RU2795199C2 - Compositions for improving oral hygiene - Google Patents

Abstract

FIELD: veterinary.

SUBSTANCE: group of inventions relates to methods for reducing bad breath in dogs (options). To implement the methods, the dog food compositions are used that include a fragment of a microorganism belonging to Lactobacillus genus in an effective amount. The said fragment is a membrane fraction obtained by preparing a membrane preparation as a sensory neutral oral care agent. The microorganism is able to bind to a microorganism from the mutans Streptococci group, and the microorganism is in a thermally inactivated form, and where the binding (i) is resistant to heat treatment for at least 20 minutes at a temperature of more than 95°C, and/or (ii) is resistant to protease treatment, wherein the protease is selected from the group consisting of pronase E, proteinase K, trypsin, and chymotrypsin, and/or (iii) is calcium dependent, and/or (iv) is formed in the range pH between 4.5 and 8.5, and/or (v) formed in the presence of saliva, and/or (vi) independent of magnesium. The above fragment is used as a sensory neutral agent against bad breath, and the microorganism is selected from the group consisting of the following Lactobacillus strains: DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673. The composition is administered in an amount effective to reduce bad breath in a dog. The composition contains 3 g of a fragment of a microorganism per 1 kg of the base composition, while the base composition includes beef, brown rice, rape seeds, linseed meal, sunflower seeds, buckwheat seeds, barley and millet, or the base composition includes soybean meal, brown rice, rapeseed, flaxseed meal, sunflower seeds, buckwheat seeds, barley and millet, or the base composition includes chicken, chicken meal, barley, peas, brown rice, crushed extruded whole soybeans and chicken fat.

EFFECT: use of the above dog food compositions containing fragments of microorganisms that have a very low, unobtrusive odor and taste provides an effective reduction of bad breath in dogs.

19 cl, 20 ex, 3 dwg

Description

The present invention relates to microorganisms or their fragments as sensory-neutral oral hygiene products, especially for the prevention of tartar, as anticaries agents and/or preparations for bad breath. The present invention further relates to compositions containing microorganisms or fragments thereof for the reduction of Streptococcus mutans. Such compositions can be used in oral hygiene compositions, for example, for the prevention of caries, or for the prevention of tartar or bad breath. They may also or instead be used to prevent or treat bad breath. Since the microorganisms and their fragments according to the present invention have a very low, unobtrusive odor and taste, they are particularly useful as sensory neutral agents for the prevention of tartar formation, caries, biofilm formation in the mouth and/or for the prevention or treatment of unpleasant mouth odor. Microorganisms and their fragments, as well as compositions containing such microorganisms and fragments, thus, can be successfully used in food and feed compositions, especially in pet foods. In addition, the present invention relates to methods for obtaining such microorganisms, fragments, compositions, food and feed.

One of the well-known problems of animals with teeth is the decay of such teeth. This problem is of particular importance for such animals, which cannot, like sharks, push their teeth out, constantly replacing them with a new set. Among the most important causes of tooth decay is caries. Under the influence of microorganisms that colonize the oral cavity, tooth enamel is continuously weakened and eventually dissolved, which leads to the formation of carious lesions. Such lesions, in turn, are suitable for further colonization by microorganisms, which worsens the problem of caries.

A problem associated with the formation of caries is the development of bad breath. Bad breath is thought to be the result of unwanted colonization by microorganisms in the oral cavity. However, no microorganism has so far been listed as the main cause of bad breath. It seems clear, however, that the formation of caries and halitosis are independent processes, since halitosis can occur in the absence of teeth and caries can occur in the absence of halitosis.

Another problem of oral hygiene, especially in animals, is the formation of tartar. Calcified deposits on the teeth are formed by microorganisms that colonize the surfaces of the teeth. As a rule, within 7-10 days, calcified deposits on the teeth grow to such an extent that they can be visible to the naked eye. Tartar forms a focus that allows the teeth to be colonized by additional microorganisms. This, in turn, can lead to the development of bad breath, tooth decay and inflammation of the gum tissue. There is thus a need to prevent the development of tartar, for example by slowing down the formation of tartar.

Therefore, it is often necessary to try to solve these problems independently. Among the most notable solutions so far in the art are toothpastes and mouthwashes. Such toothpastes and mouthwashes typically contain antimicrobial agents effective to reduce or inhibit the activity of tooth-colonizing microorganisms, especially caries-causing and/or halitosis-causing microorganisms. However, due to the lack of correlation between caries and halitosis formation, many substances effective against caries are ineffective for preventing or treating halitosis, and vice versa. In addition, the effectiveness against tartar needs to be improved.

Another problem is that substances effective as antimicrobial agents often have a strong odor or taste of their own that is thought to be repulsive to consumers and especially pets. Thus, oral hygiene compositions often contain additional olfactory agents to mask or alter the taste or odor inherent in the antimicrobial agents. However, such additional agents are also not easily accepted by consumers due to their even stronger own taste or smell. This leads to an undesirably low adherence of consumers to the application of manufacturers' recommendations for such oral hygiene compositions.

The problem of adherence to therapy is especially important with children and animals. Both tend to develop clever ways to dispose of or misuse oral hygiene compositions, such as by ingestion, so that an effective antimicrobial agent or agents cannot fully exert their intended effect. The resistance, especially of pets such as cats and dogs, to any form of treatment is often complained of. In addition, such animals are known to avoid medication, including antimicrobially effective oral hygiene compositions, that they would otherwise take, even when masked in food. In addition, some methods for oral application of hygiene compositions are not available to animals, as they, for example, are not able to gargle. In addition, some oral procedures are particularly stressful for animals and require anesthesia. For example, animals such as cats and dogs will vehemently resist the removal of tartar.

Thus, oral hygiene is considered tiresome for humans, including children, and for animals, including pets, and the like. For this reason, the present invention intends to alleviate the above problems of the prior art. In particular, the present invention is directed to improving compliance with oral hygiene guidelines for humans and animals, especially pets.

In accordance with the present invention, it is important to further adhere to a therapy regimen such that the agent or agents used for the prevention of tartar, caries and/or for the prevention or treatment of bad breath have a low intrinsic taste and aroma. Thus, rather than masking or altering the taste and odor of such agents by adding additional strong tasting or strong smelling agents, the present invention proposes a different approach. The intrinsic taste and odor of oral hygiene agents has not yet been taken into account or given much importance. In addition, it has surprisingly been found that the agents that are available are effective for both prevention of tartar, caries and/or prevention or treatment of bad breath without having a strong taste and smell of their own. It has also surprisingly been found that such agents can be used to improve adherence to oral hygiene therapy instructions and in particular to reduce oral hygiene avoidance behavior in humans, including children, and in animals, including pets. In addition, the present invention provides oral hygiene compositions having different flavors or aromas than those previously required to mask or change the taste or smell of antitartar agents, anti-caries agents and/or anti-malodorous agents. .

The present invention relates to microorganisms or their fragments as sensory neutral means of oral hygiene. The invention further relates to compositions containing microorganisms or fragments thereof for the reduction of Streptococcus mutans. Such microorganisms and fragments thereof, as well as such compositions, can be used to treat or prevent tartar. Such microorganisms, fragments or compositions may also be used to treat or prevent caries. They may also be used for or instead of preventing or treating bad breath. Since the microorganisms and their fragments in accordance with the present invention have a very low, unobtrusive odor and taste, they are particularly suitable as sensory neutral agents for the prevention of caries, the formation of biofilm in the oral cavity, including the formation of tartar and/or for the prevention or treating bad breath. Microorganisms and their fragments, as well as compositions containing them, can thus be used with advantage in food and feed compositions, especially in pet foods. In addition, the invention relates to methods for obtaining such microorganisms, fragments, compositions, food and feed.

According to the present invention, the microorganism or fragment thereof useful as a sensory neutral oral hygiene agent is preferably a lactic acid bacterium.

As indicated above, it has now surprisingly been found that lactic acid bacteria can be used as a sensory neutral oral hygiene agent, especially for the treatment and prevention of dental calculus, as anti-caries drugs and/or anti-malodorous drugs. This could not be expected since lactic acid bacteria are known to have a generally strongly acidic or acidic taste and/or odor, for example in yogurt and other dairy products.

In accordance with the present invention, a lactic acid bacterium is any microorganism taxonomically of the order Lactobacillales and preferably of the family Lactobacillaceae. A microorganism according to the present invention is considered to belong to a specified taxonomic group if it is more similar to a strain within that taxonomic group or a lower taxonomic rank than to any type of strain belonging to a taxonomic group different from the group in question or its lower rank where said analysis is based on a comparison of their genetic material, excluding plasmids and non-integrated viruses. Thus, a microorganism is considered to belong to the family Lactobacillaceae if, as defined in the previous sentence, it is genetically more similar to a typical strain of a species or genera in the family Lactobacillaceae compared to typical strains of a species or genera belonging to another family in the order Lactobacillales.

Similarity in accordance with the present invention was evaluated using the Needleman-Wunsch global alignment algorithm. This algorithm is a standard algorithm for finding the optimal alignment, including breaks in two nucleic acid sequences along their entire length. The Needleman and Wunsch algorithm was published in 1970 by J. Mol. Biol. 48, 443-453, in which the gap penalty in n positions is calculated by the formula

penalty for introducing a deletion into an alignment + (n-1) × penalty for continuing the deletion.

There is no penalty for hanging ends.

The penalty for introducing a deletion into an alignment in the context of the present invention is 10.0. The penalty for continuing a deletion in the context of the present invention is 0.5. A substitution matrix for comparing nucleic acid sequences in the context of the present invention is a unitary DNA identity transformation matrix that assigns 1 point for each identical base "substitution" and -10,000 for all other base substitutions. Sequence alignment can be done with these parameters, for example, using the public tools offered by EBI.

A particular advantage inherent in microorganisms of the family Lactobacillaceae is that they can generally be considered safe for human and animal consumption. For example, lactic acid bacteria have been used for a long time in food production, for example through milk processing. In addition, they are easy to handle due to their low risk to human or animal health, and they are easily grown in large quantities, for example, in batches of 500 liters of culture medium. Particularly preferred culture methods will be described below.

Among the members of the Lactobacillaceae family, such lactic acid bacteria are especially preferred according to the present invention, which belong to the genus Lactobacillus, Paralactobacillus, Pediococcus or Sharpea, among which microorganisms of the genus Lactobacillus are most preferred. Such microorganisms have been widely used in food and feed production, they are easy to handle and can be obtained in large quantities. In addition, in the Lactobacillaceae family, especially among the genus Lactobacillus, microorganisms can be obtained that are both sensory neutral and effective as oral hygiene products, in particular antitartar agents, anti-caries agents and/or anti-malodor drugs. from mouth.

The lactic acid bacteria of the present invention are preferably rod-shaped or spherical, varying from long and thin to short curved rods, preferably non-motile and/or non-spore-forming and producing lactic acid as the main or sole product of enzymatic metabolism. The genus Lactobacillus to which the microorganism of the present invention belongs is preferably divided according to the following characteristics into three main subgroups, it being contemplated that the Lactobacillus species of the present invention may belong to each of the three main subgroups:

(a) homofermentative lactobacilli

(i) producing lactic acid, preferably L-, D- or DL-isomer(s) of lactic acid in an amount of at least 85% from glucose via the Embden-Meyerhoff pathway;

(ii) growing at 45°C but not at 15°C;

(iii) having a rod-shaped form, and

(iv) having glycerol-teichoic acids in the cell wall;

(b) homofermentative lactobacilli

(i) producing lactic acid, preferably the L- or DL-isomer(s) of lactic acid via the Embden-Meyerhoff pathway;

(ii) growing at 15°C, showing variable growth at 45°C;

(iii) short rod-shaped or coryneform and

(iv) having ribitol and/or glycerol-teichoic acids in the cell wall;

(c) heterofermentative lactobacilli

(i) producing lactic acid, preferably the DL isomer of lactic acid in an amount of at least 50% from glucose via the pentose phosphate pathway;

(ii) producing carbon dioxide and ethanol;

(iii) showing variable growth at 15°C or 45°C;

(iv) having the form of long or short rods, and

(v) having glycerol-teichoic acids in the cell wall.

Based on the characteristics described above, the microorganisms preferred in accordance with the present invention can be identified as belonging to the group of lactic acid bacteria, in particular to the genus Lactobacillus.

In a preferred embodiment of the invention, the microorganism according to the invention has metabolic fingerprints selected from the group consisting of:

(i) it metabolizes D-lactose but not L-sorbose and/or D-sucrose and/or D-inulin,

(ii) it metabolizes inulin,

(iii) it metabolizes L-sorbose but not D-lactose and/or D-sucrose and/or inulin, and

(iv) it metabolizes L-sorbose, D-lactose and inulin.

Preferably, the microorganism according to the present invention has metabolic fingerprints selected from the group consisting of:

(i) it metabolizes D-lactose but not L-sorbose, D-sucrose and inulin,

(ii) it metabolizes L-sorbose, D-lactose and inulin but not D-sucrose,

(iii) it metabolizes L-sorbose but not D-lactow, D-sucrose and inulin, and

(iv) it metabolizes L-sorbose, D-lactose, D-sucrose, but not inulin.

Of course, the microorganism according to the present invention is not limited to metabolizing the aforementioned Sugars of metabolic fingerprint samples, but may be able to metabolize additional sugars.

In the context of the present invention, the term "microorganism" does not only refer to those microorganisms that, when placed under appropriate culture conditions, can be propagated (viable microorganisms). It is a particular advantage of the present invention that, instead of such viable microorganisms, appropriate thermally inactivated or lyophilized microorganisms can also be used. This is particularly useful because some consumers may object to the idea of consuming viable microorganisms, or administering such viable microorganisms to children or pets, even though they believe such viable microorganisms are beneficial to their health. Since the present invention relates to increasing compliance with oral care instructions, it is of particular advantage that even such unfounded objections can be taken care of. Thus, unless expressly stated in this description and the accompanying examples, drawings and claims, no distinction is made between viable, thermally inactivated or lyophilized microorganisms.

According to the present invention, thermally inactivated cells are preferably obtained by autoclaving viable microorganism cells at 121° C. for at least 20 minutes in the presence of saturated steam at 2 bar atmospheric pressure. Such production of thermally inactivated microorganisms can be achieved using standard laboratory equipment, since the autoclaving process is a standard method known in the field of microbiology and related biotechnological engineering. This is a very fast technique and has been proven to inactivate microorganisms of the Lactobacillaceae family and in particular the Lactobacillus genus.

Alternatively, thermal inactivation of microorganisms in accordance with the present invention can be achieved by freezing such cells to a temperature of -20°C. Such freezing is also easy to perform using standard laboratory equipment.

Regardless of the mode of thermal inactivation, i.e. by autoclaving or by freezing, in accordance with the present invention, it is preferable that the concentration of viable microorganisms would be reduced during treatment by at least 85%, 90% or 95%, and particularly preferably by at least 97%, 98%, 99% and particularly preferably 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99, 8% or 99.9%. Particularly preferred is such autoclaving or freezing that results in a decrease in the concentration of viable microorganisms by at least 3 orders of magnitude (i.e., 1000 times), even more preferably at least 4 orders of magnitude, and even more preferably at least 5 orders of magnitude.

A particular advantage of the microorganisms of the present invention is that they retain their usefulness as a sensory neutral oral hygiene agent, preferably an antartar preparation, an anti-caries agent and/or an anti-malodor preparation even after such inactivation in an autoclave or freezing. In such a thermally inactivated form, the microorganisms according to the present invention are particularly easy to store even at 20° C. in a standard atmosphere of 80% relative humidity at this temperature. This storage stability is of particular importance since the inactivated microorganisms of the present invention allow the production of food or feed containing such inactivated forms without the compromising expiration date.

The same benefits can be obtained in accordance with the present invention using lyophilized microorganisms in accordance with the present invention, i. lyophilized microorganisms of the Lactobacillaceae family and especially lyophilized lactic acid microorganisms of the genus Lactobacillus. Lyophilization in accordance with the present invention is preferably carried out for at least 2 hours at room temperature, i.e. at temperatures from 16°C to 25°C.

In addition to or as an alternative to the microorganisms of the present invention, for example in viable, thermally inactivated or lyophilized form, fragments thereof may be used. Fragmentation of microorganisms can be easily carried out using standard methods known in the art, in particular through cell lysis and/or pasteurization. The method used for lysis or fragmentation of the microorganism in accordance with the present invention, including thermally inactivated and/or lyophilized forms, is not particularly important. Appropriate methods may be selected, for example, from chemical methods, including enzymatic treatment, preferably with one or more proteases, for example proteinase K, lipases or glycosidases; non-limiting examples for other chemicals are: ionophores, detergents, e.g. sodium dodecyl sulfate, acids or alkalis, non-limiting examples for physical agents are: high pressure, like a French press, osmolarity, and grinding, e.g. using glass or steel beads . Particularly preferred methods for the preparation of thermally inactivated and/or lyophilized microorganisms according to the present invention and fragments thereof are described in WO 2006/027265 A1, pages 25-28 and WO 2009/149816 A1, pages 28-34. "A fragment of a microorganism in accordance with the present invention" encompasses any part of a cell of a microorganism in accordance with the present invention. Preferably, said fragment is a membrane fraction obtained by preparing a membrane preparation. Membrane preparations of binding microorganisms belonging to the family Lactobacillaceae, preferably the genus Lactobacillus, can be obtained by methods known in the art, for example, using the method described in Rollan et al. Int. J. Food Microbiol. 70 (2001), 303-307, Matsuquchi et al, Clin. Diagn. Lab. Immunol. 10 (2003), 259-266 or in Stentz et al. Appl. Environ. microbiol. 66 (2000), 4272-4278 or Varmanen et al. J. Bacteriology 182 (2000), 146-154. In addition, whole cell preparations are also contemplated. Preferably, the fragment of the binding microorganism described in the present invention in accordance with the present invention retains the ability to specifically bind to Streptococcus mutans and more preferably Streptococcus mutans, which is described in detail in the present invention.

The microorganisms used in the present invention, i. lactic acid bacteria, preferably of the Lactobacillaceae family or the Lactobacillus genus, including the corresponding thermally inactivated or lyophilized forms and fragments thereof, are preferably used as anticaries agents. In accordance with the present invention, the term "anti-caries agent" is defined as any agent which, by its presence in the oral cavity of a human or animal, including domestic animals such as dogs, cats, rats, mice, hamsters, guinea pigs or monkeys, reduces the risk of developing new carious lesions. A particular advantage of the present invention is that the microorganism used as an anticaries agent need not be an effective antimicrobial by itself. Thus, it is not necessary according to the present invention that the microorganisms or their fragments used as anticaries agents reduce the number of caries-generating microorganisms, in particular microorganisms belonging to the mutans Streptococci group, by co-cultivation.

Therefore, another advantage according to the present invention is that the microorganisms used as anticaries agents have a very limited effect on the normal oral flora of humans or animals, especially the domestic animal. Suitable microorganisms will be described below.

The microorganism used in the present invention, i.e. lactic acid bacteria, preferably of the Lactobacillaceae family or the lactobacillus genus, including the corresponding thermally inactivated or lyophilized forms and fragments thereof, are preferably used as antitartar agents. For the purposes of the present invention, the term "tartar agent" is defined as any agent which, by its presence in the oral cavity of a human or animal, including pets, reduces the risk of developing tartar, or which retards the further development of tartar. It is not required that the tartar agents of the present invention actually eliminate tartar already formed on the tooth.

In addition, the microorganisms used in the present invention, i. lactic acid bacteria, preferably of the Lactobacillaceae family or the lactobacillus genus, including the corresponding thermally inactivated or lyophilized forms and fragments thereof, are preferably used as preparations against bad breath. In accordance with the present invention, the term "bad breath" means any bad odor emanating from the oral cavity. Thus, the term is used in accordance with the present invention, both for mild forms of bad breath, also called "bad breath", which is not a disease, as well as for such forms of bad smell, which, for example, due to its intensity or aroma, are considered symptoms. diseases; these latter forms of bad breath are also referred to as halitosis. Clinically significant forms of bad breath, i.e. halitosis, can have various causes. Most notable for all forms of halitosis are infections caused by microorganisms colonizing the oral cavity, where such a microorganism digests organic material such as dead epithelial cells, other microorganisms, or food or feed residues. Preferably, the present invention delays the onset of halitosis and/or reduces the intensity or flavor of halitosis, whether clinically inconsistent with bad breath or clinically significant halitosis. However, unless expressly mentioned in the present invention, the term "bad breath" does not cover bad odors originating outside the oral cavity, such as in the esophagus or stomach. Such unpleasant odors, for example, may become noticeable during burping.

