the stress levels of companion animals. Blood stress hormone concentrations, including epinephrine, norepinephrine, dopamine, cortisol and C-reactive protein, can be measured to determine stress levels and their reduction or maintenance. These hormones are recognized biological stress markers and can be easily measured using techniques known to those skilled in the art. Additionally, the direct measurement of the adrenal magnitude as an in vivo marker of the activity of the hypothalamic-pituitary-adrenal axis can be performed as a CT image.

In addition, the maintenance or improvement of the health of the skin or fur of domestic animals, including atopic skin diseases, can be measured using skin and fur evaluations conducted by two people with proper training. Examples of the criteria examined during these assessments include the following:

a) Hair loss index: each subject that performs the test is assigned a hair loss index picking up the hair produced during a standardized brushing session. The hair is stored and weighed, and the subjects who perform the test are compared with the control subjects.

b) Subjective evaluations of skin and fur: members formed from a panel subjectively assess the condition of the skin and the hair, assessing the change of hair, dandruff, shine, uniformity, smoothness and density.

c) Functional evaluation of the skin: the barrier function of the skin can be evaluated by passing a gauze soaked in acetone on the surface of the skin. This technique effectively destabilizes the skin barrier by eliminating the monocellular layers and associated lipid fractions of the corneal layer of the epidermis. Barrier destabilization is quantified by measuring the increase in transepidermal water loss (TEWL) and the degree of redness of the damaged site using methods known to those skilled in the art. Redness scores (erythema) are obtained using the camera and lighting system described above. TEWL readings and redness scores are obtained immediately before and after destabilization, and in evaluation criteria of 5 and 24 hours to assess the protective and healing properties of the skin.

The treatment or prevention of diarrhea in pets can be measured using stool scores. According to the present invention, the scores for depositions can be recorded daily according to the following guidelines and compare the control and test groups before and after ingestion with the bacteria.

Score: 5 Extremely dry

This deposition is hard and does not adhere to surfaces. The deposition will roll if pushed. No indentation occurs when collecting the deposition. Deposition is often defecated in groups of individual bowel movements rather than in a single unit. The deposition retains its original form after being collected.

Score: 4 Firm (ideal deposition)

This deposition is firm, well defined and cylindrical. This deposition does not break easily when picked up. This deposition may leave residue on surfaces and gloves. This deposition is often defecated as a unit. The deposition retains its original form after being collected.

Score: 3 Soft and defined

This deposition is soft, however, it has definite forms. This deposition will break easily and, without a doubt, will leave residue on surfaces and gloves. Deposition often loses its original form after being picked up. This deposition is often present with another score but may comprise the entire sample of the deposition.

Score: 2 Soft, shapeless

This deposition is soft and will not have a cylindrical shape. The form often associated with a "2" is a "cow dung" form. This deposition will lose its original shape when picked up and will leave, without a doubt, residue on the surfaces and gloves. The score of this deposition is often present with another score but may comprise the entire sample of the deposition. This sample of deposition can be spread over an area of several centimeters.

Score: 1 Liquid

Deposition with this score will always seem liquid and there may or may not be matter in the form of particles. This deposition will often be defecated in groups of batteries rather than in a complete unit. In this sample of deposition there are often mucus present. This sample of deposition is very difficult to collect and there is always residue on the surfaces and gloves. This sample of deposition can be spread over an area of several centimeters.

In addition, other observations are recorded, including: blood in the stool; foreign objects in the stool; or mucosa in the stool.

In addition, the treatment of gastrointestinal infections in companion animals may comprise the improvement of the microbial ecology of companion animals. Improving the microbial ecology of companion animals preferably comprises reducing pathogenic bacteria in the feces of companion animals. The levels of pathogenic bacteria present in the feces of domestic animals can be quantified using the standard plate counting technique known to those skilled in the art. More preferably, the pathogenic bacteria are selected from the group consisting of Clostridia, Escherichia, Salmonella, bacteroides and mixtures thereof. Among the non-limiting examples of suitable strains of pathogenic bacteria are: C. perfringens, C. difficile, Eschericia coli, Salmonella typhimurium and mixtures thereof.

The method of using the bacteria of the present invention may also include the treatment, whether prophylactic or therapeutic, of the urinary tract of mammals, preferably companion animals. Non-limiting examples of urinary tract treatment include treatment or prevention of urinary tract infections, treatment or prevention of kidney diseases, including urinary tract stones, treatment or prevention of bladder infections and the like. Without intending to impose any theory, it is believed that the Lactobacilli bacteria of the present invention are useful for preventing these ailments, as a result of their ability to degrade oxalic acid, as demonstrated in vitro. Oxalic acid is a byproduct of urinary metabolism, and it can form insoluble precipitates that cause infections in the kidney, bladder and other places in the urinary tract. By degrading oxalic acid and, consequently, potentially preventing its precipitation and accumulation in the urinary tract, the bacteria of the present invention can treat and prevent infections and other urinary tract ailments. Oxalic acid degradation can be measured in vitro using the oxalic acid test kit, catalog number 755699 marketed by Boehringer Mannheim / R-Biopharm.

The Lactobacilli of the present invention can be used in a method to improve and maintain the health of companion animals comprising the improvement of fiber digestion. The improvement of fiber digestion is desirable as it stimulates the growth of said probiotic bacteria, in addition to the beneficial endogenous microflora, which contributes to the suppression of some potentially pathogenic bacteria. In addition, a decrease in the amount of toxic metabolites and harmful enzymes that are the result of colonic fermentation has been documented in humans (Tomomatsu, H. "Health effects of oligosaccharides", (1994) Food Technol, 48, 61- 65). Fiber digestion can be determined using the method described in Vickers et al., "Comparison of fermentation of selected fructooligosaccharides and other fiber substrates by feline colonic microflora", (2001) Am. J. Vet. Res. 61 (4), p. 609-615, with the exception that instead of inoculating using diluted fecal samples, each experiment used pure cultures of the bacterial strains of interest. 6

The feline probiotic strains of the present invention can be used to reduce the smell of feces and urine and, concomitantly, in the hygienic litter box, reducing the production in the feces and urine of odor-producing compounds. Non-limiting examples of compounds that produce odors include ammonia, indoles, phenols, amines, branched-chain fatty acids and volatile sulfur-containing compounds. Without attempting to impose any theory, it is believed that reducing the levels of these compounds in the feces or urine of a companion animal reduces odors associated with feces or urine. In addition, for pets that use a hygienic litter box, the smell in the litter box is consequently reduced.

The method of use of the lactic acid bacteria of the present invention typically involves oral consumption by the animal. Oral consumption may be part of the normal dietary intake, or a supplement to it. Oral consumption is usually performed at least once a month, preferably at least once a week, more preferably at least once a day. The lactic acid bacteria of the present invention can be administered to the companion animal in a therapeutically effective amount, to maintain

or improve the health of the animal, preferably a companion animal. As used herein, the term "therapeutically effective amount" referred to lactic acid bacteria means the amount of bacteria sufficient to provide the effect or advantages to a host animal that requires treatment, but low enough to avoid adverse effects such as toxicity, irritation, or allergic responses, in accordance with a reasonable list of advantages / risks, when used in the manner of the present invention. The specific "therapeutically effective amount" will vary depending on factors such as the particular condition to be treated, the physical condition of the animal, the duration of the treatment, the nature of the concomitant treatment (if applicable), the specific pharmaceutical form used, the vehicle used, the solubility of the pharmaceutical form and the specific dosage.

Preferably, lactic acid bacteria are administered to the companion animal at a dose of 1.0E + 04 to 1.0E + 14 CFU per day, more preferably 1.0E + 06 to 1.0E + 12 CFU per day. Preferably, the composition may contain at least 0.001% of 1.0E + 04 to 1.0E + 12 CFU / g of lactobacilli lactic acid bacteria obtainable by isolation from feline GI tract removed and washed. Lactic acid bacteria can be administered to the animal in a viable way, either as dead cells, or distillates, isolated material or other fractions of the fermentation products of the lactic acid bacteria of the present invention, or any mixture thereof. .