In the context of the present invention, the term "oral cavity" means such a cavity that extends from the lips and teeth to the uvula, but not including it.

The mouth defines the oral cavity of mammals, preferably humans or animals such as pets, consisting of the oral mucosa (gums, lips, cheeks, palate and floor of the mouth), tongue and teeth (including artificial structures).

In addition, in accordance with the present invention, the term "sensory neutral" is defined as such an agent that can replace up to 2 wt. % of wholemeal wheat flour so that no more than 5 out of 10 trained experts, when considering the taste and aroma of such a mixture of wheat flour and microorganisms and / or fragments in accordance with the present invention, will note significant changes, as judged on the basis of a scale from "no difference", "slightly changed", "markedly changed" to "significantly changed". Preferably up to 5 wt. % of whole wheat flour can be replaced by microorganisms and/or their fragments, so that no more than 5 out of 10 experts consider the taste and/or smell of such a mixture to be significantly altered.

In accordance with the present invention, thus also provides a composition containing

- a binding microorganism or a fragment thereof in an amount that is both (a) sensory neutral and (b) effective for reducing Streptococci mutans in the oral cavity of a human or animal, and/or effective for reducing bad breath, wherein the microorganism is a lactic acid bacteria .

It has now surprisingly been found that in order to exhibit the anticaries effect of a sensory neutral agent, it is sufficient to use binding lactic acid bacteria to reduce Streptococci mutans in the oral cavity. As described above, such binding lactic acid bacteria, thermally inactivated or lyophilized forms, as well as fragments of such binding lactic acid bacteria can be used as an anticaries agent without an antimicrobial effect, i.e. without killing the cariogenic microorganisms themselves or inhibiting their growth. Instead, it is sufficient that the binding microorganism, or fragment thereof, binds to one or more mutans Streptococci species. Such binding can lead to agglutination of bound Streptococci mutans, which are in turn cleared from the oral cavity through normal saliva swallowing and eating and drinking. As described in WO 2008/074473 A2, the removal of Streptococci mutans from the oral cavity significantly reduces the risk of biofilm formation in the oral cavity and on the teeth, thereby indirectly reducing the risk of caries. This is especially true for those binding microorganisms that bind to Streptococcus mutans, as described in WO 2006/027265 A1. However, it has not hitherto been known that such binding microorganisms can be used as sensory neutral agents, ie at a sensory neutral concentration as described above and detailed below. In addition, it has not been known so far that such binding to Streptococci mutans and especially Streptococcus mutans can reduce the risk of developing bad breath.

The composition according to the present invention thus preferably comprises a microorganism or fragment thereof capable of specifically binding to a bacterium belonging to the group mutans Streptococci, where the specific binding is

(i) heat-resistant, and/or

(ii) resistant to protease treatment and/or

(iii) calcium dependent and/or

(iv) formed in the pH range between 4.5 and 8.5 and/or;

(v) formed in the presence of saliva.

The term "specifically binding" in the context of the present invention means that a binding microorganism or fragment thereof, preferably a microorganism (or corresponding fragment) belonging to the Lactobacillaceae family and more preferably the Lactobacillus genus, binds to one or more strains belonging to the mutans of Streptococci, preferably Streptococcus mutans, but does not associate with most others, preferably none of the other species belonging to the genus Streptococcus. In particular, the binding microorganism or fragment thereof preferably does not bind to bacteria belonging to the species Streptococcus oralis and/or Streptococcus mitis and/or Streptococcus salivarius. Even more preferably, the binding microorganism also does not bind to bacteria belonging to the species Streptococcus salivarius, more preferably belonging to the subspecies thermophilus. More preferably, the binding microorganism or fragment thereof does not bind Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and/or Streptococcus salivarius DSMZ 20567. And even more preferably, the binding microorganism or fragment thereof would also not bind to Streptococcus salivarius ssp. Thermophilus.

The specific binding reaction includes binding and preferably aggregating Streptococcus mutans or other Streptococci mutans cells as described in the present invention as a binding microorganism in accordance with the present invention, including its fragment in the oral cavity. This specific binding results, as a consequence, in the flushing of the associated cells, for example, by means of saliva or rinsing or rinsing the mouth and the like, as described in the present invention. Preferably, the specific binding reaction of the binding microorganism according to the present invention and fragments thereof with Streptococcus mutans and/or other Streptococci mutans prevents such Streptococcus cells from attaching to the surface of the tooth or teeth, or, not being bound by such a theory, may lead to detachment of Streptococcus cells from surface of the tooth or teeth. As a result, the specific binding reaction results in the flushing of bound Streptococcus cells from the mouth, thereby reducing the causative agent of the biofilm and thus preventing and/or curing caries.

It is believed that the binding microorganism, or fragment thereof, can specifically bind to the streptococcal I/II antigen, which is also known as the B, IF, P1, SR, MSL-1 or PAc antigen. However, the binding microorganism, or fragment thereof, can bind to any other S. mutans protein or surface structure, thereby aggregating S. mutans and flushing it out of the mouth, as described in the present invention. It is known that Streptococcus mutans is associated through the specified streptococcal antigen I/II with plaque. Accordingly, when a binding microorganism according to the present invention can bind to, for example, said streptococcal I/II antigen, Streptococcus mutans or other appropriate Streptococcus mutans is difficult to bind to the tooth surface, thus helping to prevent and/or treat caries.

The pellicle is a transparent thin coating containing proteins and lipids found in saliva. It forms within seconds of brushing the tooth surface. Pellicle formation is the first step in plaque formation. Dental plaque is soft deposits that build up on the teeth. A plaque can be defined as a microbial community complex, with more than 10 ¹⁰ bacteria per milligram. Experts estimate that up to 400 different types of bacteria can be found in the plaque. In addition to bacterial cells, the plaque contains a small number of epithelial cells, leukocytes, and macrophages. The cells are contained in the extracellular matrix, which is formed from bacterial products and saliva. The extracellular matrix contains proteins, polysaccharides and lipids. One of the proteins present in saliva is agglutinin, which on the one hand is believed to lead to partial removal of Streptococci mutans from the oral cavity, however, on the other hand, it is believed to facilitate adhesion of Streptococci mutans to the surface of the teeth, thereby facilitating the initial attachment Streptococcus cells to the teeth and thus the onset of caries.

Preferably, the aforementioned binding microorganism belonging to the lactic acid bacteria group - or a fragment thereof - is capable of specifically binding to Streptococcus mutans serotype c (DSMZ 20523) and/or serotype e (NCTC 10923) and/or serotype f (NCTC 11060) and/ or Streptococcus sobrinus DSMZ 20742 and/or Streptococcus ratti DSMZ 20564 and/or Streptococcus cricetus DSMZ 20562 and/or Streptococcus feras DSMZ 20646 and/or Streptococcus macacae DSMZ 20714.

This means that the aforementioned binding microorganism belonging to the group of lactic acid bacteria, or a fragment thereof, preferably binds to at least one microorganism selected from the group consisting of Streptococcus mutans serotype c (DSMZ 20523), serotype e (NCTC 10923) serotype f ( NCTC 11060), Streptococcus sobrinus DSMZ 20742, Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ 20562, Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ 20714. bacteria, or a fragment thereof would bind to any combination, grouping or subgroup of the above bacteria. Even more preferably, the above binding microorganism or its fragment belonging to the group of lactic acid bacteria would be associated with all of the above bacteria. In accordance with the present invention, a "serotype" is the antigenic properties of a bacterial cell, preferably a Streptococcus mutans or Streptococcus sobrinus cell, which have been determined by serological methods known in the art.

As described above, specific binding of the binding microorganism or fragment thereof to Streptococci mutans and preferably Streptococcus mutans is preferably heat-resistant. Accordingly, binding is not abolished if the binding microorganism of the present invention is treated with heat, for example, at a temperature above 55°C, even more preferably more than 65°C, particularly preferably more than 95°C, and most preferably at 121°C. After cooling, the ability of a binding microorganism according to the present invention, or a fragment thereof, to specifically bind to Streptococci mutans is determined as described in the present invention.

The appropriate temperature may depend on the particular species of binding microorganism, but can be readily determined by one of ordinary skill in the art through routine experimentation, for example, by incubating the appropriate cells at various temperatures and determining the number of binding cells or fragments thereof that are still able to specifically bind. with Streptococci mutans and/or Streptococcus mutans using the methods demonstrated in the present invention. Typically, the heat treatment is continued for a period of time of at least 1 minute. Preferably, the heat treatment is continued for a period of at least n minutes, in which n is an integer in the range of 2 to 60, with n=20 being particularly preferred. However, there is in principle no upper limit to the incubation time. However, preferably no more than 4, 3, 2 or 1 hour(s). Most preferred is a heat treatment of at least 20 minutes at 121° C. in saturated steam having an atmospheric pressure of 2 bar. Thus, the thermally inactivated form of the binding microorganism according to the present invention, obtained in an autoclave, is particularly preferred. The most preferred heat treatment is considered to be the abolition of any protein function and any cell viability, thus distinguishing the microorganism according to the present invention from other microorganisms in that it is still able to bind specifically to S. mutans. Thus, it is very useful for any food, feed, drink or composition in accordance with the present invention in the event that it is desired that the microorganism should not be alive.

The specific binding of the binding microorganism or fragment thereof is further preferably characterized by its resistance to protease treatment. Preferably, the binding is resistant to treatment with one or more proteases selected from the group consisting of pronase E, proteinase K, trypsin, and chymotrypsin. These proteases show no specificity and thus are considered to degrade any protein found on the surface of the cells of the microorganism. Other proteases known to have preferences for certain amino acid residue sequences are elastase, thrombin, aminopeptidase I, carboxypeptidase, dostripain, endoproteinase, papain, cathepsin B, pepsin, gastrixin, chymosin, cathepsin D. The latter proteases can also be used to test whether a specific binding of a binding microorganism or fragment thereof to S. mutans or other Streptococcus mutans would be resistant to the latter more specific proteases. Thus, after treatment with a protease as described in the examples of WO 2008/074473 A2 or WO 2006/027265 A1, which are included herein, the binding microorganism or fragment thereof is still able to specifically bind to Streptococcus mutans/mutans Streptococci.

In addition, the specific binding of the binding microorganism or fragment is further preferably characterized by its dependence on calcium. Preferably, specific binding occurs in the presence of a calcium ion concentration in the range of 0.05 mM to 500 mM, preferably between 1 mM and 100 mM. Particularly preferred calcium concentrations are between 2 and 30 mM. The dependence of specific binding on calcium can be investigated as described in the examples of WO 2008/074473 A2 or WO 2006/027265 A1, which are included in the present description. In addition, specific binding to the binding microorganism or fragment is preferably maintained in the pH range from 4.0 to 9.0, preferably from 4.0 to 7.0, in particular, the pH value at which specific binding takes place is still preferably is 4.5. Quantification of the maintenance of specific binding outside the pH range described above is shown in the above examples.

The specific binding is preferably independent of magnesium. Thus, there is no need for magnesium ions or magnesium salts to be present.

Another preferred characteristic of specific binding is its presence in the presence of saliva. Saliva is an exogenous secret that is synthesized in the salivary glands. It is a complex liquid containing, in addition to about 99% water, many organic and inorganic compounds. The physiological ingredients of saliva are in particular enzymes, for example amylases, carbonic anhydrases, lysozyme, peroxidases or proteins, for example mucin, lactoferrin, proline-rich proteins, cystatins, histatins or staterins or soluble IgA. Thus, although various potentially interfering substances are present in saliva, the specific binding of the microorganism in accordance with the present invention is not disturbed or impeded. For specific binding studies in the presence of saliva, saliva is preferably used which preferably contains the Streptococcus species described above. If, however, Lactobacillus rhamnosus species are tested for specific binding to S. mutans in the presence of saliva, it is preferred that Streptococcus salivarius ssp. thermophilus were missed. Specific binding was analyzed as described in the present invention. The above characteristics of the binding microorganism or its fragment belonging to the group of lactic acid bacteria makes it a reliable and effective agent for the prevention and/or treatment of caries, since they are mainly administered in various forms to the mouth, including the oral cavity and teeth, where, in in particular, saliva is present, including some proteases and low pH values after ingestion of carbohydrate-containing foods. In addition, heat resistance is beneficial when the microorganism according to the present invention is added as an additive to a food or feed during the production of said food or feed. Namely, food or feed is often subjected to thermal sterilization, pre-cooking, pasteurization, etc., which negatively affects the viability of microorganisms.

A binding microorganism according to the present invention and/or a corresponding fragment thereof, is capable of specifically binding to one or more Streptococci mutans and preferably Streptococcus mutans, wherein the specific binding is

(i) resistant to heat treatment, and

(ii) resistant to protease treatment; and

(v) formed in the presence of saliva.

Such binding microorganisms and their fragments combine the above-mentioned advantages. In particular, since their specific binding is resistant to heat treatment, they can be included in a product that is heat treated for sterilization, thereby extending the shelf life of the product without damaging the oral hygiene properties provided to the product by microorganisms or their fragments, preferably without impairing efficacy against tartar, efficacy against caries, or efficacy against mouth malodor of the product. In addition, due to protease resistance, binding microorganisms and their fragments can be incorporated into various products of various compositions even before such products are subjected to heat treatment to denature the protein. And it is important that the formation of specific binding in the presence of saliva allows the use of binding microorganisms and their fragments in products that do not remove saliva from the oral cavity. This ability, in turn, increases compliance, especially in children and animals, including pets, since such users are generally unwilling to use a product that would lead to dry mouth.

For these reasons, the binding microorganism according to the present invention and/or the corresponding fragment is even more preferably capable of specifically binding to one or more Streptococci mutans and preferably Streptococcus mutans, where the specific binding is

(i) resistant to heat treatment for 20 minutes at 121°C in saturated steam having an atmospheric pressure of 2 bar, and

(ii) resistant to treatment with one or more enzymes selected from pronase E, proteinase K, trypsin and chymotrypsin, and

(v) formed in the presence of saliva.

Preferably, the specific binding of the binding microorganism according to the present invention and/or the corresponding fragment is also

(iii) calcium dependent and

(iv) is formed in the pH range from 4.5 to 8.5.

A method for determining the binding of binding microorganisms and their fragments to mutans Streptococci is described in Lang et al (2010) Journal of Dental Research 89(2) 175-179, which is included in the present invention.

Preferably, the specific binding of a microorganism or fragment thereof can be analyzed as follows:

(a) growing said binding microorganism to stationary phase, or, if a fragment is to be tested, obtaining such a fragment,

(b) mixing said binding microorganism or fragment with mutans Streptococcus grown to stationary phase,

(c) incubating the mixture obtained in step (b) under conditions allowing the formation of aggregates from said microorganism and said Streptococcus, and

(d) detection of aggregates by precipitation.

Preferably, the bacteria belonging to the Streptococci mutans group used in such an assay are Streptococcus mutans. For specific binding to Streptococci mutans, preferably no binding can be found to at least one, preferably at least two or more, preferably at least three, and even more preferably all microorganisms selected from the group consisting of Streptococcus salivarius spp. . thermophilus, Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus sanguinis DSMZ 20567.

In particular, binding microorganisms belonging to the group of lactic acid bacteria, preferably of the Lactobacillaceae family and even more preferably of the Lactobacillus genus, or corresponding fragments, are preferably mixed with mutans Streptococci in a cell-to-cell ratio of 3:1 to 60:1 (mutans Streptococci : binding microorganism ). Both lactic acid bacteria binding bacteria and mutans Streptococci are grown in culture fluid to stationary phase. Preferably, the optical density is measured photometrically at a wavelength of 600 nm. These ratios correspond to the ratio of colony-forming units from 1:50 to 1:2.5. Preferably OD ₆₀₀ =1 in 1 ml corresponds to 3×10 ⁸ colony forming units of mutans Streptococci. Preferably OD ₆₀₀ =1 in 1 ml corresponds to 7×10 ⁹ colony-forming units of lactic acid bacteria. Preferably, for analysis of the aggregation reaction by sedimentation, the bacteria are in a volume of 2 ml in 15 ml tubes from Falcon. If necessary, the culture suspension was diluted with PBS buffer to obtain the volume ratios mentioned above while maintaining a final volume of 2 ml.

Preferably, the mixture of Streptococcus and binding lactic acid bacteria or fragments thereof is stirred for about 15 seconds and then left alone for at least 5, 10, 15 minutes, and more preferably for 20 minutes at room temperature, i. any temperature between 16°C to 25°C. Aggregation was observed as an immediate turbidity of the suspension, and after at least 20 minutes aggregation is visible in the form of aggregates that settled as a visible precipitate, while aggregating mixtures of non mutans Streptococci remained in suspension. As a control for autoaggregation of the respective bacterial binding or fragments thereof and the Streptococcus mutans strain can be analyzed by exclusion of either Streptococcus mutans or binding microorganisms/fragments.

Binding microorganism and Streptococcus mutans aggregation according to the assay described above can be quantified by isolation of formed aggregates as described in Lang et al (2010) Journal of Dental Research 89(2) 175-179, or by centrifugation, e.g., at 500× g for 30 seconds. Subsequently, the amount of aggregation can be determined by measuring the amount of non-aggregated cells that remain in the supernatant. The determination may be performed by any suitable method known to the person skilled in the art. Preferably, the determination is made by removing a certain volume of supernatant, eg 1 ml. Subsequently, the optical density of the removed supernatant can be measured at any suitable wavelength known to those skilled in the art, for example at 600 nm. The measured value, after subtracting the value of the corresponding control sample without lactobacilli, represents the number of cells that have not yet been aggregated.

A method for determining specific binding in accordance with the present invention is described in example 4 in WO 2008/74473 A2, with corresponding instructions for growing the respective microorganisms described in example 1 of this publication. Both examples are given in the present invention for the purpose of disclosing a method for determining specific binding in accordance with the present invention.

In addition, a dye, preferably a fluorescent dye, can be used to address the problem of possible autoaggregation. Thus, in a more preferred embodiment, specific binding can be analyzed as follows:

(a) growing said microorganism to stationary phase;

(b) mixing said microorganism with bacteria belonging to the mutans Streptococci group grown to stationary phase and which have been stained using a suitable dye, preferably a fluorescent dye;

(c) incubating the mixture obtained in step (b) under conditions allowing the formation of aggregates from said microorganism and bacteria of the mutans Streptococci group, and

(d) detection of aggregates by detecting a dye, preferably a fluorescent dye.

In addition, in a preferred embodiment, the bacteria belonging to the mutans Streptococci group used in such an assay are Streptococcus mutans.

Preferably, the aggregation assay can be carried out such that the binding microorganism and Streptococci mutans are first grown to stationary phase as described above. Preferably, the optical density is measured photometrically at a wavelength of 600 nm. Preferably OD ₆₀₀ =1 in 1 ml corresponds to 3×10 ⁸ colony forming units of the corresponding Streptococcus mutans. Preferably OD ₆₀₀ =1 in 1 ml corresponds to 7×10 ⁹ colony-forming units of binding microorganisms, in particular of the lactobacillaceae family and even more preferably of the lactobacillus genus, as described in the present invention.

Subsequently, mutans Streptococci stains. In yet another preferred embodiment, binding microorganisms are stained while Streptococci are not stained. Since any suitable dye may be used, fluorescent dyes known to those skilled in the art may preferably be used. Preferably a specific or non-specific fluorescent dye can be used, for example CFDA-SE to stain intact cells, other useful dyes include carboxyfluorescein diacetate acetoxymethyl ether, BCECF AM and calcein AM. In particular, the cells are harvested, for example by centrifugation, preferably at 3200×g for 5 minutes. The resulting pellet can then be resuspended in any suitable buffer known to the person skilled in the art, preferably PBS buffer. The buffer volume can be calculated so that the resulting suspension would have an OD ₆₀₀ , eg 4.2/ml. The suspension can then be mixed with a suitable dye, eg a fluorescent dye, preferably 5,6-carboxyfluorescein diacetate, succinimide ester (CFDA-SE), more preferably 2 µl of CFDA-SE solution (Invitrogen). The cells can then be incubated for a suitable period of time, as is known to those skilled in the art, eg, for 2 hours, at a suitable temperature, as is known to those skilled in the art, eg, 37°C. In the next step, the stained cells can be collected, for example by centrifugation. Preferably, centrifugation is carried out at 3200×g for 5 minutes. These cells can then be resuspended in a suitable buffer, as known to those skilled in the art, for example 2 ml of PBS buffer.

For aggregation, the binding microorganisms are preferably mixed with mutans Streptococci in volume ratios of 3:1 to 1:3 (mutans Streptococci : binding microorganisms). More preferably, the volume ratio of the mixture was 1:1. These ratios correspond to the ratio of colony-forming units from 1:50 to 1:150. For analysis of the aggregation reaction by measuring staining, preferably fluorescence, the binding microorganism and Streptococci mutans are used in any suitable volume known to those skilled in the art, Preferably in a volume of 50 μl. Preferably, mixing is carried out in a microtiter plate, such as a 96-well microtiter plate. Subsequently, the mixture may be shaken, preferably for 12 minutes at full speed. The mixture can then be centrifuged, for example for 10 seconds at 500×g. The supernatant may then be removed and the pellet may be resuspended in any suitable buffer known to those skilled in the art, preferably PBS buffer in any suitable volume, eg 100 µl. The color of the suspension can be measured in a mixture using any suitable means known in the art. Preferably, in the case of fluorescence, fluorescence can be detected in a fluorescent reader, for example at an excitation wavelength of 495 nm and for emission at 525 nm. As a control, the binding microorganism itself and the Streptococci stained mutans themselves can be analyzed. Any background staining, such as fluorescence, can be measured for the Streptococci mutans tested on their own and preferably can be subtracted from the value for aggregation with the appropriate binding microorganism. An aggregation effect is present if the background staining, e.g. fluorescence, measured as described in the present invention above, is subtracted from the measured staining, e.g. the present invention is higher, and the resulting value is at least higher than zero. More preferably, an aggregation effect is present if the value obtained is reproducibly above zero in a series of tests carried out as described above. "Series of experiments" means at least two, preferably three, more preferably 4, and most preferably 5 trials.

An alternative method for testing binding can be performed as described in example 5 in WO 2008/074473 A2, which is incorporated into the present invention by reference.

For fragments of binding microorganisms, binding is generally determined for both viable and inactivated binding microorganisms, as described above. The number of fragments to be used to determine binding or to prepare a food or feed composition according to the present invention in place of binding microorganism cells is preferably the same as the amount of cell wall material of a viable or thermally inactivated binding microorganism. For example, the peptidoglycan content of the binding microorganism can be measured using appropriate dyes and the same number of fragments based on the measurement of such dyes can be used.

Said binding microorganisms are preferably lactic acid bacteria belonging to the genus Lactobacillus, more preferably the species Lactobacillus, as described in the present invention. Even more preferably, said Lactobacillus belongs to the species Lactobacillus paracasei or Lactobacillus rhamnosus. However, Lactobacillus species are not limited to them. Said binding microorganisms may preferably be "isolated" or "purified". The term "isolated" means that the material is removed from its original environment, such as the natural environment if they are of natural origin. For example, naturally occurring microorganisms, preferably Lactobacillus species, separated from some or all of the coexisting materials in the natural system, is isolated. Such a microorganism may be part of the composition, and may still be considered isolated, since in this composition it is not part of its natural environment. Thus, a microorganism grown in pure culture is still considered an isolated microorganism. In addition, a fragment of a microorganism is considered "isolated" in accordance with the present invention, if it is separated from at least part of the material of the corresponding microorganism.

The term "purified" does not require absolute purity, but rather, it is intended as a relative definition. Individual microorganisms isolated from a library are usually purified to microbiological homogeneity, i.e. they grow as single colonies when streaked onto agar plates using methods known in the art. Preferably, the agar plates used for this purpose are selective for lactic acid bacteria, and are particularly preferred for Lactobacillus species. Such selective agar plates are known in the art.

To obtain fragments, it is preferable to destroy viable or thermally inactivated cells of the binding microorganisms by methods known in the art, for example, sonication, French press or ball milling, and separate the fragments from other cell debris by centrifugation. The fragment pellet can then be washed and centrifuged again to obtain a fragment pellet.

Mikroorganismen und Zellkulturen GmbH ("DSMZ") на 26 августа 2004, и обладающие следующими номерами депонирования DSMZ 16667, 16668, 16669, 16670, 16671, 16672 или 16673. DSMZ находится в Mascheroder Weg 1b, D-38124 Braunschweig, Germany. Вышеупомянутые номера депонирования DSMZ были сделаны в соответствии с условиями Будапештского договора о международном признании депонирования микроорганизмов для целей процедуры выдачи патентов.More preferably, the above binding microorganism belonging to the group of lactic acid bacteria is selected from the group consisting of Lactobacillus paracasei or Lactobacillus rhamnosus, respectively, having DSMZ accession number 16667 (L. paracasei ssp. paracasei Lb-Ob-KI), DSMZ number DSMZ accession number 16668 (L. paracasei ssp. paracasei Lb-Ob-K2), DSMZ accession number DSMZ 16669 (L. paracasei ssp. paracasei Lb-Ob-K3), DSMZ accession number DSMZ 16670 (L. paracasei ssp. paracasei Lb- Ob-K4), DSMZ accession number DSMZ 16671 (L. paracasei ssp. paracasei Lb-Ob-K5), DSMZ accession number DSMZ 16672 (L. rhamnosus Lb-Ob-K6) and DSMZ accession number DSMZ 16673 (L. rhamnosus Lb -Ob-K7) or a mutant or derivative thereof, wherein said mutant or derivative retains the ability to specifically bind to Streptococci mutans. The term "Lactobacillus paracasei or Lactobacillus rhamnosus having a DSMZ accession number" refers to cells of a microorganism belonging to the species Lactobacillus paracasei or Latobacillus rhamnosus deposited in the Deutsche Sammlung

Mikroorganismen und Zellkulturen GmbH ("DSMZ") on August 26, 2004, and possessing the following deposit numbers DSMZ 16667, 16668, 16669, 16670, 16671, 16672 or 16673. DSMZ is located in the Mascheroder WEG 1B, D-38122 4 Braunschweig, Germany. The above DSMZ deposit numbers have been made in accordance with the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Granting Procedure.

The "mutant or derivative" of the above binding microorganism belonging to the group of lactic acid bacteria, preferably the cells of the deposited Lactobacillus paracasei or Lactobacillus rhamnosus, preferably have the same characteristics as the respective deposited strains, i.e. they retain the ability to specifically bind to Streptococci mutans, preferably with binding characteristics as described in the present invention. For example, the specified derivative can be obtained using genetic engineering. In the context of the present invention, the term "genetically engineered" is used in the broadest sense for methods known to the person skilled in the art to change the desired nucleic acids in vitro and in vivo so that genetic modifications have an effect and genes are changed. using recombinant DNA technology. Accordingly, it is preferred that these methods include cloning, sequencing and transformation of recombinant nucleic acids. Suitable vectors for this purpose, including expression vectors for Lactobacillus species, such as those described in EP 0506789 B1, EP 0316677 B1, EP 0251064 B1, EP 0218230 B1, EP 0133046 B1 and WO 89/01970.

Primers, enzymes, additional host cells for cloning intermediate constructs, and the like can be used and are known to those skilled in the art. Preferably, the genetically modified mutants comprise cells of a binding microorganism belonging to the group of lactic acid bacteria, preferably of the lactobacteriaceae family, even more preferably of the lactobacillus genus, and most preferably of one of the deposited Lactobacillus species carrying the recombinant nucleic acid, either contained in the bacterial chromosome or on one or more plasmids, or contained in the bacterial chromosome, and/or on one or more plasmids. Said recombinant nucleic acids are preferably foreign to the above binding microorganism belonging to the group of lactic acid bacteria. The term "foreign" means that the polynucleotide or nucleic acid molecule is either heterologous to the host cell, meaning derived from a cell or organism with a different genetic environment, or is homologous to the host cell but located in a different genetic environment. than the natural counterpart of said nucleic acid molecule. In this case, the heterologous polynucleotide may be under the control of its own promoter or under the control of a heterologous promoter. The above-described vector or nucleic acid molecule present in a host cell can either be integrated into the genome of the host cell or it can be stored in some form outside the chromosome. In this regard, it should also be understood that the above-described nucleic acid molecule can be used to restore or create a mutant gene using homologous recombination. The plasmid can be low, medium or high plasmid copy number. These genetically engineered mutants may carry nucleic acids encoding glucanase or mutanase that are capable of disrupting the mutan-specific 1,3-glycosidic bonds of the sucrose subunits. Fungal glucanases, for example, are described in Fuglsang et al., J. Biol. Chem. 275 (2000), 2009-2018. It is also contemplated that the genetically modified mutants contain cells carrying nucleic acids encoding antibodies that are preferentially secreted or anchored to the bacterial cell wall. The term "antibody" includes intact antibodies as well as antibodies, fragments thereof, such as separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')2. The term "antibody" also includes humanized antibodies, bifunctional antibodies, and antibody constructs such as single chain Fv, (scFv), or antibody fusion proteins. It is also intended in the context of the present invention that the term "antibody" includes antibody constructs that can be expressed in cells derived from the above deposited microorganisms, for example, antibody constructs that can be transformed using, in particular, vectors using methods known in this field of technology. In particular, such antibody constructs are expected to specifically recognize, for example, the streptococcal ML antigen. Such an approach is, for example, described in Krueger et al., Nat. Biotechnol. 20 (2002), 702-706 or Shiroza, Biochim Biophys Acta 1626 (2003), 57-64.

Secretion of the expressed antibody is preferably achieved by operably linking the nucleic acid encoding the antibody to a secretory signal sequence. Anchoring to the bacterial cell wall can be achieved through the use of the sortase enzyme mechanism. Specifically, Gram-positive bacterial surface proteins are associated with the bacterial cell wall by a mechanism that involves cleavage of a conserved Leu-Pro-X-Thr-Gly (LPXTG) motif, which occurs during assembly of the peptidoglycan cell wall. Thus, a nucleic acid molecule encoding an antibody can be fused to a sequence encoding the aforementioned conserved motif, which is used by sortase to anchor proteins to the bacterial cell wall.

In addition, the aforementioned binding microorganism is believed to belong to the group of lactic acid bacteria that are genetically modified to carry nucleic acid molecules encoding reuterin, which is an antimicrobial substance particularly effective against Streptococcus mutans. Reuterin, for example, is described in Talarico et al., Chemother. 33 (1989), 674-679.

A binding microorganism mutant belonging to the lactic acid bacteria group, preferably a mutant of deposited strains of Lactobacillus, is preferably obtained by artificial mutation. In accordance with the present invention, the term "mutated" means one or more permanent modifications of the genetic material, i. nucleic acids caused, for example, by natural or physical agents or chemical compounds/substances/agents such as EMS or ENU. These modifications include point mutations, such as transactions or transversions, deletion/insertion/insertion of one or more bases in the nucleic acid/gene/chromosome, thereby modifying the nucleic acid/gene/chromosome, which can lead, in particular, to aberrant gene expression/ transcription/translation or inactive gene products, constitutively active/inactive gene products leading, for example, to dominant-negative effects. Preferably, the mutation results in an increase in the specific binding capacity of Streptococci mutans. Thus, it is also preferred that mutant cells of the microorganism which carry one or more mutations in one or more desired genes, or in which one or more mutations in one or more desired genes have been induced by methods known to those skilled in the art. It is also known in the art that mutated or genetically modified bacterial cells can be selected using any suitable method/phenotype.

In the context of the present invention, a mutant having an increased ability to specifically bind to Streptococci mutans can be tested according to the methods described above. The term "mutant", however, also includes cells of the aforementioned binding microorganism belonging to the group of lactic acid bacteria, preferably cells of the deposited microorganism, which carry natural, spontaneous mutations in their genome, ie in the bacterial chromosome. "Spontaneous mutations" are mutations that occur naturally, ie. without direct human genetic manipulation, or under the influence of mutagens. Selection of spontaneous mutants can be achieved by culturing the strain and selecting the desired variant, for example, for the ability of the bacterium variant to show improved binding to Streptococci mutans. Methods for selecting spontaneous mutants are well known in the art (see e.g. Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)). For example, such mutations may occur during culture, for example during the normal process of cell division in conjunction with DNA replication, or during passage and/or maintenance of a mutant of the aforementioned binding microorganism belonging to the group of lactic acid bacteria.

However, even though the genetic manipulation of microorganisms may confer beneficial or even highly beneficial properties on the microorganism, it is alternatively preferred that the binding microorganism and preferably all microorganisms used in accordance with the present invention are not genetically modified microorganisms as defined in Article 2(2) of Directive 2001/18/EC in the relevant version in force on the date of filing of this application. However, microorganisms produced by the genetic modification techniques listed in Annex 1B of said Directive 2001/18/EC according to the present invention are preferably not considered to be genetically modified microorganisms as defined in Article 2(2) of Directive 2001/18/EC . By eliminating genetically modified microorganisms, rational and irrational consumer fears can be avoided, thereby increasing consumer oral hygiene therapy adherence.

According to the present invention, the binding microorganism, preferably of the Lactobacillaceae family and even more preferably of the lactobacilli genus, most preferably one of the aforementioned especially preferred strains, does not contain genetic material that has been altered in a way that does not occur naturally as a result of crossing and/or natural recombination. Such methods include

(1) recombinant nucleic acid techniques, involving the generation of new combinations of genetic material by introducing a nucleic acid molecule produced by any means outside the body into any virus, bacterial plasmids or other vector systems and incorporating them into a host organism in which they are not naturally present. meet, but in which they are able to continue their reproduction;

(2) techniques involving direct introduction into the body of inherited material obtained from outside the body, including microinjection, macroinjection, and microencapsulation;

(3) cell fusion, including protoplast fusion, or hybridization techniques, where cell life with novel combinations of inherited material is formed by the fusion of two or more cells through processes that do not occur naturally.

Natural processes such as conjugation, transduction and transformation, however, are preferably not excluded in accordance with the present invention. In addition, preferably non-excludable techniques in accordance with the present invention are, provided that they do not include the use of recombinant nucleic acid molecules or genetically modified organisms other than those obtained by one or more of the techniques/methods described herein:

(1) mutagenesis by spontaneous or induced spontaneous mutation, and

(2) cell fusion, including the fusion of protoplasts of microorganisms capable of exchanging genetic material within the framework of traditional breeding methods.

In accordance with the present invention, it is thus preferred that the binding microorganism, and preferably also any other microorganism used in accordance with the present invention, contain no genetic material from an organism of the archaebacteria or eukaryote superkingdom, with the exception, of course, of such genetic material that naturally may be found in strains of the same species, or less preferably at least the same genus as the microorganism used in accordance with the present invention. Furthermore, it is preferable that the microorganism used in accordance with the present invention does not contain the genetic material of a microorganism found only in a phylum other than Firmicutes, and even more preferably does not contain genetic material found only in, or derived from, microorganisms from class other than bacilli, even more preferably does not contain genetic material that can only be found in, or derived from microorganisms of the order other than Lactobacillales.

If the microorganism used in accordance with the present invention, and in particular the binding microorganism, belongs to the Lactobacillaceae family, then it is preferable that the corresponding microorganism does not contain genetic material found only in, or derived from, microorganisms of a family other than Lactobacillaceae. Even more preferably, if the microorganism according to the present invention, and in particular the binding microorganism, belonged to the genus Lactobacillus, then the corresponding microorganism would not contain genetic material found only in, or derived from, microorganisms of a genus other than lactic acid bacteria.

The composition according to the present invention is preferably an anti-cariogenic food or feed composition or an anti-cariogenic pharmaceutical composition. Thus, the invention relates to the use of the aforementioned binding microorganism or a fragment thereof for the preparation of an anticariesogenic composition, preferably a pharmaceutical or cosmetic composition for the treatment or prevention of caries caused by Streptococci mutans and/or Streptococcus mutans.

The term "composition" as used in accordance with the present invention indicates compositions that contain at least one binding microorganism - possibly in thermally inactivated or lyophilized form, or a fragment thereof, preferably a deposited microorganism, as described above - optionally in thermally inactivated or lyophilized form - or a fragment of the specified microorganism. It is intended that the compositions used in accordance with the present invention contain the above ingredients in any combination. They may optionally contain at least one additional ingredient suitable for the prevention and/or treatment of caries. Accordingly, they may optionally contain any combination of the additional ingredients described below. The term "ingredients suitable for the prevention and/or treatment of caries" encompasses compounds or compositions and/or combinations thereof which either inhibit the binding of Streptococci mutans to dental surfaces, to plaque and/or which inactivate Streptococci mutans. More preferably, said term encompasses compounds or compositions and/or combinations thereof that can inhibit adhesion of Streptococci mutans to tooth surfaces, inhibit the activity of glycosyltransferases from Streptococci mutans, inhibit or inactivate Streptococci mutans, inhibit agglutinin-dependent binding of Streptococci mutans, and/or inhibit sucrose. -dependent binding mutans Streptococci, as will be described below.

The composition of the present invention is preferably a pet oral hygiene composition, and even more preferably a tartar prevention or reduction composition. Such compositions are known, for example, from EP 0141645 A2, WO 0150882 A2, WO 2001/070043 A2, WO 02/078462 A1, WO 2004/082518 A2, WO 2005/092087 A2 and WO 2010/052467 A2. Food and feed products described in these documents, in particular in the examples given in these documents, are included in this description as examples of preferred base compositions. These base compositions according to the present invention are additionally introduced by incorporation of a binding microorganism according to the present invention or a fragment thereof.

A particular advantage of the present invention is that under the action of the binding microorganism or fragment thereof, i. By specific binding to Streptococcus mutans, and most preferably through specific binding to Streptococcus mutans, tartar build-up can be delayed or slowed down without having to rely on the action of germicides or other chemicals that kill microorganisms in the oral cavity. It is suspected that the normal microflora of the oral cavity is beneficial to humans and animals, for example, as such microorganisms, normal microflora will compete with pathogens for food, thereby limiting the growth of pathogens, thus avoiding infections. The present invention allows the normal microflora of the oral cavity to remain substantially undisturbed and still confer oral hygiene properties, preferably preventing or slowing down the formation of tartar.

Another advantage of the present invention in the prevention and treatment of tartar is that the present invention does not require the presence of calcium salt scavenging agents such as zinc sulfate, soluble pyrophosphates, sodium tripolyphosphate and soluble diphosphonates. Such agents remove calcium from the oral cavity, thereby removing a key component of tartar. However, tooth enamel is primarily composed of hydroxyapatite, which is a mineral with a high calcium content. Thus, the removal of calcium from the oral cavity leads to further weakening of the teeth, which is undesirable. The present invention, on the other hand, allows for the treatment or prevention of tartar formation without calcium removal, which is essential for maintaining healthy tooth enamel.

In order to achieve the best results in the prevention of tartar formation, the compositions of the present invention therefore include a binding microorganism or a fragment thereof, in which the binding microorganism belongs preferably to the Lactobacillaceae family, more preferably to the Lactobacillus genus, and most preferably to one of the above deposited strains of binding microorganisms, and additionally contains agents for removing calcium salts in such a concentration that the binding of binding microorganisms or their fragments to mutans Streptococci is reduced by no more than 10%, as determined by nephelometry, and preferably does not contain such agents. In such preferred compositions, calcium salt removers are preferably selected from the group of: zinc sulfate, zinc chloride, soluble pyrophosphates, sodium tripolyphosphate and soluble diphosphonates.

The present invention also provides the use of a binding microorganism, or a fragment thereof, as described above, for the preparation of a medicament for the prevention or treatment of dental calculus, preferably in a child or pet. As mentioned above, the binding microorganism may also be in a thermally inactivated form, in particular in an autoclaved form or in a lyophilized form. The binding microorganism preferably belongs to the Lactobacillaceae family, even more preferably the Lactobacillus genus, even more preferably the Lactobacillus paracasei or Lactobacillus rhamnosus species. In particular, the binding microorganism according to the present invention may be selected from the above strains of L. paracasei or L. rhamnosus, respectively, having any DSMZ accession number from 16667 to 16673, or a mutant or derivative thereof.