Preferably, lactic acid bacteria, or a purified or isolated fraction thereof, are used to prepare a composition intended to maintain or improve the health of an animal. As indicated above, the composition may be part of the normal dietary intake, or a supplement. When the composition is part of the normal dietary intake, the composition may be in the form of dry animal feed, for example, biscuits or croquettes, processed cereal food, wet animal feed, yogurts, sauces, chewing foods, candies and the like .

Such compositions may comprise additional components. Other components are advantageous to be included as compositions used in the present invention, but are optional for the purposes of the present invention. For example, food compositions are preferably nutritionally balanced. In one embodiment, said food compositions may comprise, calculated with respect to the dry substance, 20% to 50% crude protein, preferably 22% to 40% crude protein, by weight of the food composition. The raw protein material may comprise any material that has a protein content of at least 15% by weight, non-limiting examples of which include vegetable proteins such as soybeans, cottonseed and peanuts, animal proteins like casein, albumin and meat tissue. Examples of meat tissue useful in the present invention include fresh meat and dried or processed flours such as fishmeal, poultry meal, meat meal, bone meal and the like. Other types of suitable raw protein sources include wheat gluten or corn gluten and proteins extracted from microbial sources such as yeast.

In addition, the food compositions may comprise, calculated with respect to the dry substance, from about 5% to about 35% fat, preferably from about 10% to about 30% fat, by weight of the food composition. Additionally, food compositions comprising the lactic acid bacteria of the present invention may also comprise from about 4% to about 25% of total dietary fiber. The compositions may also comprise a multiple source of starch, as described in WO99 / 51108.

The compositions of the present invention may also comprise a carbohydrate source. Grains or cereals such as rice, corn, milo, sorghum, barley, alfalfa, wheat, and the like are illustrative sources. In addition, the compositions may also contain other materials such as dried whey and other dairy by-products.

Compositions comprising the bacteria of the present invention may also comprise a prebiotic. The term "prebiotic" includes substances or compounds that are fermented by the intestinal flora of the domestic animal and, therefore, stimulate the growth or development of lactic acid bacteria in the gastrointestinal tract of the domestic animal at the expense of pathogenic bacteria. The result of this fermentation is a release of fatty acids, especially short chain fatty acids in the colon. This has the effect of reducing the pH value in the colon. Non-limiting examples of suitable prebiotics include oligosaccharides, such as inulin and its hydrolysis products commonly known as fructooligosaccharides, galacto-oligosaccharides, xyl-oligosaccharides or oligo derived from starch. Prebiotics can be supplied in any way that is appropriate. For example, the prebiotic can be supplied in the form of plant material that contains the fiber. Suitable plant materials include asparagus, artichokes, onions, wheat or chicory, or residues of these plant materials. Alternatively, prebiotic fiber can be supplied as an inulin extract, for example chicory extracts are suitable. Suitable inulin extracts can be obtained through Orafti SA of Tirlemont 3300, Belgium under the trade name "Raftiline". For example, inulin can be supplied in the form of Raftiline (g) ST which is a fine white powder containing between about 90% and about 94% by weight of inulin, up to about 4% by weight of glucose and fructose, and between approximately 4 and 9% by weight sucrose. Alternatively, the fiber may be in the form of a fructooligosaccharide such as that obtained from Orafti SA of Tirlemont 3300, Belgium under the trademark "Raftilose". For example, inulin can be supplied in the form of Raftilose (g) P95. On the other hand, fructooligosaccharides can be obtained by hydrolyzing inulin, by enzymatic methods, or by using microorganisms.

For dry pet food, extrusion cooking is a suitable process, although baking and other suitable processes can be used. When cooked by extrusion, dry pet food generally has the form of croquettes. If a prebiotic is used, the prebiotic can be mixed with the other ingredients of dry pet food, before processing. A suitable process is described in European Patent Application No. 0850569. If a probiotic microorganism is used, it is best that the body is covering the dry food for pets, or is filling it. European Patent Publication Number EP-0 862 863 describes a suitable process.