Most preferably, the compositions according to the present invention are food or feed compositions, especially for children or pets. Such food or oral hygiene food compositions are sometimes referred to as functional food or functional food. The terms "food" and "feed" are used in accordance with the present invention regardless of the nutritional value of the respective compositions, and thus are not limited to specific nutritional purposes, although food and feed compositions in accordance with the present invention may be adapted for such specific purposes. The terms "food" and "food" thus indicate that the respective compositions are suitable for placement in the oral cavity and for ingestion.

Of particular importance in accordance with the present invention are food or feed compositions for animals, preferably for pets, and most preferably for cats, dogs, rats, hamsters and guinea pigs. In such animals, caries formation is particularly notorious and also difficult to treat, as any dental treatment, such as tartar removal, requires the pet to be anesthetized. The present invention is particularly suitable for preventing the need for such stressful treatment.

The food composition according to the present invention is preferably a pet food, i. composition for the nutrition of animals bred or kept but not normally used for human consumption in the European Union. Preferably, the "pet" according to the present invention is a mammal of the order Carnivora, even more preferably the suborder Canis or the suborder Feline, and most preferably the canine or feline families. In addition, domestic animals of the order Rodents are preferred, with mice, rabbits, hamsters and guinea pigs being particularly preferred animals.

For pet foods, the composition according to the present invention is preferably compound feed, complete feed, complementary feed or feed with mineral supplements. In accordance with the present invention, the term "compound feed" means a mixture of at least two starting materials, containing or not containing feed additives for oral feeding of animals in the form of a complete or supplementary feed. The term "complete feed" means compound feed which, by virtue of its composition, is sufficient for a daily diet. The term "complementary food" in accordance with the present invention means a compound feed which has a high content of certain substances, but which, due to its composition, is sufficient for a daily diet only if it is used in combination with other feeds. The term "food with mineral supplements" means supplementary food containing at least 40% minerals. Finally, the term "feed" in the sense of the present invention means products of vegetable or animal origin whose main purpose is to satisfy the nutritional requirements of the animal in their natural state, fresh or preserved, and products obtained by industrial processing thereof, whether organic or inorganic. substances, whether or not containing feed additives, which are intended to be used in the oral feeding of animals, either directly as such, or after processing, or in the preparation of compound feed, or as a carrier for premixes. The term "oral feeding of an animal" means the introduction of food into the gastrointestinal tract of an animal through the mouth in order to meet the nutritional needs of the animal and/or maintain the productivity of normally healthy animals.

In accordance with the present invention, the composition containing the binding microorganism may also be an oral care composition. Preferred examples of oral care compositions according to the present invention are: toothpaste, dentifrices, tooth powders, oral gels, mouthwashes, denture substance, mouth sprays, lozenges, oral tablets, chewing gums, dental floss or dental tape and, in particular for animals, chewing products.

A preferred drug composition according to the present invention contains not only a binding microorganism or a fragment thereof, but also an orally acceptable carrier, such a carrier may be any suitable vehicle that can be applied to the oral cavity in a safe and effective manner, so that the binding microorganism of the present invention and/or a fragment thereof may bind to one or more strains of Streptococci mutans, and preferably one or more strains of Streptococcus mutans, thereby exerting an anti-tartar and/or anti-cariogenic and/or anti-malodor effect. The drug composition may be a composition with one or more phases.

In particular, the oral care agent of the present invention may be a paste, gel, or liquid composition, unless otherwise indicated. The dentifrice composition may be in any desired form, such as bulky streaky, superficially streaky, multi-layered, gel-coated paste, or any combination thereof. The dentifrice composition may be contained in physically separated compartments of the dispenser and released in parallel. Compositions for caring for teeth, for example, are described in EP 0617608 B1.

Preferred dentifrice compositions are described in Examples 21 to 24 of WO 2008/074473 A2. In addition to the ingredients described above, the dentifrice compositions of the present invention may contain a variety of optional dentifrice ingredients, some of which are described below. Optional ingredients include, for example, but not limited to, adhesives, foaming agents, flavors, sweeteners, additional antitartar agents, additional abrasives, and colorants. These and other optional components are further described in, for example, US Pat.

For example, a toothpaste may include one or more surfactants, chelating agents, fluoride sources, tooth whitening actives and tooth color modifying agents, thickeners, humectants, flavors and sweeteners, alkali metal bicarbonate, various carriers, and /or other active agents.

One of the preferred optional agents used in accordance with the present invention is a surfactant, preferably selected from the group consisting of sarcosinate surfactants, isethionate surfactants and taurate surfactants. Preferred for use in the present invention are the alkali metal or ammonium salts of these surfactants. Most preferred in the present invention are the sodium and potassium salts of the following: lauroyl sarcosinate, myristoyl sarcosinate, palmitoyl sarcosinate, stearoyl sarcosinate, and oleoyl sarcosinate.

Another preferred additional agent is a chelating agent such as tartaric acid and its pharmaceutically acceptable salts, citric acid and alkali metal citrates and mixtures thereof. Chelating agents are capable of complexing with the calcium found in bacterial cell walls. Chelating agents can also destroy plaque by removing calcium from the calcium bridges that help keep this biomass intact. However, as indicated above, there is a preferred maximum concentration of such agents or such compositions that are intended for particularly gentle dental treatment.

Typically, there is an additional water-soluble fluoride compound present in dentifrices and other oral compositions in an amount sufficient to produce a concentration of fluoride ion in the composition at 25° C. and/or when used from about 0.0025% to about 5.0% by weight, preferably from about 0.005% to about 2.0% by weight, to provide additional anticaries efficacy. A wide variety of materials containing fluoride ions can be used as sources of soluble fluoride in compositions in accordance with the present invention. Examples of suitable fluoride ion yielding materials are found in US Pat. No. 3,535,421 and US Pat. No. 3,678,154. Representative sources of fluoride ions include stannous fluoride, sodium fluoride, potassium fluoride, sodium monofluorophosphate, and many others. Tin fluoride and sodium fluoride are particularly preferred, as well as mixtures thereof.

The oral hygiene compositions of the present invention may also include tooth whitening actives, including whitening or oxidizing agents such as peroxides, perborates, percarbonates, peroxyacids, persulfates, metal chlorites, and combinations thereof. Suitable compounds include hydrogen peroxide, urea peroxide, calcium peroxide and mixtures thereof. The preferred percarbonate is sodium percarbonate. Other suitable bleaching agents include potassium, ammonium, sodium and lithium persulfates and perborate mono- and tetrahydrates, and sodium pyrophosphate peroxyhydrate. Suitable metal chlorites include calcium chlorite, barium chlorite, magnesium chlorite, lithium chlorite, sodium chlorite and potassium chlorite. The preferred chlorite is sodium chlorite. Additional whitening actives may be hypochlorite and chlorine dioxide.

In addition to tooth whitening agents as whitening agents, tooth color modifying agents may be considered among the oral care actives useful in the present invention. These substances are suitable for changing the color of the teeth to suit the consumer. These substances include particles which, when applied to the surface of teeth, modify that surface by absorbing and/or reflecting light. Such particles provide an improvement in appearance when a film containing such particles is applied to the surfaces of a tooth or teeth.

When making toothpastes or gels, it is necessary to add some thickening agents to obtain the desired consistency of the composition, to provide the desired active release characteristics in use, to ensure storage stability and to ensure the stability of the composition, etc. Preferred thickening agents are carboxyvinyl polymers, carrageenan, hydroxyethyl cellulose, ^LAPONIT® (manufactured by Rockwood Additives Limited) and water-soluble cellulose ether salts such as sodium carboxymethyl cellulose and sodium carboxymethyl hydroxyethyl cellulose. Natural gums such as karaya gum, xanthan gum, gum arabic and gum tragacanth can also be used. Colloidal magnesium aluminosilicate or fumed silica can be used as part of the thickener to further improve texture.

Another optional component of the oral carrier of the topical composition according to the present invention is a humectant. The humectant serves to prevent the toothpaste composition from hardening when exposed to air, giving the composition a moist mouthfeel, and for specific humectants, imparting a desirable sweet taste to the toothpaste composition. The humectant on a pure moisturizing basis typically comprises from about 0% to about 70%, preferably from about 5% to about 25%, by weight of the composition of the present invention. Suitable humectants for use in the compositions of the present invention include edible polyhydric alcohols such as glycerol, sorbitol, xylitol, butylene glycol, polyethylene glycol and propylene glycol, especially sorbitol and glycerin.

Flavorings and sweetening agents may also be added to the compositions. Suitable flavors include wintergreen oil, peppermint oil, spearmint oil, clove oil, menthol, anethole, methyl salicylate, eucalyptol, cassia, 1-menthyl acetate, sage, eugenol, parsley oil, oxanone, alpha irisone, marjoram, lemon , orange, propenyl guaetol, cinnamon, vanillin, thymol, linalool, cinnamaldehyde glycerol acetal, known as CGA, and mixtures thereof. Flavorants are typically used in the compositions in an amount of from about 0.001% to about 5% by weight of the composition.

Sweeteners that may be used include sucrose, glucose, saccharin, glucose, levulose, lactose as described above, mannitol, sorbitol, fructose, maltose, xylitol, saccharin salts, thaumatin, aspartame, D-tryptophan, dihydrochalcones, acesulfame salts and cyclamate, especially sodium cyclamate and sodium saccharin and mixtures thereof. The infant formula composition preferably contains from about 0.1% to about 10% of these agents, preferably from about 0.1% to about 1% by weight of the composition. Preferably, the pet food according to the present invention has only a low content of said Sugars which can be metabolized by Streptococci mutans and preferably by Streptococcus mutans to avoid the cariogenic activity of Streptococci mutans. Thus, the content of sucrose, cane sugar, caramel, corn syrup, corn syrup, glucose, fructose and sorbitol is preferably kept low, with a maximum content of 20 wt. % of total infant food or pet's preferred food; maximum content 5 wt. % is even more preferred, and the maximum content of 2 wt. % is most preferred. It is also preferred that the content of each of saccharin, glucose, levulose, lactose, mannitol, maltose and xylitol is less than 20 wt. %, more preferably less than 5 wt. % and most preferably less than 2 wt. %, and preferably without xylitol in pet food in general.

The oral hygiene medicinal composition of the present invention may also include an alkali metal bicarbonate salt. Alkali metal bicarbonate salts are soluble in water and, if not stabilized, tend to release carbon dioxide in an aqueous system. Sodium bicarbonate, also known as baking soda, is the preferred alkali metal bicarbonate salt. The composition in accordance with the present invention may contain from about 0.5% to about 30%, preferably from about 0.5% to about 15%, and most preferably from about 0.5% to about 5% alkali metal bicarbonate salt. The water used in the preparation of commercially acceptable oral compositions should preferably be low in ions and free of organic impurities. Water typically comprises from about 10% to about 50%, and preferably from about 20% to about 40%, by weight of the aqueous toothpaste composition of the present invention. These amounts of water include free water that is added plus that added with other materials such as sorbitol. Titanium dioxide can also be added to compositions in accordance with the present invention. Titanium dioxide is a white powder that imparts opacity to compositions. Titanium dioxide is typically present in about 0.25% to about 5% by weight of the dentifrice composition.

The pH of the compositions according to the present invention is preferably controlled by the use of buffering agents. Buffer agents used in the present invention refer to agents that can be used to adjust the pH of the compositions in the range from about 4.5 to about 9.5, preferably from 4.5 to 8.5. Buffering agents include sodium monophosphate, trisodium phosphate, sodium hydroxide, sodium carbonate, sodium acid pyrophosphate, citric acid, and sodium citrate. Buffering agents can be added in an amount of from about 0.5% to about 10% by weight of the compositions in accordance with the present invention. The pH of the dentifrice composition is measured from a 3:1 aqueous slurry of dentifrice, eg 3 parts water to 1 part toothpaste.

Other optional agents that may be used in the compositions of the present invention include dimethicone copolyols selected from alkyl and alkoxy dimethicone copolyols, such as C12 to -C20 alkyl dimethicone copolyols, and mixtures thereof. Highly preferred is cetyl dimethicone copolyol, which is sold under the trade name Abil EM90. The dimethicone copolyol is typically present in an amount of from about 0.01% to about 25%, preferably from about 0.1% to about 5%, more preferably from about 0.5% to about 1.5% by weight. Dimethicone copolyols help in providing positive tooth feel effects. Other useful carriers include biphasic dentifrice formulations such as those disclosed in US Pat.

Compositions in accordance with the present invention may also contain other active agents such as antimicrobial agents. Among such agents are water-insoluble non-cationic antimicrobial agents such as halogenated diphenyl ethers, phenolic compounds including phenol and its homologues, mono- and polyalkyl and aromatic halophenols, resorcinol and its derivatives, bisphenolic compounds and halogenated salicylanilides, benzoic acid esters and halogenated carbanilides. Water-soluble antimicrobials include quaternary ammonium salts and bis-biguanide salts, among others. Triclosan monophosphate is an additional water-soluble antimicrobial agent. Quaternary ammonium agents include those in which one or two of the substituents on the quaternary nitrogen have a carbon chain length (typically an alkyl group) of about 8 to about 20, typically about 10 to about 18 carbon atoms, and the remaining substituents (typically an alkyl or benzyl group) have fewer carbon atoms, for example, from about 1 to about 7 carbon atoms, usually methyl or ethyl groups. Dodecyltrimethylammonium bromide, tetradecylpyridinium chloride, domifene bromide, N-tetradecyl-4-ethylpyridinium chloride, dodecyldimethyl-(2-phenoxyethyl)ammonium bromide, benziddimethylstearylammonium chloride, cetylpyridinium chloride, quaternized 5-amino-1,3-bis(2-ethylhexyl)- 5-methyl-hexahydropyrimidine, benzalkonium chloride, benzethonium chloride and methylbenzetonium chloride are examples of typical quaternary ammonium antibacterial agents. Other compounds are the bis[4-(11-amino)-1-pyridinium]alkanes described in US Pat. No. 4,206,215. Other antimicrobial agents such as copper bisglycinate, copper glycinate, zinc citrate, and zinc lactate may also be included. Enzymes are another type of active substance that can be used in compositions in accordance with the present invention. Useful enzymes include those that belong to the category of proteases, lytic enzymes, plaque matrix inhibitors and oxidases: proteases include papain, pepsin, trypsin, ficin, bromelain; cell wall lytic enzymes include lysozyme, plaque matrix inhibitors include dextranases, mutanases, and oxidases include lactate oxidase, galatose oxidase, uric acid oxidase, peroxidases including horseradish peroxidase, myeloperoxidase, lactoperoxidase, chloroperoxidase. Oxidases also have bleaching/cleaning activity, in addition to antimicrobial properties. Such agents are described in US Pat. No. 2,946,725 and US Pat. No. 4,051,234. Other antimicrobial agents include chlorhexidine, triclosan, triclosan monophosphate, and aromatic oils such as thymol. Triclosan and other agents of this type are described in US Pat.

The term "chewing gum" as defined herein means a confectionery composition that is suitable for chewing and that contains any suitable amount of elastomer known to those skilled in the art, preferably in an amount of 2% or more by weight of the composition. Suitable lozenge and chewing gum components are described in, for example, US Pat. No. 4,083,955; US Pat. No. 6,770,264 or US Pat. No. 6,270,781. Preferred lozenges are described in examples 19 and 20 of WO 2008/074473 A2. A preferred chewing gum composition is described in Example 25 of WO 2008/074473 A2.

Compositions in accordance with the present invention preferably contain an elastomer or a mixture of several different elastomers. Elastomeric materials are generally known in the art, but illustrative examples include styrene-butadiene rubber (SBR); synthetic gums, polyisobutylene and copolymers of isobutylene and isoprene; natural gums, chicle, natural rubber, gelutong, balata, gutta-percha; lechispi; pluck and their mixtures. The compositions used in accordance with the present invention preferably contain from about 2% to about 30%, more preferably from about 5% to about 25% by weight of the elastomer. These concentrations are determined by the desired final texture of the chewing gum, since when the total elastomer level is below about 2%, the base composition loses elasticity, chewy texture and grip properties, while above about 30% the formulation becomes hard, rubbery and chewy. difficult. Elastomer solvents are also preferably present in the composition used in accordance with the present invention, since they help soften the elastomer component. Preferred examples of elastomer solvents for use in the present invention include pentaerythritol partially hydrogenated rosin ester, pentaerythritol rosin ester, partially dimerized rosin glycerol ester, polymerized rosin glycerol ester, tall oil glycerol ester, wood rosin or gum rosin, partially hydrogenated glycerol ester rosin, partially hydrogenated rosin methyl ester, and mixtures thereof. The compositions used in accordance with the present invention preferably contain from about 2% to about 50%, more preferably from about 10% to about 35% by weight of the elastomer solvent.

Lozenges according to the present invention can be prepared, for example, using methods known in the art for forming compressed tablets, wherein the disaccharide is dispersed over a compressed solid carrier, optionally in combination with some suitable tabletting agent such as lubricants (for example , magnesium stearate) and compressed into tablets. The solid carrier for such tablet formulations may be a salivary soluble solid such as cold water soluble starch or a monosaccharide such that the lozenge will readily dissolve in the mouth to release its disaccharide acid in the saliva solution for contact and absorption through mucous membrane of the oral cavity / nasopharynx, when placing the lozenge in the mouth. The pH of the above formulations may range from about 4 to about 8.5. The lozenges of the present invention may also be prepared using other techniques known in the art for preparing solid unit dosage formulations.

The mouthwash or mouthrinse of the present invention may contain EtOH or may be free of EtOH and may contain other active ingredients such as antimicrobial agents such as chlorhexidine. A preferred mouth wash or mouth rinse according to the present invention may be as follows:

A Peppermint oil 1.2 parts.

Arnica tincture 3.0 parts

Myrrh tincture 3.0 parts

Tween 5.0 parts

B Alcohol 90% 50.0 parts

C Sodium benzoate 0.2 parts

Sweetener (such as aspartame) 0.02 parts

Distilled water up to 100.0

A must be well mixed, B is added while stirring and C is added later. The resulting clear liquid must be filtered within 48 hours of preparation. Another preferred mouth rinse is described in Example 26 of WO 2008074473 A2.

Regardless of the dosage form, liquid or solid, in one of the preferred embodiments of the present invention, the dosage form is retained in the oral cavity of the consumer, preferably the oral cavity of the animal for a period of time sufficient to promote contact of the microorganism or analogue or fragment of the aforementioned microorganism belonging to the group lactic acid bacteria, with the oral cavity of the patient.

The terms "dental floss" and "dental tape" as used in the present invention refer to a material for displacing and removing decaying food particles that accumulate in the interdental and subgingival surfaces, and for expelling and removing bacteria, plaque and/or dental stones that accumulate in the oral cavity. The dental floss or dental tape may further comprise, in addition to the microorganisms of the present invention as described herein, cleaning agents, abrasives, tartar control ingredients, bleaches, surfactants and/or active ingredients such as as fluorides, antimicrobials, chemotherapeutic agents or antibiotics. Other additional agents are antiplaque agents, flavoring agents and colorants. The floss or dental tape may be in any suitable form known to those skilled in the art, such as PTFE (Teflon) dental floss, as described, for example, in US Pat. , US Pat. U.S. Patent 5,765,576 or U.S. Patent 5,911,228 as an interdental surgical monofilament device are described, for example, in US patent 3800812, US patent 4974615, US patent 5760117, US patent 5433226, US patent 5479952, US patent 5503842, US patent 5755243, US patent 5884639, US patent 60035 25 or U.S. Patent 6,027,592, or in the form of tapes from biocomponents. Preferably, the floss or dental tape is in the form of an elastomeric coated surgical monofilament as described in, for example, US Pat.

Cosmetic compositions for oral care as described in the present invention, in particular, antitartar compositions, anti-caries compositions and anti-malodor compositions can be used in the range of oral administration to humans, as well as in the range of oral administration in veterinary medicine, preferably mammals. non-human, more preferably for pets. If the composition is used for oral administration in the veterinary range, the composition may contain other ingredients suitable for such administration, as known to a person skilled in the art.

The pharmaceutical composition according to the present invention preferably further comprises a pharmaceutically acceptable carrier or excipient.

Pharmaceutical compositions contain a therapeutically effective amount of the above microorganisms or their fragments, and can be prepared in various forms, for example, in solid, liquid, powder, aqueous, lyophilized forms.

The pharmaceutical composition can be administered with a pharmaceutically acceptable carrier to a patient, preferably a human or animal, and most preferably a child or pet. The term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeias for use in animals and in particular in humans. The preferred pharmaceutical composition used in accordance with the present invention does not contain lactose in the range of 1% (w/w) and 6% (w/w). In addition, it is preferable that the pharmaceutical composition would contain no more than 1% (wt./wt.) lactose, for example, would contain less than 1%, preferably less than 0.9% (wt./wt.), 0.8% ( wt./mass.) lactose, etc., or that the pharmaceutical composition would contain more than 6%, 7%, 8%, etc. (w/w) lactose. Alternatively, but also preferred, the pharmaceutical composition is lactose-free.

The term "carrier" refers to a diluent, adjuvant, excipient or carrier with which the drug is administered. Such a carrier is pharmaceutically acceptable, i. is non-toxic to the recipient at the dosage and concentration used. An isotonic, hypotonic or mildly hypertonic vehicle and one having a relatively low ionic strength, such as provided by a sucrose solution, is preferred. Such pharmaceutical carriers may be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous solutions of glucose and glycerol can also be used as liquid carriers, in particular for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, skimmed milk powder, glycerin, propylene glycol, water, ethanol, and the like. The excipient may contain lactose as described above, but most preferably it does not contain lactose. The composition, if necessary, may also contain small amounts of wetting or emulsifying agents or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral compositions may include standard carriers such as pharmaceutical grade maltodextrin, mannitol, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington Pharmaceutical Sciences, E.W. Martin. Skimmed milk, skimmed milk powder, products that do not contain milk or lactose can also be used. Skimmed milk powder is usually suspended in phosphate buffered saline (PBS), autoclaved or filtered to remove protein and live contaminants, and then freeze-dried, heat-dried, vacuum-dried, or freeze-dried. Some other examples of substances that can serve as pharmaceutical carriers are sugars such as glucose and sucrose, starches such as cornstarch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetates, powdered gum tragacanth, malt, gelatin; talc, stearic acid; magnesium stearate, calcium sulfate, calcium carbonate, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; agar, alginic acid; pyrogen-free water, isotonic saline, cranberry extracts and phosphate buffer solution, skimmed milk powder, as well as other non-toxic compatible substances used in pharmaceutical preparations such as vitamin C, estrogen and echinacea, for example. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, lubricants, excipients, substances used in the manufacture of tablets, stabilizers, antioxidants and preservatives may also be present.

Preferably, the oral formulation contains lactose, as described in the present invention, and most preferably does not contain lactose. Various carriers and/or excipients suitable for oral administration, which are well known in the art, can be used for the purposes of the present invention. The non-cariogenic composition may optionally also contain various known additives such as, for example, preservatives, hardeners, lubricants, emulsifiers, stabilizers, essences and the like. Such compositions will contain a therapeutically effective amount of the above compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the appropriate form for administration to the patient. The formulation must match the route of administration.

The preferred composition in accordance with the present invention does not contain lactose in the range from 1% (wt./mass.) to 6% (wt./mass.). In addition, it is preferable that the composition contains no more than 1% (wt./wt.) lactose, for example, it would contain less than 1%, preferably less than 0.9% (wt./wt.), 0.8% ( wt./mass.) lactose, etc., or that the composition would contain more than 6%, 7%, 8%, etc. (w/w) lactose. Alternatively, but preferably, the composition is lactose-free.

In an additional aspect, a composition according to the present invention can be obtained by including the step of formulating a binding microorganism, a fragment thereof, with a cosmetically, orally or pharmaceutically acceptable carrier or excipient. Preferably, this microorganism would be a deposited microorganism, as described in the present invention above, or its mutant, derivative, analog or fragment thereof. The preferred composition in accordance with the present invention does not contain lactose in the range from 1% (wt./mass.) to 6% (wt./mass.). In addition, it is preferable that the composition contains no more than 1% (wt./wt.) lactose, for example, it would contain less than 1%, preferably less than 0.9% (wt./wt.), 0.8% ( wt./mass.) lactose, etc., or that the composition would contain more than 6%, 7%, 8%, etc. (w/w) lactose. Alternatively, but preferably, the composition is lactose-free.

A composition according to the invention, preferably a food or feed composition, including pharmaceutical compositions, contains a binding microorganism as described above, optionally in a thermally inactivated or lyophilized form, in an amount of from 10 ² to 10 ¹² cells, preferably from 10 ³ to 10 ⁸ cells per mg in solid form of the composition. In the case of the liquid form of the composition, the number of microorganisms is from 10 ² to 10 ¹³ cells per ml. However, for a particular composition, the number of microorganisms may be different, as described in the present invention.

Preferably, the concentration of binding microorganisms in the composition in accordance with the present invention would be from 0.01 wt. % up to 10 wt. % relative to the total weight of the composition. Even more preferably, the concentration of binding microorganisms in the composition in accordance with the present invention would be from 0.025 wt. % up to 2 wt. % As described above, when fragments are used instead of binding cells, the concentration of fragments is chosen so that the peptidoglycan content of the binding cells is the same.

In accordance with the present invention, the term food includes all edible and drinkable foods and drinks. Accordingly, the microorganism or fragment thereof may be included in food or drink. These are, for example, gum, spray, drinks, candy, baby food, ice cream, frozen dessert, sweet salad dressing, dairy products, cheese, cottage cheese, lactose-free yogurt, fermented milk, cream or whipped cream, and the like.

Milk-based products are provided within the scope of the present invention. Milk, however, is understood to mean animal products such as cows, goats, sheep, buffaloes, zebras, horses, monkeys, or camels, and the like. The milk may be in its native state, reconstituted milk, skimmed milk, or milk supplement with compounds necessary for bacterial growth or for post-processing of fermented milk, such as fats, yeast extract proteins, peptone and/or surfactant, for example. The term milk is also applied to what is commonly referred to as plant milk, i.e. extracts of plant material that have been processed or not, such as legumes (soybeans, chickpeas, lentils, etc.), or oilseeds (rapeseed, soybeans). , beans, sesame, cotton and the like), the extract of which contains proteins in solution or in colloidal suspension, which coagulate through chemical action, through acid fermentation and/or heating. Finally, the word milk means mixtures of animal milk and vegetable milk.

Where the binding microorganism of the present invention or a fragment thereof is added to yogurt and the like having a similar content, it is sufficient to add the microorganism according to the present invention at a concentration of about 10 ⁵ -10 ⁷ cells/ml, or an equivalent amount of its fragment. In such a case, it is possible to completely prevent or inhibit biofilm formation by Streptococci mutans, preferably Streptococcus mutans, and thus prevent or slow down the development of tartar, halitosis or caries induced by cariogenic strains, without significant side effects on the quality of the drink itself.

Such an edible beverage or feed can be prepared in a general manner for the production of foods, beverages or feeds, including adding the active ingredient to a raw or prepared food, beverage or feed material. The food, drink or feed according to the present invention can be molded and pelletized in the same manner as is commonly used for food, drink and feed. Shaping and granulation includes granulation methods such as fluidized bed granulation, agitation granulation, extrusion granulation, roller granulation, gas flow granulation, injection molding granulation, hot granulation, spray granulation, and injection granulation, coating methods such as panning, fluidized bed coating and dry coating, dry sponge method, excess steam method, foam matting method, expansion methods such as microwave incubation method and extrusion methods with extrusion pelletizing machines and extruders.

The food, drink or feed of the present invention includes any food, drink or feed that contains the binding microorganism of the present invention or a fragment thereof as an active ingredient. The active ingredient in a food, drink or feed is not specifically limited to any concentration as long as the resulting food, drink or feed can exert its activity to specifically bind to Streptococci mutans. The concentration of the active ingredient is preferably from 0.001 to 100% by weight, more preferably from 0.01 to 100% by weight and most preferably from 0.1 to 100% by weight of food, drink or feed containing such an active ingredient, or in relation to to the number of cells of those described in the present invention.

Specific foods or drinks in which the active ingredient is added include, for example, juices, soft drinks, soups, teas, fermented milk drinks, dairy products such as fermented milk products, ice cream, butter, cheese, processed milk and skimmed milk, meat foods such as ham, sausage, and hamburger, foods such as fish cake, egg products such as seasoned egg rolls and egg curds, confectionery products such as cookies, jelly, snacks, and chewing gum, bread, pasta, pickles, smoked products, dried fish and seasonings. The food or drink form includes, for example, powdered foods, sheet foods, bottled foods, canned foods, autoclaved foods, encapsulated foods, tablet foods, and liquid foods.

A food or drink according to the present invention that is intended to be swallowed by infants, i. containing a binding microorganism or a fragment thereof with activity to specifically bind to Streptococci mutans, is preferably an infant formula. Such nutritional composition for infants includes modified milk prepared for infants, milk with hydrolyzed proteins, modified milk for specific nutrition or baby food, and meals prepared for infants. The form of infant formula includes, but is not limited to, special powdered milk, dried and sprayed and baby food, and also includes common products such as ice cream, fermented milk, and jelly for infants to swallow.

The nutritional composition for infants, as well as nutritional products or feed compositions for animals and especially pets in accordance with the present invention mainly consist of protein, lipids, saccharides, vitamins and/or minerals. In the nutritional composition, the active ingredient interferes with these components. Protein includes milk proteins such as skim milk, casein, cheese whey, whey protein concentrate and whey protein isolates and fractions thereof such as alpha-s-casein, beta-casein, alpha-lactoalbulin and beta-lactoglobulin. Additionally, egg white such as egg yolk protein, egg white and egg albumin, or soy protein such as defatted soy protein, isolated soy protein and concentrated soy protein can be used. Besides these proteins, such as wheat gluten, fish meat proteins, bovine meat proteins and collagen can also be used satisfactorily. Additionally, fractions of these proteins, peptides from acidic or enzymatic treatment of them, or free amino acids can also be satisfactorily used. Free amino acids can serve as nitrogen sources and can additionally be used for specific physiological activities. Such free amino acids include, for example, taurine, arginine, cysteine, cystine, and glutamine. For dogs, amino acids include arginine, methionine, histidine, phenylalanine, isoleucine, threonine, leucine, tryptophan, lysine, and valine. For cats, taurine is also important. Lipids include animal fats and oils such as milk fat, lard, meat fat and fish oil, vegetable oils such as soybean oil, rapeseed oil, corn oil, coconut oil, palm oil, palm kernel oil, safflower oil, perilla oil, linseed oil. oil, evening primrose oil, medium-long fatty acid triglycerides and cottonseed oil, fats and oils produced by bacteria and fractionated oils of the foregoing, their hydrogenated oils and their interesterified oils. The amount of lipids to be mixed varies depending on the application.

In addition, omega-6 fatty acids and omega-3 fatty acids are preferred ingredients in foods and feeds, especially pet foods. Particularly preferred are linoleic acid, preferably in the form of corn, soy, rapeseed, safflower and sunflower oils, whole grains and/or poultry fats; arachidonic acid, preferably in the form of poultry fat, meat, egg yolk and/or fish oil; gamma-linoleic acid, preferably in the form of blackcurrant seed oil, borage oil and/or evening primrose oil; dihomogammalinolenic acid, preferably in the form of spleen, kidney, adrenal and/or metabolized from gla (gamma-linolenic acid); alpha-linolenic acid, preferably in the form of linseed oil, canola oil, soybean and/or walnut oil; eicosapentaenoic acid, preferably in the form of cold fish and their oil; docosahexaenoic acid, preferably in the form of cold fish and their oil.

Saccharides include, for example, one or more starches, soluble polysaccharides, dextrins, monosaccharides such as sucrose, lactose as described herein, maltose, glucose and fructose, and other oligosaccharides. Preferred saccharides include glucose, fructose, honey, galactose, lactose, sucrose, maltose, dextrins, glycogen and starch. However, as described above, the maximum content of glucose, fructose, honey, galactose, lactose, sucrose and maltose is preferably 20 wt. % of the total weight of the composition, more preferably 5 wt. % of the total composition and most preferably 2 wt. % of the total weight of the composition. Even more preferably, the aforementioned maximum content of 20 wt. %, more preferably 5 wt. % and most preferably 2 wt. % refers to the total amount of all substances of the group: cane sugar, caramel, corn molasses, corn syrup, glucose, fructose, galactose, glucose, honey, lactose, levulose, maltose, mannitol, saccharin, sorbitol, xylitol and sucrose, and most preferably, to the total number of mono- and disaccharides.

Also preferred are dietary fibers, preferably selected from cellulose, hemicellulose, pectin, vegetable gums and a mixture of solutions of gums, beet pulp, guar gum, gum arabic, xanthan gum and locust bean gum. The total amount of such saccharides is preferably 40 to 80% by weight of the total solids in the nutritional composition. In addition, artificial sweeteners such as aspartame can be used satisfactorily. The amount of artificial sweetener is suitably 0.05 to 1.0% based on the total weight of solids in the nutritional composition.

Vitamins include, but are not limited to: lycopene as the main component and additionally include, for example, vitamins such as vitamin A, vitamin B, vitamins C, D, E and vitamin K, folic acid, pantothenic acid, nicotinamide , carnitine, choline, inositol and biotin as long as such vitamins can be administered to children or pets. Such vitamins preferably comprise from 10 mg to 5 g%, based on the total weight of solids in the infant nutritional composition. Preferred vitamins for nutritional compositions for infants and/or animals, preferably pets, include:

Vitamin A (retinol) and/or beta-carotene as a precursor. The vitamin is preferably present in the form of liver, fish oil, carrots, green leafy vegetables, egg yolks and/or yellow fruits, or as a synthetic form of vitamin A and/or beta-carotene.

Vitamin D (calciferol). The vitamin is preferably present in the form of halibut and/or cod liver oil, sea fish, cheese, yogurt and/or egg, or as a synthetic form of vitamin D.

Vitamin E (tocopherol). The vitamin is preferably present in the form of germ, corn, nuts, seeds, spinach and/or other green leafy vegetables, asparagus, vegetable oils, or as a synthetic form of vitamin E.

Vitamin K (naphthoquinone). The vitamin is preferably present in the form of cabbage, cauliflower, spinach and/or other green leafy vegetables, cereals, soy and/or other vegetables, or as a synthetic form of vitamin K. Preferably, the food or feed does not contain menadione.

Vitamin B1 (thiamine). The vitamin is preferably present as wheat germ, rice and/or other whole grains, lean meat (especially pork), liver, fish, yeast, dried beans, peas and/or soybeans, or as synthetic forms of vitamin B1.

Vitamin B2 (riboflavin). The vitamin is preferably present as lean meat, liver, fish, eggs, yeast, cheese, legumes, nuts and/or green leafy vegetables, or as a synthetic form of vitamin B2.

Vitamin B3 (niacin). The vitamin is preferably present in the form of liver, lean meat, poultry, fish, nuts, yeast, legumes, asparagus, seeds and/or green leafy vegetables, or as synthetic forms of vitamin B3.

Vitamin B5 (pantothenic acid). The vitamin is preferably present in the form of eggs, fish, lean beef, legumes, yeast, broccoli and/or other vegetables of the cabbage, white and/or sweet potato families, or as synthetic forms of vitamin B5.

Vitamin B6 (pyridoxine). The vitamin is preferably present in the form of meat, fish, eggs, bananas and/or whole grains, or as synthetic forms of vitamin B6.

Vitamin B8 (biotin). The vitamin is preferably present as raw egg yolk, liver and/or vegetables, or as synthetic forms of vitamin B8.

Vitamin B9 (folic acid, folate). The vitamin is preferably present in the form of carrots, yeast, liver, egg yolk, melon, apricots, pumpkin, beans, rye, whole wheat and/or green leafy vegetables, or synthetic forms of vitamin B9.

Vitamin B12 (cobalamin). The vitamin is preferably present as fish, liver, meat, poultry, eggs and/or cheese, or as synthetic forms of vitamin B12.

Vitamin C (ascorbic acid). The vitamin is preferably present in the form of lemon fruit juice or pulp, berries, tomato, cauliflower, potatoes, green leafy vegetables and/or green peppers or synthetic forms of vitamin C. Supplements in an appropriate form, preferably calcium ascorbate, are preferred from -for their beneficial effects on dogs suffering from chronic joint and musculoskeletal disorders. In puppies, they help prevent the development of such disorders.

In addition, minerals include calcium, magnesium, potassium, sodium, iron, copper, zinc, phosphorus, chlorine, manganese, selenium, and iodine. Such minerals preferably comprise from 1 mg to 5 g based on the total weight of solids in the infant nutritional composition.

In addition to these components described above, the infant nutritional composition, as well as the food or feed compositions for animals, preferably pets, used in accordance with the present invention can be mixed with any component, preferably mixed with nutritional compositions, for example, dietary fibers. , nucleotides, nucleic acids, flavors and dyes.

The food or drink used in accordance with the present invention can be used as a health food or drink or a functional food or drink to prevent and/or treat caries and/or to prevent or treat bad breath and/or to prevent formation or treatment of tartar.

When the food or drink according to the present invention is ingested, the amount to be taken is not limited in any particular way. The amount to be ingested is generally in the range of 0.1 to 50 g, preferably 0.5 g to 20 g per day, based on the total amount of the active ingredient.

The food or drink is continuously ingested in this amount for a period of one day to 5 years, preferably 2 weeks to one year. In the present patent, the amount consumed can be adjusted to an appropriate range depending on the severity of the symptoms of the individual taking the food or drink, their age and body weight, and the like.

The composition in accordance with the present invention may contain, in addition to the binding microorganism or its fragment, cereals, bran, cake flour, feed raw materials of animal origin, other food raw materials and refined products. Cereals may include, celery, wheat, barley, oats, rye, brown rice, buckwheat, italian millet, chinese millet, deccan grass, corn, and soybeans. The bran may include rice bran, defatted rice bran, low grade flour bran, wheat germ, barley bran, selected feed pellets, corn bran and corn germ. Seed meal includes, for example, soybean meal, soybean powder, flaxseed meal, cottonseed meal, peanut meal, sunflower meal, coconut meal, palm meal, sesame meal, sunflower meal, rapeseed meal, kapok meal, and mustard flour. Raw materials for animal feed include, for example, fish meal, imported foods, whole foods, coastal foods, fish digest, meat powder, meat and bone meal, blood meal, hydrolyzed hair, bone meal, slaughterhouse by-products, feather meal, mulberry pupae silkworm, skimmed milk, casein, whey powder and krill. Other feed raw materials include, for example, plant stems and leaves such as alfalfa, hey cube, alfalfa leaf products, locust bean leaf powder, corn processing industry by-products such as corn gluten meal, corn densifier and liquid corn extract, starch, sugar, yeast, fermentation by-products such as beer sludge, malt root, liquor sludge and soy sauce sludge and agricultural by-products such as citrus processing residues, bean curd residue, coffee grounds and cocoa residue, cassava, fava beans, guar flour, seaweed, spirulina and chlorella. Purified products include, for example, proteins such as casein and albumin, amino acids, starch, cellulose, saccharides such as sucrose and glucose, minerals and vitamins.

The composition in accordance with the present invention may further contain one or more additives. Such food additives can be prepared by the general process for preparing food, beverage and feed additives. If necessary, additives for general use in food, drink or feed, such as those described in the Food Additive Handbook (The Japan Food Additives Association; issued on January 6, 1997) can be added satisfactorily, including sweeteners, colorants , preservatives, thickeners and stabilizers, antioxidants, color fixers, bleaches, antiseptics, gum base, bitters, enzymes, bleaches, acidifiers, seasonings, emulsifiers, enhancers, manufacturing agents, flavorings and spice extract. In addition, conventional saccharides, starch, inorganic substances, vegetable powders, excipients, disintegrators, lubricants, binders, surfactants, plasticizers and agents mentioned above for pharmaceutical tablets can be added satisfactorily.