For wet foods, the processes described in US Patents 4,781,939 and US 5,132,137 can be used to produce meat-like products. Other methods can also be used to produce products in pieces; For example, cook in a steam oven. Alternatively, bread-type products can be manufactured, emulsifying a suitable meat material to produce a meat emulsion, adding a suitable gelling agent, and heating the meat emulsion before filling cans or other containers with it. The usual wet food compositions may comprise 5% to 15% protein, 1% to 10% fat and 1% to 7% fiber. Ingredients that, in a non-limiting manner, can be used in wet food compositions, include chicken, turkey, beef, white fish, chicken broth, turkey broth, beef broth, chicken liver, rice residues for brewing, ground corn, fishmeal, eggs, beet pulp, chlorides, flax flour, lamb, beef by-products, chicken by-products, and mixtures thereof.

In another embodiment, supplementary compositions such as cookies, chewing products and other treats may comprise, calculated with respect to the dry substance, from 20% to 60% protein, or from 22% to 40% protein, by weight of the supplementary composition. In another example, the supplementary compositions may comprise, calculated with respect to the dry substance, from 5% to 35% fat, or from 10% to 30% fat, by weight of the supplementary composition. Food and supplementary compositions intended for feline consumption are well known in the art.

Pet food may contain other active agents such as long chain fatty acids and zinc. Among the suitable long chain fatty acids are alpha linoleic acid, gamma linoleic acid. linoleic acid, eicosapentanoic acid, and docosahexanoic acid. Fish oils are suitable sources of eicosapentanoic acids and docosahexanoic acid.

Borage oil, blackcurrant seed oil and night primrose oil are suitable sources of gamma linoleic acid. Safflower oils, sunflower oils, corn oils and soybean oils are suitable sources of linoleic acid. These oils can also be used in the coating substrates mentioned above. Zinc can be supplied in various suitable ways, for example as zinc sulfate or zinc oxide. On the other hand, many ingredients commonly used in pet food are sources of fatty acids and zinc. It has been observed that the combination of chicory, as a prebiotic source and an oil rich in linoleic acid, for example soybean oil, brings unexpected advantages, suggesting that there is a synergy effect.

In cases where the composition is in the form of meat sauce, the composition may comprise at least 10% broth or consommé, whose non-limiting examples include the vegetable, beef, chicken or ham consommé. Typical sauce compositions may comprise 0.5% to 5% crude protein, 2% to 5% crude fats, and 1% to 5% fiber.

Additional, non-limiting examples of supplements suitable for use in the present invention include powders, oil suspensions, milk suspensions, cheeses, cocoa butter based compositions, and pills or capsules. When the composition is in the form of a pill, suitable binding agents are required for the pill to remain in a compressed solid form. Non-limiting examples of suitable binding agents include natural gums such as xanthan, pectins, lecithins, alginates and others known to those skilled in the art. When the composition is in capsule form, preferably the composition is encapsulated using techniques known to those skilled in the art. Non-limiting examples of suitable encapsulation materials include polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginates and gelatin. Yogurt-based compositions may comprise 1% to 5% protein, 10% to 20% carbohydrates, 1% to 5% fiber, 1% to 5% fat and 50% to 90% of a liquid vehicle like milk.

Examples

The following examples are provided to illustrate the invention and are not intended at all to limit the scope thereof.

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Example 1: Isolation of lactic acid bacteria from the feline GI tract

Intestinal samples of felines were obtained from healthy cats submitted to local veterinarians for euthanasia on the initiative and with the approval of their owners. All animals were healthy and disease free. The colon, middle colon, blind and ileum of each cat were dissected to expose the mucosa.

The supernatants were removed after agitation of the mucous tissue (with vortex shaker for 1 minute) and after mechanical homogenization of the tissue. Each supernatant was plated on Mann Rogosa Sharpe's agar (MRS). The plates were incubated anaerobically using the Anerocult GasPak system for 48 hours at 37 ° C. Colonies isolated from the plates reseeded on each MRS and re-incubated anaerobically under the same conditions. Isolated colonies were reseeded 4 more times to purify a single strain. Colony morphology and microscopic appearance were evaluated. In suitable isolates, the Gram reaction and catalase activity were tested. The identification of gram positive bacilli, catalase negative, was performed using the API test (API 50CHL, BioMerieux). The collected cells were washed twice with 0.05 M phosphate buffer (pH 6.5) and cysteine-HCl (500 mg / l) followed by sonic treatment. With centrifugation, cell debris was removed. The supernatants were incubated with NaF (6 mg / ml) and Na iodoacetate (10 mg / ml) for 30 minutes at 37 ° C. The reaction was stopped by incubation with hydroxylamine-HCl (pH6.5) for 10 minutes at room temperature. Color development was monitored after the addition of HCl (4M), FeCl3.6H2O (5% (w / v) in 0.1 M HCl) and fructose-6-phosphate (Na salt). The formation of acetyl phosphate from fructose-6-phosphate was evidenced by the reddish color formed by the ferric chelate of its hydroximate.

Example 2: Review of antimicrobial activity

Each of the bacterial strains of lactic acid was incubated anaerobically in an MRS medium. 2 µl of each culture were placed on MRS agar plates and incubated anaerobically overnight. Salmonella typhimurium and enteropathogenic E. Coli (ExPEC) were grown overnight and 100 µl inoculated in molten agar (1% v / v). This indicator culture was poured onto the surface of the inoculated MRS plates. After overnight incubation, the zones of inhibition around the probiotic colony were measured. All experiments were performed in duplicate on three separate occasions. In addition, the incorporation to the agar of the 2% beta-glycerophosphate buffer allowed the evaluation of the contribution of acid production to the pathogenic inhibition observed in vitro.

The data presented in Table 2 clearly demonstrates that the lactic acid bacteria strains of the present invention obtainable by isolation from the feline GI tract, removed and washed, possess considerable in vitro antimicrobial activity, indicative of a potential probiotic activity.

Table 2

 AHF122A

AHF223C AHF5119

S. typhimurium

9.34 7.5 9,665

EXPEC

 11.84 7.67 11.17

Example 3: In Vitro Survival and Colonization Measurements pH Tolerance Bacteria cells were collected from cultures overnight, washed twice in phosphate buffer (pH

6.5) and resuspended in an MRS / TPY medium adjusted with 1M HCl at pH 2.5. The cells were grown anaerobically at 37 ° C and their survival was measured at intervals of 0, 30, 60, 120, 240 and 360 minutes using the platelet count method known to those skilled in the art. Table 3 summarizes these data by strain.

Table 3 Survival of strains in a medium of low pH (pH 2.5). All data are UFC log counts.

Time (min.)

Strain

0 30 60 120 180 360

NCIMB 41287

9.31 9.13 8.95 8.88  8.84  8.52

NCIMB 41288

8.85 8.79 8.89 8.65  8.71  8.59

NCIMB 41289

9.25 9.06 8.97 9.10  9.00  8.88

Bile resistance

Bacterial strains were applied by stretch marks on MRS agar supplemented with 0.5%, 1% and 5% (w / v) porcine bile (Sigma). Plates were incubated at 37 ° C under anaerobic conditions and growth was recorded after 48 hours. An expert observer compared the growth with that of control plates, and the growth of the colonies was

5 described as follows:

Negative (0) - no growth;

+ (1) - Blurred translucent growth (<33% of control plates with 0% bile); ++ (2) - Clear growth, but not as good as the controls (> 33% but <66%); +++ (3) - Growth equivalent to that of controls (> 66%).

10 Once the growth of the colonies in the presence of bile salts has been compared with the controls, numerical values of 0, 1, 2 or 3 are assigned to the growth descriptors (-; +; ++, +++ respectively ), and then expressed as percentages, where 3 represents 100%.

Table 4 shows that the Lactobacilli of the present invention clearly demonstrate a resistance to bile salts, and are capable of growing and forming colonies at a level of at least 66% in most cases, 15 when exposed to 0 , 3% porcine bile salts. Table 4 Survival of strains at various concentrations of swine bile.