Sweeteners include brazzein; curculin; erythritol; glycyrrhizin; glycerin, E422; hydrogenated starch hydrolyzate; inulin; isomalt, E953; lactitol, E966; arhat; mabinlin; maltitol, E965; maltooligosaccharides; mannitol, E421; miraculin; monatin; monellin; pentadine; sorbitol, E420; stevia extracts or steviol glycosides, especially stevioside, rebaudioside A, rebaudioside C, dulcoside A, rubusoside, steviolbioside H and rebaudioside B; tagatose; thaumatin, E957; xylitol, E967, acesulfame potassium, E950; elites; aspartame, E951, aspartame-acesulfame salt, E962; cyclamate, E952; dulcin; glucin; neohesperidin dihydrochalcone, E959; neotame, P-4000, saccharin, E954; sucralose, E955; licorice; xylose and crayfish (fruit Momordica grosvenori).

Colors include carotenoids and turmeric resin, flavanoid, caramel color, spirulina color, chlorophyll, purple sweet potato color, purple yam color, perilla color, and blueberry color.

Preservatives include, for example, sodium sulfite, benzoates, benzoin extract, sorbates and propionates.

Thickeners and stabilizers include, for example, gums such as gum arabic and xanthan gum, alginates, chitin, chitosan, aloe extract, guar gum, hydroxypropyl cellulose, sodium casein, corn starch, carboxymethyl cellulose, gelatin, agar, dextrin, methyl cellulose, polyvinyl alcohol, microfiber cellulose, microcrystalline cellulose, seaweed cellulose, sodium polyacrylate, sodium polyphosphate, carrageenan, or yeast cell walls.

Antioxidants include, for example, vitamin C, sodium ethylenediaminetetraacetate, calcium ethylenediaminetetraacetate, erythorbic acid, oryzanol, catechin, quercetin, clove extract, enzyme-treated rutin, apple extract, sesame seed extract, dibutylhydroxytoluene, fennel extract, horseradish extract, celery aqueous extract, tea extract, tocopherols, rapeseed, coffee bean extract, sunflower seed extract, ferulic acid, butylhydroxyanisole, blueberry leaf extract, propolis extract, pepper extract, garden balsam extract, gallic acid, eucalyptus extract, and rosemary extract.

Color fixing agents include, for example, sodium nitrite. Bleaches include, for example, sodium sulfite.

Antiseptics include, for example, o-phenylphenol. The gum base includes, for example, methyl acetyl ricinoleate, urushi wax, rosin ester, elemi gum, urukuri wax, kauri gum, carnauba wax, glycerol fatty acid ester, spermaceti wax, copai balm, copal gum, rubber, rice bran wax, cane wax , shellac, gelutong, sucrose fatty acid ester, natural rubber depolymerization, paraffin wax, fir balsam, propylene glycol fatty acid esters, powdered cellulose, powdered rice husk, jojoba oil, polyisobutylene, polybutene, microcrystalline wax, mastic, beeswax and phosphate calcium. Bitters include, for example, iso-alpha-bitter acid, caffeine, kawaratake extract (Coriolus versicolor), quinine extract from red Peruvian bark, philodendron bark extract, gentian root extract, spice extracts, enzymatically modified naringin, Jamaican cassia extract, theabromin, naringin, cassia extract, Poland extract, isodonis extract, olive tea, bitter orange (Citrus aurantium), hops and wormwood extract. Enzymes include, for example, amylase, trypsin or rennet. Bleachers include, for example, urushi wax and Japanese wax. The acidifier includes, for example, adipic acid, itaconic acid, citric acid, succinic acid, sodium acetate, tartaric acid, carbon dioxide, lactic acid, phytic acid, fumaric acid, malic acid, and phosphoric acid. Seasonings include, for example, amino acids such as asparagine, aspartic acid, glutamic acid, glutamine, alanine, isoleucine, glycine, serine, cysteine, tyrosine, leucine and proline, nucleic acids such as sodium inosinate sodium uridinate, sodium guanylate, sodium cytidilate , calcium ribonucleotide and sodium ribonucleotide, organic acids such as citric acid and succinic acid, potassium chloride, dilute sodium chloride brine, commercial potassium chloride, whey salt, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, and chlorella extract.

Enhancers include, for example, zinc salts, vitamin C, various amino acids, 5-adenylic acid, ferric chloride, hesperidin, various calcified calcium, various uncalcified calcium, dibenzoyltriamine, calcium hydroxide, calcium carbonate, thiamine hydrochloride, Dunaliella carotene, tocopherol, nicotinic acid, carrot carotene, palm oil carotene, calcium pantothenate, vitamin A, hydroxyproline, calcium pyrophosphate iron dihydrogen phosphate, iron pyrophosphate, ferritin, heme iron, menaquinone, folic acid and riboflavin.

Manufacturing agents include, for example, process aids such as acetone and ion exchange resins.

Flavors include, for example, vanilla essence and spice extracts, including, for example, capsicum extract.

These various additives may be added to the active ingredient, depending on the route of administration, in accordance with the present invention.

It is contemplated that the composition according to the present invention, preferably a food or feed composition or a pharmaceutical composition, includes the aforementioned binding microorganism belonging to the group of lactic acid bacteria as a probiotic microorganism. Namely, in addition to the probiotic effect, the above probiotic microorganism belonging to the group of lactic acid bacteria can be used to treat and/or prevent the formation of a biofilm caused by Streptococci mutans. The amount of said probiotic microorganism is high enough to substantially positively modify the condition being treated, preferably caries, tartar and/or bad breath, but low enough to avoid serious side effects (with a reasonable benefit/risk ratio) within the framework of sound medical practice. judgments. The effective amount of said probiotic microorganism will depend on the particular goal to be achieved, the age and physical condition of the patient being treated, the severity of the underlying disease, the duration of treatment, the nature of the concomitant therapy, the particular microorganism used. An effective amount of said probiotic microorganism will thus be the minimum amount that will provide the desired specific binding to Streptococci mutans. The presence of, for example, 1×10 ⁹ bacteria, both viable and non-viable whole cells, in 0.05 ml of phosphate-buffered saline solution, or in 0.05 ml of agar suspension or dry equivalent weight of cell wall fragments, is effective when administered in amounts from about 0.05 ml to about 20 ml. Undoubted practical success lies in the fact that probiotic organisms can be administered in a convenient form, such as oral route, depending on the route of administration, it may be necessary to cover the active ingredients that contain said probiotic organisms with a material in order to protect said organisms from the action of enzymes, acids and other natural conditions that can inactivate these organisms. In order to introduce probiotic organisms in a way other than parenteral administration, they must be coated, or administered with material to prevent inactivation. For example, probiotic organisms can be administered together with enzyme inhibitors or in liposomes. Pancreatic enzyme inhibitors include the trypsin inhibitor, diisopropylfluorophosphate (DFP) and trasylol. Liposomes include P40 water-in-oil-in-water emulsions, as well as conventional and specially designed liposomes that transport lactic acid bacteria or their by-products to the urogenital surfaces. Dispersions can also be obtained, for example, in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Generally, dispersions are prepared by incorporating various sterilized probiotic organisms into a sterile carrier which contains a basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which result in a powder of the active ingredient plus any additional desired ingredient from a previously filter-sterilized solution thereof. Additional preferred methods of preparation include, but are not limited to, lyophilization and thermal drying.

The composition of the present invention also includes products intended for oral or buccal administration, which contain an acceptable pharmaceutical carrier as described in the present invention, in which or on which, the cells of the aforementioned microorganism belonging to the group of lactic acid bacteria are added in fresh, concentrated or dried form , For example. These products may be provided as an oral suspension, gel, nebulizer, capsule, hard gelatin capsule, syrup, or any other herbal form known to those skilled in the art.

When the probiotic organisms are suitably protected as described above, the active compound may be administered orally, for example, with an inert diluent or with a digestible edible carrier, or it may be enclosed in hard or soft gelatin capsules, or it may be compressed into tablets intended for in order to pass through the stomach (i.e. with enteric), or it can be included directly in the diet. For oral therapeutic administration, probiotic microorganisms can be incorporated into excipients and used in the form of oral tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, cachets, and the like. Compositions and preparations in accordance with the present invention are prepared such that the oral unit dosage form contains, for example, approximately 1×10 ⁹ viable and non-viable cells per ml, for example, lactobacilli per ml. The probiotic organism is formulated for convenient and effective administration in effective amounts with a suitable acceptable carrier for pharmaceutical or nutritional purposes in unit dosage form as described above. The unit dosage form may, for example, contain the main active compound in an amount of approximately 10 ⁹ viable or non-viable, for example, lactobacilli per ml. In the case of compositions containing additional ingredients such as prebiotics, dosages are determined based on the usual dose and route of administration of said ingredients.

Preferred ingredients in the food and feed composition of the present invention are: alfalfa, alfalfa powder concentrate, dehydrated alfalfa meal, alfalfa nutritional concentrate, alpha lipoic acid, animal hydrolysate, animal fat, arginine, artificial flavors, ascorbic acid, asparagus, Bacillus subtilis, bacon, bacon flavor, barley, barley greens, barley malt, basil, beans, beef, meat and bone meal, beef broth, beef offal, beef seasoning, beef liver, beef meat, beef tallow, beet pulp, beet pulp (sugar removed), beetroot, beta-carotene, BHA, Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium thermophilum, biotin, blue 2, blue 2 and other colors, blueberries, bone meal, borage oil, dry brewer's yeast, brewer's yeast , brewer's yeast extract (fermentable residues of Saccharomyces cerevisiae), brewer's rice grits, broccoli, brown rice, brown rice flour, calcium, calcium ascorbate, calcium carbonate, calcium chloride, calcium iodate, calcium pantothenate, calcium phosphate, calcium propionate, calcium sulfate , Cane Sugar Molasses, Canola Flour, Canola Oil, Canthaxanthin, Caramel, Caramel Color, Carmine, Carrageenan, Carrageenan Gum, Carrot Powder, Carrot, Casein, Catfish, Catfish Dishes, Celery, Cellulose, Cellulose Powder, Cheese Powder, Chelated cobalt, chelated copper, chelated iron, chelated potassium, chicken, chicken broth, chicken by-products - organs only, chicken by-products, chicken by-products dishes, chicken by-products - organs only, chicken natural cartilage, chicken fat, natural chicken fat, chicken aromatic additive, chicken giblets, chicken liver, chicken liver hydrolyzate, chicken liver flavor, chicken flour, chicken a la naturel, chicken broth, chicory extract, choline chloride, chondroitin sulfate, cinnamon, citric acid, citric acid and rosemary, citric acid and rosemary extract, citrus pectin, clove oil, cobalt amino acid chelate, cobalt carbonate, cobalt proteinate, codfish, copper, copper amino acid chelate, copper amino acid complex, copper oxide, copper proteinate, copper sulfate, corn, corn bran , corn flour, corn germ meal, corn gluten, corn gluten meal, corn grits, corn flour, corn oil, corn starch, modified corn starch, corn syrup,

crushed barley, crushed pearl barley, cranberries, cranberry powder, chicken debons, lamb debons, turkey debons, dehydrated alfalfa, dehydrated alfalfa flour, dehydrated carrots, dehydrated potatoes, glucose, DHA, dicalcium phosphate, DL-alpha-tocopherol acetate, DL- methionine, dried animal hydrolysate, dried apples, dry extract of Bacillus lichemformis fermentation, dry extract of fermentation of Bacillus subtilis, dried beet pulp, dried beet pulp ((sugar removed), dried blueberries, dried brewer's yeast, dried buttermilk, dried capsicum, dried carrots, cellulose, dried cheddar cheese, dried cheese, dried cheese powder, dried chicken cartilage, dried chicken liver, dried chicken broth, dried chicory root, dried citrus pulp, dried cooked turkey, dried cranberries, dried eggs, dried egg powder, dried egg product, dried egg product, dried garlic, dried ginger, dried grape pomace, dried green beans, dried kelp, dried kelp flour, dried liver hydrolysates, dried meat by-products, dried paprika, dried parsley flakes, dried peas, dried beet pulp plain beets, dried potatoes, dried spinach, dried sweet potatoes, dried tomato pomace, dried carrot vegetable fibers, dried whey, dried yams, duck, hard flour, semolina enriched with thiamine mononitrate, egg noodles, egg fragments, egg products, eggs , Enterococcus faecium, Ethoxyquin, Eucalyptus Oil, Ferrous Sulfate, Fiber, Fish, Fish Broths, Fish Meal, Natural Fish Meal, Fish Oil, Flaxseed Meal, Flaxseed, Flaxseed Meal, Folic Acid, Folic Acid Pyridoxine Hydrochloride, Food Starch , fresh juice, fresh chicken, fresh chicken offal, fructooligosaccharides, fumaric acid, garlic, garlic extract, garlic flavor, garlic oil, garlic powder, gelatin, ginger, ginger extract, glucosamine, glucosamine hydrochloride, glycerin, glycerin, glycerol monostearate, glycine , green beans, green tea, crushed corn, crushed flax seeds, crushed psyllium seeds, crushed rice, crushed wheat, crushed wheat flour, crushed whole grain barley, crushed whole grain corn, crushed whole grain sorghum, crushed whole grain wheat, crushed peas , crushed whole grain wheat, crushed yellow grain corn, guar gum, gum arabic, halibut, herring flour, herring oil, hydrochloric acid, inositol, iodine, iodized salt, iron amino acid chelate, amino acid iron complex, iron oxide, iron proteinate, ferrous sulfate, L-alanine, L-arginine, L-ascorbyl-2-polyphosphate, L-ascorbyl-2-polyphosphate a, L-carnitine, L-lysine, L-lysine monohydrochloride, L-tryptophan, Lactobacillus acidophilus, lamb, lamb broth, mutton offal, mutton hydrolysate, mutton fat, mutton liver, mutton meal, mutton broth, mutton tripe, lecithin, lentils, lettuce, liver, locust bean gum, lutein, lycopene, lysine, mackerel, magnesium oxide, malt extract, malted barley flour, manganese sulfate, manganese amino acid chelate, manganese oxide, manganese proteinate, manganese sulfate, manganese oxide, manganese oxide, calcium iodate, manganese proteinate, manganese sulfate, maple syrup, marigold extract, marigold flour, natural meat and bone meal, meat - liver meal, organ meats, menadione dimethylpyrimidinol bisulfite, menadione sodium bisulfite complex, menadione vitamin K3, menhaden fish meal, menhaden fish oil, milk, tocopherols, mixed carrot vegetable fibers, modified food starch, modified starch, molasses, monocalcium phosphate , Monosodium Phosphate, Natural and Artificial Chicken Flavors, Natural and Artificial Flavors, Natural Chicken Flavors, Natural Color, Natural Flavor, Natural Poultry Flavor, Natural Smoke Flavor, Niacin, Niacin and Ferrous Sulfate, Low Fat Yogurt, Oat Bran, oat fiber, oatmeal, oatmeal, oats, ocean fish, ocean fish meat, ocean fish with white meat, omega fatty acids, onion extract, onion powder, pantothenate, paprika oil resin, parsley, parsley flakes, parsley oil, parsley powder , partially hydrogenated soybean oil, pasta, pea fibers, pea protein, peanut shells 10.8%, pearl barley, peas, peppers, petroleum jelly, phylloquinone vitamin K1, phosphoric acid, pork broth, pork offal, pork liver, potassium sorbate, potassium amino acid complex, potassium chloride, potassium citrate, potassium iodide, potassium iodide, potassium sorbate, potato, potato fibers, potato starch, poultry, poultry offal meal, poultry offal, poultry fat, poultry giblets, poultry liver, powdered cellulose, powdered cellulose 11.1%, propionic acid, propyl gallate and citric acid, propylene glycol, pyridoxine hydrochloride, psyllium, rabbit, rabbit offal, rabbit broth, red 3, red 40 and other colors, red pepper, riboflavin, rice, rice bran, rice flour, rice gluten, rice husk, rice protein concentrate, rosemary, rosemary extract, rosemary extract and citric acid, rye, sage, salmon, salmon broth, salmon flour, salmon oil, salt, sea salt, selenium, shrimp, smoke flavor, sodium alginate, sodium ascorbate, sodium bisulfate, sodium carbonate, sodium caseinate, sodium chloride, sodium hexametaphosphate, sodium metabisulfate, sodium nitrite for color retention, sodium phosphate, sodium propionate, sodium selenite, sodium aluminate, silicon, sodium tripolyphosphate, sodium tripolyphosphate, sorbic acid, sorbitol, soy flour, soy husk, soy lecithin, soy protein concentrate, soy protein isolate, soy oil, soy husk, soy flour, soy meal, soy oil, mint, spinach, spirulina, starch, defatted bone meal, sucrose , sufficient water for processing, sugar, sun-dried alfalfa flour, meal, sunflower oil, sweet potato powder, sweet potato, lard, tapioca starch, taurine, sodium tetrapyrophosphate, textured vegetable protein, thiamine, thiamine hydrochloride, thiamine mononitrate, thyme, titanium dioxide, titanium dioxide pigment, tomato flakes, tomato paste, tomato pulp, tomatoes, trace elements (calcium sulfate), trace elements (copper sulfate), trace elements (potassium chloride), trace elements (sodium tripolyphosphate), trace elements (zinc oxide), trace elements (zinc proteinate), trace elements (zinc sulfate), tricalcium phosphate, tuna, tuna flour, turkey, turkey broth, turkey offal meal, natural turkey, turkey broth, turmeric, veal, veal broth, vegetable oil, venison, offal deer meat, venison liver, venison meal, venison meat, venison broth, vitamin A, vitamin D3 and vitamin A acetate, vitamin B1, vitamin B12, vitamin B12 and D3, vitamin B2, vitamin B6, vitamin C, vitamin D3, vitamin D3 and E, vitamin E, vitamin K, water, watercress, water, enough for processing, watercress and spinach, wheat, wheat bran, wheat flour, wheat germ, wheat gluten, wheat semolina, wheat chaff, wheat starch, whey, white meat fish, white meat fish, white meat fish meal, whole brown rice, whole carrot, whole cranberry, whole egg, whole garlic, whole grain corn, whole grain wheat, crushed whole barley, crushed whole brown rice, crushed oats, crushed whole wheat, whole rice, whole sweet potato, whole wheat, whole wheat flour, wild rice, xanthan gum, yeast culture, yellow yellow 5, yellow 6, yellow squash, yellow zucchini, yucca schidigera, yucca schidigera extract, zinc amino acid chelate, zinc amino acid complex, zinc oxide, zinc proteinate, zinc proteinate and zinc sulfate.

The most preferred components are: alpha-lipoic acid, preferably in an amount sufficient to promote the formation of healthy skin and/or coat; for antibiotic purposes, the preferred ingredients are alpha-lipoic acid, dried garlic, garlic extract, garlic oil and garlic powder, lecithin, preferably as antioxidants; crushed barley, crushed round barley, crushed whole grain corn, crushed whole grain sorghum, herring oil, mackerel, menhaden fish oil, ocean fish, whole brown rice, and whole sweet potatoes are preferred sources of energy; preferred fiber sources are dehydrated carrots, dried apples, crushed whole barley grains, oat bran, oatmeal, oatmeal, crushed whole barley, crushed whole oats; cobalt proteinate (source of chelated cobalt), copper proteinate, iron proteinate, manganese proteinate, manganese proteinate, trace elements and zinc proteinate are preferred mineral sources; preferred sources of omega-3 fatty acids are borage oil, canola oil, canola oil (with mixed tocopherol preservatives), and flaxseed; rosemary extract and citric acid are preferred preservatives; preferred sources of protein are beef meal, chicken meal, dried peas, halibut, lamb meal, menhaden fish meal, ocean fish, peas, venison meal, whole brown rice, and crushed whole wheat; preferred sources of vitamins are calcium ascorbate, carrots, dried carrots, mixed tocopherols, whole carrots: preferred to replace plain water, and preferred flavorings are beef broth, chicken broth, lamb broth, mutton broth and turkey broth.