BILIS PERCENTAGE (%)

Strains

0 0.3 0.5 one 2 5 7.5 10

NCIMB 41287

+++ + + - - - - -

NCIMB 41288

+++ +++ +++ ++  ++ ++  ++ +

NCIMB 41289

+++  +++ +++  +++ ++ ++ + +

In addition, to assess any difference in the ability of strains to colonize the GI tract of cats, the

20 bacterial strains were applied on MRS agar supplemented with 0.5%, 1% and 2% feline bile (w / v). Feline bile was obtained from cats undergoing endoscopy in a clinical setting during a non-terminal procedure. Plates were incubated at 37 ° C under anaerobic conditions and growth was recorded after 48 hours. An expert observer compared the growth with that of control plates, and the growth of the colonies was described as follows:

25 Negative (0) - no growth;

+ (1) - Blurred translucent growth (<33% of control plates with 0% bile);

++ (2) - Clear growth, but not as good as the controls (> 33% but <66%);

+++ (3) - Growth equivalent to that of controls (> 66%).

Once the growth of the colonies in the presence of bile salts has been compared with the controls, it

30 assigns to the growth descriptors numerical values of 0, 1, 2 or 3 (-; +; ++, +++ respectively), and then expressed as percentages, where 3 represents 100%.

Table 5 shows that the Lactobacilli of the present invention clearly demonstrate a resistance to bile salts, and are capable of growing and forming colonies at a level of at least 66% in most cases, when exposed to a 0, 5% feline bile salts.

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Table 5 Survival of strains at various concentrations of feline bile.

BILIS PERCENTAGE (%)

Strains

0 0.5 one 2

NCIMB 41287

+++  +++  +++ +++

NCIMB 41288

+++ +++ ++ ++

NCIMB 41289

+++  +++  +++ +++

Cellular adhesion to the intestinal epithelium

The human epithelial cell line HT-29 was used to evaluate the adhesion properties of selected strains. Epithelial cells were routinely cultured as monolayer in tissue culture flasks of 75 cm2 at 37 ° C in a humidified atmosphere containing 5% CO2 in Dulbecco's Minimal Essential Media (DMEM) containing 10% fetal calf serum (FCS) , pen / strep, glutamine and fungizona. For experimental purposes, epithelial cells were seeded at a concentration of 5 x 105 cells / ml (3 ml total volume) per well in 6 culture plates (Sarstedt). After a 7 day incubation, to allow differentiation, the epithelial monolayers were washed with an antibiotic-free medium containing 10% FCS. Bacterial suspensions plus / in antibiotic-free DMEM were added to each well and the cells were incubated for 90 minutes at 37 ° C. After incubation, the monolayers were washed three times with PBS. Lysis of epithelial cells was performed in deionized water, and the number of bacteria adhered to was counted using the plate count method known to those skilled in the art. Adhesion was expressed as a percentage of the initial number of bacteria on the plate. Lactobacillus AHF122A had an adhesion level of 39.5%, while Lactobacillus AHF223C had an adhesion level of 13.9%. Lactobacillus AHF5119 had a level of adherence of 36.7%.

Example 4: Sequence of 16s-23s intergenic polynucleotides

The isolated feline Lactobacilli were centrifuged in tubes and the resulting microgranules were dissolved in 100 µl of extraction solution and 25 µl of tissue preparation solution (Sigma, Kit XNAT2), incubated for 10 minutes at room temperature. The samples were then incubated for 5 minutes at 95 ° C and then 100 µl of neutralization solution (kit XNAT2) was added. The genomic DNA solution was quantified using a Nanodrop spectrophotometer, and stored at 4 ° C.

PCR was performed using intergenic spacer primers (IGS), IGS L: 5’-GCTGGATCACCTCCTTTC-3 ’and IGS

A: 5’-CTGGTGCCAAGGCATCCA-3 ’Bridgidi et al. 2000, System Appl. Microbiol., 23, 391-399 (2000)). The cycle conditions were 94 ° C for 3 min (1 cycle), 94 ° C for 30 sec, 53 ° C for 30 sec, 72 ° C for 30 sec (28 cycles). The PCR reaction contained 4 µl (50 ng) of DNA, PCR mixture (XNAT2 kit), 0.4 µM primer IGS L and R (MWG Biotech, Germany). PCR reactions were performed with an Eppendorf thermocycle apparatus. The PCR products (10 µl) were passed together with a molecular weight marker (100 bp Ladder, Roche) on a gel stained with 2% EtBr agarose in TAE, to determine the IGS profile.

Lactobacilli (single band) PCR products were purified using the Promega Wizard PCR purification kit.

The purified PCR products were sequenced using the primer sequences (cited) for the intergenic spacer region. Sequence data were contrasted with the NCBI nucleotide data bank to determine the identity of the strain by nucleotide homology.

After sequencing, the sequences obtained for the four deposited strains were compared with the online “BLAST” sequence database, available at http://www.ncbi.nlm.nih.gov/BLAST/ to determine homology with other bacterial sequences 16s-23s deposited. The best correspondence for AHF122A was the strain Lactobacillus salivarius subsp. Salicinius (AB102859) with a homology score of 94%. For the AHF223C, the most similar was the strain Lactobacillus animalis LA51 (AY526615) with a homology score of 93%. The closest to AHF5316 was Lactobacillus reuteri DSM 20016 (AF080100) with a homology score of 98%.

Example 5: Example compositions

Examples 1 to 4 are examples of dry croquette compositions comprising the Lactobacilli probiotics of the present invention.

Ingredient

Weight percentage

Ex. 1

Ex 2 Ex 3 Ex 4

Cereal grains

Up to 100 Up to 100 Up to 100 Up to 100

Poultry by-products flour

43.5 40 Four. Five 35

Poultry Fat

1.28  1.02 1.16  1.35

Egg products

2.4 2.1 2.5 2.2

Thick ground chicken liver

1.0  1.0 1.0  1.0

Dry Brewer's Yeast

1.0  1.0 1.0  1.0

Monosodium Phosphate

1.0  1.0 1.0  1.0

Calcium carbonate

0.8  0.8 0.8  0.8

Potassium chloride

0.6 0.6 0.6 0.6

Vitamins

0.4 0.4 0.4 0.4

Choline chloride

0.3 0.3 0.3 0.3

Minerals

0.3  0.3 0.3  0.3

DL-Methionine

0.1 0.1 0.1 0.1

Sodium chloride

0.03  0.03 0.03  0.03

Probiotic (1 x 1010 cfu / g NCIMB 41287 in sunflower oil)

one 0.5 - 0.6

Probiotic (1 x 1010 cfu / g NCIMB 41288 in sunflower oil)

- 0.5 one 0.4

Examples 5 to 7 are examples of wet food compositions for companion animals comprising the Lactobacilli probiotics of the present invention.

Ingredient

Weight percentage

Ex 5

Ex 6 Ex 7

Water

Until 38 Until 47 Up to 50

Poultry Liver

Until 25 Until 20 Until 15

Poultry Products

25 twenty twenty

Rice for brewing

5 7 10

Egg products

3 2.5 1.5

Poultry Fat

2.9 3.0 3.2

Chicken soup

0.6 0.7 0.9

Taurine

 0.1 0.1 0.1

Vitamins

 0.05 0.1 0.1

Minerals

 0.05 0.1 0.1

Probiotic (1 x 1010 cfu / g NCIMB 41289)

4 5 6

Examples 8 to 10 are examples of compositions with yogurt supplement comprising the Lactobacilli probiotics of the present invention.

Ingredient

Weight percentage

Ex 8

Ex 9 Ex 10

Milk

 38 42 48

Sugar

 12 12 10

Modified starch

1.0 0.8 0.8

Prebiotic

 0.25 0.3 0.5

Probiotic (1 x 1010 cfu / g NCIMB 41287)

4 5 6

Although certain embodiments of the present invention have been illustrated and described, it is obvious to the person skilled in the art that it is possible to make different changes and modifications without thereby leaving the scope of the invention. Accordingly, the following claims are intended to cover all such changes and modifications contemplated within the scope of the present invention.

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Claims (26)

one.

A strain of lactic acid bacteria of the genus Lactobacilli obtainable by isolation from feline gastrointestinal tract removed and washed, which has probiotic activity, in which said strain is selected from the group consisting of Lactobacillus salivarius ss salicinius NCIMB 41287 (AHF 122A) , Lactobacillus animalis NCIMB 41288 (AHF 223C), Lactobacillus reuteri NCIMB 41289 (AHF 5119), mutants thereof and mixtures thereof.