The ingredients are not the most, but also highly preferred: alfalfa concentrate powder, dehydrated alfalfa flour, chicken fat, chicken liver, fresh juice, ocean fish meat and pearl barley as sources of energy; green beans, crushed whole peas, oats and pea fibers as fiber sources; cobalt amino acid chelate, copper amino acid chelate, copper amino acid complex, dried algae flour, iron amino acid chelate, manganese amino acid chelate, manganese sulfate, potassium amino acid complex, zinc amino acid chelate as a mineral source; chicory extract, ginger extract and yucca schidigera extract as prebiotics; food preservatives are garlic, rosemary and sage, alfalfa food concentrate, catfish, catfish meal, cod, duck, eggs, crushed whole wheat, herring, pea protein, rabbits, shrimp, tuna, venison, venison meat, fish with white meat, white meat fish, white meat fish meal, whole wheat are preferred sources of protein, beta-carotene and folic acid are preferred sources of vitamins, chicken broth, rabbit broth, turkey broth, veal broth and venison broth are preferred substitutes for regular water; and preferred flavors.

The standard food of the food composition according to the present invention, preferably a pet food, preferably contains one or more ingredients from the group consisting of vitamin B-12, biotin, calcium carbonate, calcium pantothenate, choline chloride, cobalt carbonate, copper sulfate, with added vitamin D3 and E, DL-methionine, dried algae, iron sulfate, folic acid, inositol, manganese oxide, manganese oxide, manganese sulfate, menadione complex and sodium bisulfite, mineral supplements, including zinc sulfate, natural flavor, nicotine acid and ferrous sulfate, niacin as a supplement, potassium chloride, potassium iodide, pyridoxine hydrochloride, riboflavin, sodium selenite, taurine, thiamine, thiamine mononitrate, thiamine mononitrate, vitamin A, vitamin A and D3, vitamin A acetate, vitamin B12, vitamin D3, water, zinc oxide and zinc sulfate.

The food used in accordance with the present invention may be any food containing the binding microorganism or its fragment as an active ingredient. The feed includes, for example, pet food for dogs, cats and rats and cattle food for cows and pigs.

Feed can be obtained by appropriate mixing of the active ingredient, as described in the present invention above, with feed raw materials, including, for example, cereals, bran, oilseed meal, feed raw materials of animal origin, other feed raw materials and refined products, as indicated above. . The preferred foods in accordance with the present invention are chewable foods. Preferred products are pig ears, bovine tendons, bovine tails, gullets, dried muscle meat, pig feet and dried pressed cowhide, buffalo skin or composite chew products containing plant materials such as fibers, bran, etc. Other chewable products are known in the art and are also preferred, such as the chewable products of US Pat. The chewy product is preferably used in the form of a bone, roll, doughnut, staple, pretzel, figure eight, or chip.

In the case of providing animals with feed according to the present invention, the amount of feed to be consumed is not particularly limited, but is preferably, for example, 0.1 mg to 50 g per 1 kg of body weight per day, preferably 0.5 mg to 20 g per day. 1 kg of body weight per day, depending on the amount of active ingredient. Feed is constantly taken in this amount for a period of from one day to 5 years, preferably from 2 weeks to one year. Again, the intake amount can be adjusted to an appropriate range depending on the species, age and body weight of the animal receiving the food, and the like.

Also preferably, the composition according to the present invention would include one or more probiotic microorganisms or products derived from them, regardless of the binding microorganism and its fragment. Preferred probiotic microorganisms and their respective products are Bacillus subtilis, Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium thermophilum, dried enzyme extract of Bacillus licheniformis, dried enzyme extract of Bacillus subtilis, Enterococcus faecium and Lactobacillus acidophilus.

Particularly preferred are food and feed compositions further containing, in addition to the binding microorganism or fragment thereof, additional microorganisms for the prevention and/or treatment of bad breath. Most preferred are food and feed compositions further containing such microorganisms as disclosed in WO 2009/149816 A1, which is included in the present invention in its entirety, of which the additional microorganism is preferably selected from the group consisting of Lactobacillus acidophilus having a DSMZ accession number DSMZ 19825 DSMZ, DSMZ 19826, DSMZ 19827.

In the following, the invention will be further described by way of selected examples and embodiments. These embodiments and examples are intended to represent specific preferred features of the present invention without limiting the scope of the present description or the scope of the claims. It should be understood that a person skilled in the art can develop additional working examples and embodiments using his general knowledge and the instructions and explanations given in the present description and documents incorporated in the present invention by reference.

Example 1: Storage and cultivation of binding microorganisms

Storage and cultivation of strains can be carried out in accordance with conventional procedures. In accordance with this present example, the strains were stored as cold storage at -80°C. 1 ml culture was grown to stationary phase (OD600: 4-8) in MRS medium and mixed with 500 µl sterile 50% glycerol solution and frozen. Cultures of mutans Streptococci were grown in TSY medium to stationary phase (OD600 1-2) and treated as above for frozen storage.

The cultivation of mutans Streptococci (S. mutans, S. sobrinus, S. ratti, S. cricetus, S. ferus or S. macacae), as well as the cultivation of lactobacilli can be carried out in 5 ml in closed Falcon tubes at 37°C without shaking for night.

In particular, the strains used in the present application were stored in the form of low temperature storage at -80°C. 1 ml of culture grown to stationary phase (OD600/ml 4-8) in MRS broth was mixed with 500 µl of sterile 50% glycerol solution and frozen.

In particular, cultures of mutans Streptococci were grown in TSY broth in stationary phase (OD600/ml 1-2) and treated as described above.

Growing Mutans Streptococci (S. Mutans (DSMZ 20523, Serotype C; NCTC 10923, Serotype E; NCTC 11060, Serotype F), S. Sobrinus DSMZ 20742, S. RATTI DSMZ 20564, S. Cricetus DSMZ 20562, SA . Ferus dsmz 20646 or S. macacae DSMZ 20714) and cultivation of lactobacilli was carried out in 5 ml in closed Falcon tubes at 37°C without shaking overnight. For fluorescent analysis, as described in example 5, S. mutans DSMZ 20523 was used.

For analysis of aggregation, lactobacilli were grown in MRS medium. In 5 ml of MRS medium, 10 μl of stock solution was inoculated and incubated for 3 days at 37° C. under aerobic conditions. The optical density of the culture was measured at 600 nm (OD600). The culture was then diluted to an OD600 of 2 with PBS buffer. mutans Streptococci were grown in 7 ml TSY medium. 7 ml of TSY medium was inoculated with 10 μl of stock solution and incubated at 37°C under anaerobic conditions.

MRS broth:

MRS-blend (Difco, USA) 55 g/l, pH: 6.5

TSY broth:

TSY-blend (Difco, USA) 30 g/l

Yeast extract (Deutsche Hefewerke, Germany) 3 g/l

Buffer:

PBS buffer:

Na ₂ HPO ₄ 2H ₂ O 1.5 g/l

KH ₂ PO ₄ 0.2 g/l

NaCl 8.8 g/l

The pH was adjusted with HCl

Example 2: Taxonomic Classification of Binding Strain and Streptococcus Strains

The taxonomic classification of the strains was carried out according to their carbohydrate fermentation pattern. It was determined using the API 50 CH system (bioMerieux, France) and analyzed using the APILAB PLUS software version 3.3.3 (bioMerieux, France).

Example 3: Cell Staining

After lactobacilli and Streptococci mutans were grown as described in Example 1, Streptococci mutans were stained with a fluorescent strain. For this, the OD600 of the culture was measured. The culture was harvested by centrifugation at 3200×g for 5 min. The pellet was resuspended in PBS buffer. The amount of buffer was calculated so that the resulting suspension when mixed had an OD600 of 4.2 ml of this suspension with 2 µl of CFDA-SE solution (Invitrogen, USA), which was prepared according to the manufacturer's instructions. Cell staining was performed by incubating the mixture for 2 hours at 37°C. Stained cells were harvested by centrifugation at 3200×g for 5 min. The cells were then resuspended in 2 ml of PBS buffer.

Example 4 Aggregation Analysis of Precipitation of mutans Streptococci

For analysis, the mixing of lactobacilli and mutans Streptococci was carried out in a volume ratio of 3:1 to 60:1 (mutans Streptococci : lactobacilli), which corresponds to a CFU ratio of 1:50 to 1:2.5.

The optical density, measured at a wavelength of 600 nm in 1 ml, was preferably 3×10 ⁸ colony forming units for mutans Streptococci and 7×10 ⁹ colony forming units for lactobacilli. Mixing was carried out in a volume of 2 ml in 15 ml Falcon tubes. The culture suspensions were diluted with PBS buffer to obtain the volume ratio mentioned above while maintaining a final volume of 2 ml. The mixture was shaken for 15 seconds. Aggregation was observed as an immediate turbidity of the suspension. The tubes were left undisturbed for 20 minutes, after which time the aggregates precipitated as a noticeable precipitate, while the non-aggregating mixtures remained in suspension. The formed aggregates were separated by centrifugation at 500×g for 30 seconds. Immediately thereafter, the number of aggregates was assessed by measuring the number of non-aggregated cells that remained in the supernatant. To do this, 1 ml of the supernatant was carefully removed to measure the optical density. Optical density was measured at 600 nm. The value after subtracting the corresponding control experiment without lactobacilli is the number of cells that did not aggregate.

As a control, the autoaggregation of the respective Lactobacillus strain and Streptococcus mutans strains was always investigated by performing a test when only Lactobacillus or Streptococcus mutans strains were added to the tube. Aggregation of S. mutans with Lactobacillus is shown in FIG. 1 (left tube) and 2.

Lactobacillus strains as described above in the present invention, in particular those deposited in the DSMZ, showed aggregation of all S. mutans serotypes without showing autoaggregation behavior.

Example 5: Fluorescence analysis of aggregation of mutans Streptococci

For the analysis of a suspension of the respective lactobacilli and the respective stained mutans Streptococcus (S. mutans DSMZ 20523 with Lb-OB-K1 (DSMZ 16667), S. mutans DSMZ 20523 with Lb-OB-K2 (DSMZ 16668), S. mutans DSMZ 20523 with Lb -OB-K3 (DSMZ 16669), S. mutans DSMZ 20523 with Lb-OB-K4 (DSMZ 16670), S. mutans DSMZ 20523 with Lb-OB-K5 (DSMZ 16671), S. mutans DSMZ 20523 with Lb-OB -K6 (DSMZ 16672), S. mutans DSMZ 20523 with Lb-OB-K7 (DSMZ 16673), S. sobrinus DSMZ 20742 with Lb-OB-KI (DSMZ 16667), S. sobrinus DSMZ with 20742 Lb-OB- K2 (DSMZ 16668), S. sobrinus DSMZ 20742 with Lb-OB-K3 (DSMZ 16669), S. sobrinus DSMZ 20742 with Lb-OB-K4 (DSMZ 16670), S. sobrinus DSMZ 20742 with Lb-OB-K5 ( DSMZ 16671), S. sobrinus DSMZ 20742 with Lb-OB-K6 (DSMZ 16672), S. sobrinus DSMZ 20742 with Lb-OB-K7 (DSMZ 16673), S. cricetus DSMZ with 20562 Lb-OB-KI (DSMZ 16667 ), S. cricetus DSMZ 20562 with Lb-OB-K2 (DSMZ 16668), S. cricetus DSMZ 20562 with Lb-OB-K3 (DSMZ 16669), S. cricetus DSMZ 20562 with Lb-OB-K4 (DSMZ 16670), S. cricetus DSMZ 20562 with Lb-OB-K5 (DSMZ 16671), S. cricetus DSMZ 20562 with Lb-OB-K6 (DSMZ 16672), S. cricetus DSMZ 20562 with Lb-OB-K7 (DSMZ 16673);

S. ratti DSMZ 20564 with Lb-OB-K1 (DSMZ 16667), S. ratti DSMZ 20564 with Lb-OB-K2 (DSMZ 16668), S. ratti DSMZ 20564 with Lb-OB-K3 (DSMZ 16669), S. ratti DSMZ 20564 with Lb-OB-K4 (DSMZ 16670), S. ratti DSMZ 20564 with Lb-OB-K5 (DSMZ 16671), S. ratti DSMZ 20564 with Lb-OB-K6 (DSMZ 16672), S. ratti DSMZ from 20,564 Lb-OB-K7 (DSMZ 16673);

S. ferus DSMZ 20646 with Lb-OB-K1 (DSMZ 16667), S. ferus DSMZ 20646 with Lb-OB-K2 (DSMZ 16668), S. ferus DSMZ 20646 with Lb-OB-K3 (DSMZ 16669), S. ferus DSMZ 20646 with Lb-OB-K4 (DSMZ 16670), S. ferus DSMZ 20646 with Lb-OB-K5 (DSMZ 16671), S. ferus DSMZ 20646 with Lb-OB-K6 (DSMZ 16672), S. ferus DSMZ from 20646 Lb-OB-K7 (DSMZ 16673);

S. macacae DSMZ 20724 with Lb-OB-K1 (DSMZ 16667), S. macacae DSMZ 20724 with Lb-OB-K2 (DSMZ 16668), S. macacae DSMZ 20724 with Lb-OB-K3 (DSMZ 16669), S. macacae DSMZ 20724 with Lb-OB-K4 (DSMZ 16670), S. macacae DSMZ 20724 with Lb-OB-K5 (DSMZ 16671), S. macacae DSMZ 20724 with Lb-OB-K6 (DSMZ 16672) and S. macacae DSMZ with 20,724 Lb-OB-K7 (DSMZ 16673)) was mixed. 50 µl of the lactobacillus suspension was added to 50 µl of mutans-stained Streptococci in a 96-well microtiter plate. The tablet was shaken at full speed for 12 minutes. The plate was then centrifuged at 500×g for 10 seconds. The supernatant was carefully removed and discarded. The pellet was resuspended in 100 µl of PBS buffer. Fluorescence of the suspension was measured using a microtiter plate fluorescence reader at 495 nm excitation and 525 nm emission. As a control, lactobacilli alone, as well as Streptococci stained mutans, were processed and measured as described. The background fluorescence measured for the respective Streptococci mutans only was subtracted from the value measured for aggregation with the respective Lactobacillus. All measurements were performed in triplicate, mutans Streptococci were aggregated by all tested lactobacilli (see Fig. 3).

Example 6 Specificity of Aggregation to Representative Oral Flora

Cultures of Lactobacillus were grown as described in example 1. Oral bacteria - namely: Streptococcus salivarius ssp. thermophilus (isolated with OrganoBalance, identified with API 50 CH (Biomerieux, France) according to manufacturer's instructions); Streptococcus oralis (DSMZ 20066); Streptococcus oralis (DSMZ 20395); Streptococcus oralis (DSMZ 20627); Staphylococcus epidermidis (DSMZ 1798); Staphylococcus epidermidis (DSMZ 20044); Streptococcus mitis (DSMZ 12643), Streptococcus sanguinis (DSMZ 20567) - grown in 5 ml BHI medium in closed 15 ml Falcon tubes at 37° C. overnight. Each of the above oral bacteria was preferably mixed in a 3:1 volume ratio with a Lactobacillus culture and aggregation was assessed as in Example 4. For each aggregate/non-aggregate test, only one of the above bacteria was preferably used to immediately determine the outcome of the test.

As a control, autoaggregation of the respective strain of Lactobacillus and strains of mutans Streptococcus was always investigated by performing a test when only Lactobacillus or oral flora strains were added to the tube.

L. paracasei sp. paracasei strains Lb-OB-K1 (DSMZ 16667), Lb-OB-K2 (DSMZ 16668), Lb-OB-K3 (DSMZ 16669), Lb-OB-K4 (DSMZ 16670), Lb-OB-K5 (DSMZ 16671 ) did not aggregate the oral bacteria mentioned above. Thus, they are "specifically binding to Streptococci mutans" as defined above. L. rhamnosus strains Lb-OB-K6 (DSMZ 16672) and Lb-OB-K7 (DSMZ 16673) aggregated Streptococcus salivarius thermophilus ssp. They are nevertheless considered "specifically binding to Streptococci mutans" according to the above less preferred definition

BHI broth:

BHI blend (Difco, USA) 37 g/l pH: 7.2

Example 7 Temperature Stability of the Aggregation Capacity of Lactic Acid Bacteria

The bacteria were grown as in Example 1. The grown cultures of lactic acid bacteria were autoclaved at 121° C. at a pressure of 2 bar in saturated steam for 20 minutes. After cooling the autoclaved cultures to room temperature, lactobacilli were mixed in a 1:3 volume ratio with grown cultures of S. mutans, and aggregation was evaluated as in example 4, including in control experiments. Aggregation was also analyzed using oral bacteria as described in Example 6.

It was found that the aggregation behavior of lactobacilli was not altered by the autoclaving procedure in relation to the tested S. mutans serotypes or oral bacteria.

Example 8 Aggregation with Heat Inactivated Lactobacilli

Lactic acid bacteria were grown as described in Example 1. mutans Streptococci were grown and stained as described in Examples 1 and 3. The grown cultures of lactic acid bacteria were adjusted to an OD600 of 2 as described in Example 1. 1 ml of this suspension was autoclaved at 121°C at pressure of 2 bar for 20 min as described above. After cooling the autoclaved cultures to room temperature, aggregation was measured as described in Example 5, including control experiments. The heat-inactivated lactobacilli still aggregated all mutans of Streptococci.

Example 9 Aggregation as a function of pH

Bacteria were grown as in Example 1. 0.5 ml of lactobacilli and 1.5 ml of S. mutans were collected by centrifugation at 3200×g for 10 min and the supernatant was discarded. Cells were resuspended to their original volume (0.5 ml and 1.5 ml, respectively) in different PBS buffers and adjusted to different pH values. The buffer pH values were adjusted to between 7.0 and 3.0 in increments of 0.5 pH units. The cultures were resuspended in an appropriate pH buffer to be used to analyze the aggregation behavior.

After that, lactobacilli were mixed in a 1:3 volume ratio with a culture of S. mutans and aggregation was evaluated as in example 4, including control experiments. No noticeable aggregation of S. mutans by lactobacilli occurred at pH values below 4.5.

Example 10 Aggregation as a function of pH

Lactic acid bacteria were grown as described in Example 1. mutans Streptococci were grown and stained as described in Examples 1 and 3. Immediately thereafter, aggregation was analyzed at various pH values. For this purpose, lactic acid bacteria as well as streptococci were resuspended in acetate buffer adjusted to the appropriate pH. Studies were carried out at pH 4.0, 4.5 and 5.0. Aggregation was analyzed as described in Example 5. No aggregation of Streptococci mutans occurred at pH values below 4.5.

Example 11 Sensitivity of Aggregation Behavior to Lyophilization

Bacteria were grown as in example 1. Aliquots of 1 ml culture of lactic acid bacteria were collected by centrifugation at 3200×g for 10 minutes. The supernatant was discarded and the pellet was lyophilized at room temperature under vacuum for two hours. The resulting dry pellet of each tested Lactobacillus strain was stored at room temperature and at 4°C for 1 day, 1 week, 2 weeks, 3 weeks and 4 weeks, respectively. After a storage period, the lyophilized pellet was resuspended in 1 ml PBS buffer, pH 7.0. Resuspended lactobacilli were mixed in a 1:3 volume ratio with fresh cultures of S. mutans and aggregation was assessed as in example 4, including controls.

The aggregation behavior of these lactic acid bacteria towards S. mutans was not altered by lyophilization or storage procedures.

Example 12: Sensitivity of Aggregation Behavior to Lyophilization

Lactic acid bacteria were grown as described in Example 1. mutans Streptococci were grown and stained as described in Examples 1 and 3. The grown cultures of lactic acid bacteria were adjusted to an OD 600 of 2 as described in Example 1. 1 ml of this suspension was lyophilized at room temperature in vacuum for two hours. Immediately thereafter, the lyophilized pellet was resuspended in 1 ml of PBS buffer. Aggregations were measured as described in the example, including control experiments.

The aggregation behavior of these lactic acid bacteria in relation to mutans Streptococci did not change after lyophilization.

Example 13 Protease Resistance Test

Bacteria were grown as in example 1. Proteases used: pronase E, proteinase K, trypsin, chymotrypsin (all obtained from Sigma, Germany). 1 ml aliquots of lactobacilli were washed in PBS buffer by harvesting cells by centrifugation at 3200×g for 10 minutes and resuspending the pellet in 1 ml PBS buffer (pH 7.0). The cells were then collected again as described above and resuspended in PBS buffer (pH 7.0) containing the appropriate proteases at a final concentration of 2.5 mg/ml. The suspension was incubated for 1 hour at 37°C. Immediately afterwards, the cells were washed and resuspended in PBS buffer (pH 7.0) as described above.