2.

The strain according to claim 1, capable of surviving and colonizing the gastrointestinal tract of a companion animal, wherein the companion animal is a cat.

3.

The strain according to claim 2, which has a growth of at least 33% when grown in the presence of 0.5% feline bile salts.

Four.

The strain according to claim 2, capable of maintaining viability after 1 hour at pH 2.5.

5.

The strain according to claim 1, wherein the lactic acid bacterial strain has a DNA sequence in the spacer region 16s-23s selected from SEQ. No. 1, SEQ. No. 2, or SEQ. # 3.

6.

The strain according to claim 1, wherein the mutant is a natural, genetically modified or induced mutant.

7.

The strain of any one of claims 1 to 6, for use in order to maintain or improve the health of a companion animal, wherein the companion animal is a cat.

8.

The strain for use according to claim 7, for the regulation or improvement of the immune system of pets, to treat or prevent autoimmune diseases in pets, to treat or prevent inflammations in pets, to maintain or improve health of the skin system and / or fur of pets, to treat or prevent atopic skin disease in pets, to improve or reduce the effects of aging on pets, to prevent weight loss during and after infection in pets, and to increase fiber digestion in pets.

9.

The strain for use according to claim 7, for treating or preventing gastrointestinal infections in companion animals.

10.

The strain for use according to claim 9, to improve the microbial ecology of companion animals.

eleven.

The strain for use according to claim 10, comprising reducing the levels of pathogenic bacteria in the feces of companion animals, wherein said pathogenic bacteria are selected from the group consisting of Clostridia, Escherichia, Salmonella, and mixtures of the same.

12.

The strain for use according to claim 8, for treating or preventing urinary tract ailments of companion animals, wherein said urinary tract ailment comprises a urinary tract infection or wherein said urinary tract ailment comprises urinary tract stones.

13.

The strain for use according to claim 12, wherein said Lactobacilli probiotics increase the degradation of oxalic acid in vitro.

14.

The strain for use according to claim 13, for reducing stress levels in companion animals, wherein said stress levels are measured by determining the level of stress hormones selected from the group consisting of epinephrine, norepinephrine, dopamine, cortisol , C-reactive protein, and mixtures thereof.

fifteen.

The strain for use according to any of claims 7 to 14, wherein the strain is in an oral dosage form for an animal in an amount from 1.0E + 04 to 1.0E + 12 CFU / animal per day.

16.

A composition comprising a strain of lactic acid bacteria according to any one of claims 1 to 6.

17.

The composition according to claim 16, wherein said composition is a food for a companion animal, wherein the composition is a wet food for animals or a dry food for animals.

18.

The composition according to claim 17, wherein the composition is a cat food.

19.

The composition according to claim 17, wherein the composition is in a form selected from the group consisting of croquettes, chewable candies and cookies.

twenty.

The composition according to claim 17, wherein said composition is a food supplement for pets.

twenty-one.

The composition according to claim 20, wherein the composition is in the form of a meat, yogurt, cheese or other dairy product sauce.

22

The composition according to claim 20, wherein the composition is in a form selected from the group consisting of capsules, tablets and pills.

23. The composition according to claim 16, which comprises at least 0.001% or from about 1.0E + 04 CFU / g to about 1.0E + 12 CFU / g of the lactic acid bacterium. 24. The composition according to claim 16, wherein the lactic acid bacteria according to any one of claims 1 to 6 are in the form of viable cells.

25.

The composition according to claim 16, wherein the lactic acid bacteria, according to any of the 10 claims 1 to 6, are in the form of non-viable cells or of active constituent fractions thereof.

26. Use of lactobacilli lactic acid bacteria obtainable by feline gastrointestinal tract isolation, excised and washed, according to any of claims 1 to 6, to reduce odors associated with the feces or urine of a companion animal.

27.

Use according to claim 26, wherein the smell is associated with a hygienic litter box of a companion animal 15.