Aggregation was analyzed as described in example 3, including control experiments. Aggregation behavior of these lactic acid bacteria in relation to S. mutans did not change due to treatment with any of these proteases.

Example 14 Protease Sensitivity of Lactobacillus Aggregation Behavior

Lactic acid bacteria were grown as described in example 1. mutans Streptococci were grown and stained as described in examples 1 and 3. Proteases used: pronase E, proteinase K, trypsin, chymotrypsin (all obtained from Sigma, Germany). 1 ml aliquots of lactobacilli were washed in PBS buffer by harvesting the cells by centrifugation at 3200×g for 10 min and resuspending the pellet in 1 ml PBS buffer (pH 7.0). Immediately thereafter, the cells were harvested again as described above and resuspended in PBS buffer (pH 7.0) containing the appropriate proteases at a final concentration of 2.5 mg l/ml. The suspension was incubated for 1 hour at 37°C. The cells were then washed and resuspended in PBS buffer (pH 7.0) as described above. Aggregation was evaluated as described in example 5, including control experiments. Aggregation behavior of lactobacilli in relation to mutans Streptococci did not change due to treatment with any of these proteases.

Example 15: Ionic dependence of aggregation behavior

Bacteria were grown as in Example 1. 1 ml aliquots of lactobacilli were washed in 1 ml of 200 mM EDTA solution twice as described above. Immediately thereafter, the cells were harvested and resuspended in 1 ml of PBS buffer (pH 7.0).

Aggregation was analyzed as described in Example 4 and a complete loss of aggregation ability was observed. Resuspension of lactic acid bacteria in 1 ml of 2 mM calcium solution after washing twice with 200 mM EDTA solution restored the aggregation ability of S. mutans. Resuspension of EDTA-washed cells in magnesium solution up to a concentration of 100 mM did not restore the aggregation ability of S. mutans.

Example 16: Ionic dependence of aggregation behavior

Lactic acid bacteria were grown as described in Example 1. mutans Streptococci were grown and stained as described in Examples 1 and 3. 1 ml aliquots of lactobacilli were washed twice in 1 ml of 200 mM EDTA solution as described above. Immediately thereafter, the cells were harvested and resuspended in 1 ml of PBS buffer (pH 7.0). Aggregation was evaluated as described in Example 5 and a complete loss of aggregation ability was observed. Resuspension of lactic acid bacteria in 1 ml of 2 mM calcium solution after washing twice with 200 mM EDTA solution restored the aggregation ability of S. mutans. Resuspension of EDTA-washed cells in magnesium solution up to a concentration of 100 mM did not restore the ability to aggregate Streptococci mutans.

Example 17 Aggregation Study in the Presence of Saliva

Bacteria were grown as in Example 1. A 2 ml aliquot of S. mutans culture was harvested as described above and resuspended in 2 ml of saliva. Saliva was provided by two volunteers and used immediately after collection. Aggregation was analyzed as described in Example 4.

The aggregation behavior of these lactic acid bacteria in relation to S. mutans did not change in the presence of saliva.

Example 18: Aggregation of mutans Streptococci in the presence of saliva

Fresh saliva was collected from volunteers. Salivation was induced by chewing sugar-free chewing gum. Volunteers collected 15 ml of saliva for each sample. Freshly collected saliva was diluted 1:2 in PBS buffer for the research procedure. Lactobacilli and mutans Streptococci were cultured as described in Example 1. mutans Streptococci were stained as described in Example 3, except that after the staining procedure, the stained cells were resuspended in saliva instead of PBS buffer. Aggregation was evaluated as described in example 5, including control experiments. The presence of saliva did not inhibit aggregation.

Isolation Example 1: Obtaining Additional Lactobacilli

Lactobacilli can be obtained from any source, for example, through a swab from the oral cavity of volunteers with a low amount of caries and/or tartar. In addition, lactobacilli can be obtained from a collection of cell cultures such as DSMZ and ATCC, or from other known sources of lactic acid bacteria, such as plants, food and feed. The resulting strains were then purified by standard microbiological techniques. Selective media for lactobacilli are described, for example, by Rogosa et al. 1951. Selective medium for the isolation of lactobacilli from the oral cavity and faeces, J. Bacteriol. 62:132-133.

Isolated strains were grown as described in Example 1. Selection for binding was performed as described in Isolation Example 2.

Selection example 2:

Stained mutans Streptococcus (DSMZ 20523) were prepared and dispensed into 96-well microtiter plates as described in Example 5 above. In addition, as described in Example 5, 50 µl of strains selected for binding growth screening according to Example 1 were added to each well of the microtiter plate and shaken at full speed for 12 minutes. Immediately thereafter, the plate was centrifuged at 500×g for 10 seconds and the supernatant was carefully removed. The pellet was resuspended in 100 µl PBS and the fluorescence of the suspension was measured as described in Example 5.

The screening strains were then tested as described in Example 5 above for binding to S. mutans DSMZ 20523, S. sobrinus DSMZ 20742, S. cricetus DSMZ 20562, C. ratti DSMZ 20564, S. ferus DSMZ 20646 and S .macacae DSMZ 20724. Positive binders were found to produce aggregates.

Food and Feed According to Example 1: Dog Puppy Food

Main Ingredients: Beef, Brown Rice, Canola Seeds, Flaxseed Meal, Sunflower Seeds, Buckwheat (Soba) Seeds, Barley, Millet.

Minor ingredients: carrots, beets, broccoli, high oleic sunflower oil, rapeseed oil, sea salt, oregano, garlic, ascorbic acid, propolis, vitamins (vitamins A, B12, D3, C, E, thiamine, riboflavin, pyridoxine , biotin, folic acid, pantothenate, niacin), minerals (chromium, sodium selenite, iron, copper, zinc citrate, cobalt sulfate).

To 1 kg of the base composition having the above major and minor ingredients, was added 3 g of dried autoclaved binding microorganisms and their fragments. The microorganism material was obtained by growing binding microorganisms as described in Example 1. That is, lactobacilli were grown on MRS medium. 5 ml of MRS medium was inoculated with 10 μl of stock solution and incubated for 3 days at 37° C. under aerobic conditions. Microorganisms were pelleted by centrifugation and washed once with PBS. The resuspended microorganisms were autoclaved as described in Example 8. The autoclaved microorganisms were dried in an oven at 75° C. overnight to give dried heat-inactivated binding microorganisms and their fragments. Puppy dog food was prepared with each of the deposited Lactobacillus DSMZ strains 16667, 16668, 16669, 16670, 16671, 16672 and 16673 separately to produce 7 separate puppy food samples.

The total content of the following parts was as follows (by weight % of the total load).

Proteins (minimum) 26.0

Fat (minimum) 14.0

Fiber (max.) 5.0

Humidity (max.) 10.5

Food for adult dogs

Main ingredient: beef, brown rice, rapeseed, linseed meal, sunflower seeds, buckwheat (soba) seeds, barley, millet.

Minor ingredients: carrots, beets, broccoli, high oleic sunflower oil, rapeseed oil, sea salt, oregano, garlic, ascorbic acid, propolis, vitamins (vitamins A, B12, D3, C, E, thiamine, riboflavin, pyridoxine , biotin, folic acid, pantothenate, niacin), minerals (chromium, sodium selenite, iron, copper, zinc citrate, cobalt sulfate).

As described for puppy dog food, 7 samples of adult dog food formulations were prepared using deposited Lactobacillus DSMZ strains 16667, 16668, 16669, 16670, 16671, 16672 and 16673 separately.

The total content of the following parts was as follows (by mass% of the total load).

Proteins (minimum) 20.0

Fat (minimum) 12.0

Fiber (max.) 5.0

Humidity (max.) 10.5

Vegetarian dog food.

Main ingredients: soybean meal, brown rice, rapeseed, linseed meal, sunflower seeds, buckwheat seeds, barley, millet.

Minor ingredients: carrots, broccoli, high oleic sunflower oil, rapeseed oil, sea salt, oregano, garlic, ascorbic acid, propolis, vitamins (vitamins A, B12, D3, C, E, thiamine, riboflavin, pyridoxine, biotin , folic acid, pantothenate, niacin), minerals (chromium, sodium selenite, iron, copper, zinc citrate, cobalt sulfate).

As described for puppy dog food, 7 samples of vegetarian food formulations were prepared using deposited Lactobacillus DSMZ strains 16667, 16668, 16669, 16670, 16671, 16672 and 16673 separately.

The total content of the following parts was as follows (by mass% of the total load).

Proteins (minimum) 20.0

Fat (minimum) 12.0

Fiber (max.) 9.0

Humidity (max.) 10.5.

All foods were fed to a separate group of 5 dogs of the appropriate age (1-8 months, 2-4 years and 2-4 years, respectively) once a day. As required for dogs, other standard foods were provided at other times of the day. However, with the exception of the foods listed above, no other foods were provided in accordance with the present invention that were advertised by the respective manufacturer as specific for oral hygiene. After 6 months, none of the dogs developed tartar. In addition, dog halitosis measured by 10 volunteers 3 hours after the last feeding was accepted as acceptable.

Food and Feed According to Example 2: Dog Chew

A rawhide cowhide strip was dipped into a culture of binding microorganism strains DSMZ 16667, 16668, 16669, 16670, 16671, 16672 and 16673, respectively. Binding microorganisms were grown as described in the section "food and feed in accordance with example 1". The rawhide strips were then molded into a bone-like shape and autoclaved as described in the Food and Feed section of Example 1.

The autoclaved strips were air dried. The dried bone-like strips were given to 5 dogs 5-8 years old who had just had their calculus removed. New dried bone-like strips were then given twice a week. Except for the dried bone-like strips, the dogs were fed as before scaling. Within 8 months, none of the dogs had tartar again. In addition, dog halitosis measured by 10 volunteers 3 hours after the last feeding was accepted as acceptable.

Food and feed according to example 3: Complementary food for dogs

Puppy food:

Key Ingredients: Chicken, chicken meal, barley, peas, brown rice, crushed extruded whole soy, chicken fat (preserved with mixed tocopherols).

Minor Ingredients: Salmon Meat Meal, Natural Chicken Liver Flavor, Dried Brewer's Yeast, Flaxseed Flour, Egg Powder, Apples, Carrots, Potassium Chloride, Dicalcium Phosphate, Minerals (e.g. Zinc Proteinate, Iron Proteinate, Copper Proteinate, Manganese Proteinate, sodium selenite, cobalt proteinate, calcium iodate), salt, choline chloride, vitamins (e.g. vitamin E, L-ascorbyl-2-polyphosphate, vitamin B12, calcium d-pantothenate, vitamin A, niacin, riboflavin, folic acid, biotin, pyridoxine hydrochloride, thiamine mononitrate, vitamin D3 supplement, calcium carbonate), yeast cultures (Saccharomyces cerevisiae), dried Enterococcus faecium fermentation product, dried Lactobacillus acidophilus fermentation product, dried Aspergillus niger fermentation extract, Trichoderma longibrachiatum fermentation extract, dried fermentation extract and fermentable remains of Bacillus subtilis.

The total content of the following parts was as follows (by mass% of the total load).

Proteins wt. % (minimum): 28.0

Fats wt. % (minimum): 15.0

Fiber wt. % (max): 3.5.

Humidity wt. % (max): 10.0.

Adult dog food:

Key Ingredients: Chicken, chicken meal, barley, peas, oats, brown rice, chicken fat (preserved with mixed tocopherols).

Minor Ingredients: Natural Chicken Liver Flavor, Dried Brewer's Yeast, Salmon Meal Meal, Egg Powder, Apples, Whole Flax Seeds, Carrots, Dicalcium Phosphate, Potassium Chloride, Salt, Minerals (i.e. Zinc Proteinate, Iron Proteinate, Proteinate copper, manganese proteinate, sodium selenite, cobalt proteinate, calcium iodate), choline chloride, vitamins (eg, vitamin E, L-ascorbyl-2-polyphosphate, vitamin B12, calcium d-pantothenate, vitamin A, niacin, riboflavin, folic acid , biotin, pyridoxine hydrochloride, thiamine mononitrate, vitamin D3 supplement), glucosamine hydrochloride, chondroitin sulfate, yeast cultures (Saccharomyces cerevisiae), dried Enterococcus faecium fermentation, dried Lactobacillus acidophilus fermentation, dried Aspergillus niger fermentation extract, Trichoderma longibra fermentation extract chiatum , dried extract of the fermentation product and fermentable residues of Bacillus subtilis.

The total content of the following parts was as follows (by mass% of the total load).

Proteins wt. % (minimum): 26.0

Fat wt. % (minimum): 13.0

Fiber wt. % (max): 3.5.

Humidity wt. % (max): 10.0.

Weight control of dog feeding:

Main Ingredients: Brown rice, chicken meal, barley, oats, peas, chicken, chicken fat (preserved as mixed tocopherols).

Minor Ingredients: Natural Chicken Liver Flavor, Dried Brewer's Yeast, Salmon Meal Meal, Egg Powder, Apples, Whole Flax Seeds, Carrots, Dicalcium Phosphate, Potassium Chloride, Salt, Minerals (i.e. Zinc Proteinate, Iron Proteinate, Proteinate copper, manganese proteinate, sodium selenite, cobalt proteinate, calcium iodate), choline chloride, vitamins (e.g. vitamin E, L-ascorbyl-2-polyphosphate, vitamin B12, calcium d-pantothenate, vitamin A, niacin, riboflavin, folic acid , biotin, pyridoxine hydrochloride, thiamine mononitrate, vitamin D3 supplement), glucosamine hydrochloride, chondroitin sulfate, yeast cultures (Saccharomyces cerevisiae), dried Enterococcus faecium fermentation, dried Lactobacillus acidophilus fermentation, dried Aspergillus Niger fermentation extract, dried Trichoderma longi fermentation extract brachiatum , dried extract of the fermentation product and fermentable residues of Bacillus subtilis.

The total content of the following parts (by mass% of the total load).

Proteins wt. % (minimum): 24.0

Fat wt. % (minimum): 10.0

Fiber wt. % (max): 3.5.

Humidity wt. % (max): 10.0.

As described for the puppy food in the "Food and Feed according to Example 1" section, 7 corresponding samples of puppy food, adult dog food and weight control formulations were prepared using deposited strains of Lactobacillus DSMZ 16667, 16668, 16669, 16670, 16671 , 16672 and 16673 separately.

High Protein Adult Dog Food:

Main Ingredients: Deboned Chicken Meat, Chicken Meal, Turkey Meal Meal, Rose Potato, Herring Whitefish, Chicken Fat (preserved with mixed tocopherols).

Minor Ingredients: Sweet Potato, Whole Egg, Turkey, Salmon Meal Meal, Salmon Anchovy Oil, Salmon, Natural Chicken Flavor, Sunflower Oil, Sun-Dried Alfalfa, Dried Brown Seaweed, Carrots, Spinach, Peas, Tomatoes, Apples, Psyllium , dark red edible seaweed, chicory root, licorice root, turmeric root, fenugreek, glucosamine HCl, cranberry, black currant, marigold flowers, L-carnitine, fennel, common corn, peppermint leaves, chamomile flowers, dandelion, garden savory, extract rosemary, chondraitin sulfate, rosehip, vitamins (eg vitamin E, choline chloride, vitamin A, vitamin D3, thiamine mononitrate, vitamin B12, folic acid, biotin), sea salt, minerals (eg iron proteinate, zinc proteinate, manganese proteinate, copper proteinate), dried fermentation product of Lactobacillus acidophilus, dried fermentation product of Enterococcus faecium.

The total content of the following parts (by mass% of the total load).

Proteins minimum: 42 wt. %

Fat minimum: 16 wt. %

Fiber max. 3 wt. %

Humidity no more: 10 wt. %

Mineral matter max: 7 wt. %

As described for the puppy food in the "Food and Feed according to Example 1" section, 7 respective dog food formulations were prepared using the deposited Lactobacillus DSMZ strains 16667, 16668, 16669, 16670, 16671, 16672 and 16673 separately.

Vegetarian dog food:

Main Ingredients: Brown Rice, Canola Seeds, Flaxseed Meal, Sunflower Seeds, Buckwheat (Soba) Seeds, Barley, Millet, Carrots, Beets, Broccoli, High Oleic Sunflower Oil.

Minor ingredients: rapeseed oil, sea salt, oregano, garlic, ascorbic acid, propolis, vitamins (e.g. vitamins A, B12, D3, C, E, K3, thiamine, riboflavin, pyridoxine, biotin, folic acid, pantothenate, niacin) minerals (eg chromium, sodium selenite, iron, copper citrate, zinc sulfate, cobalt).

The total content of the following parts was (in mass % of the total load.)

Proteins minimum: 20 wt. %

Fat minimum: 12 wt. %

Fiber max: 9 wt. %

Humidity max: 10.5 wt. %

As described for the puppy food in the "Food and Feed according to Example 1" section, 7 respective dog food formulations were prepared using the deposited Lactobacillus DSMZ strains 16667, 16668, 16669, 16670, 16671, 16672 and 16673 separately.

Additional implementation options:

1. A binding microorganism or fragment thereof, wherein the microorganism or fragment thereof capable of binding to at least one, preferably at least two, and more preferably at least three strains of Streptococcus of the mutans Streptococcus group, and wherein the binding exhibits resistance to thermal processing and/or resistance to protease processing and/or is calcium dependent and/or is formed within a pH range above 4.0 and/or is independent of magnesium and/or is formed in the presence of saliva as an oral oral care agent.

2. The binding microorganism or fragment thereof according to claim 1, where the binding is resistant to heat treatment at a temperature of ≥55°C, preferably at ≥65°C, more preferably at 95-121°C and most preferably at a temperature of 121°C.

3. The binding microorganism or fragment thereof according to claim 1, wherein the binding is resistant to heat treatment for ≥15 minutes, preferably 15-120 minutes, preferably 15-30 minutes, most preferably 20 minutes.

4. A binding microorganism or fragment thereof, according to embodiment 3, wherein the binding is resistant to heat treatment in saturated steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably 2 bar.

5. The binding microorganism or its fragment according to claim 1, where the binding is resistant to heat treatment in saturated water vapor at a pressure of 1-5 bar, preferably 1-3 bar, more preferably 2 bar, at a temperature of 95-121°C, more preferably at 121° C. for ≥15 minutes, preferably 15-120 minutes, preferably 15-30 minutes, most preferably 20 minutes.

6. The binding microorganism or fragment thereof according to claim 1, wherein the binding is resistant to heat treatment, where the heat treatment is carried out in saturated water vapor at a pressure of 2 bar at a temperature of 121°C for 20 minutes.

7. The binding microorganism or fragment thereof according to claim 1, wherein the binding is resistant to protease treatment with pronase E, proteinase K, trypsin and/or chymotrypsin and preferably is also resistant to protease treatment with elastase, thrombin, amnopeptidase I, carboxypeptidase, domtrypain , endoproteinase, papain, cathepsin B, pepsin, gastriscin, chymosin and/or cathepsin D.

8. A binding microorganism or fragment thereof, according to embodiment 7, wherein the binding is resistant to protease treatment with pronase E, proteinase K, trypsin and chymotrypsin.

9. A binding microorganism or fragment thereof, according to embodiment 7, wherein the binding is resistant to protease treatment with elastase, thrombin, aminopeptidase I, carboxypeptidase, domtrypain, endoproteinase, papain, cathepsin B, pepsin, gastrixin, chymosin, and cathepsin D.

10. The binding microorganism or its fragment according to claim 1, where the binding is resistant to heat treatment in saturated steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably 2 bar, at a temperature of 95-121°C, more preferably at 121°C, for ≥15 min, preferably 15-120 min, preferably 15-30 min, most preferably 20 min, and also resistant to protease treatment with pronase E, proteinase K, trypsin and/or chymotrypsin, and preferably , are also resistant to protease treatment with elastase, thrombin, aminopeptidase I, carboxypeptidase, domtrypain, endoproteinase, papain, cathepsin B, pepsin, gastrixin, chymosin and/or cathepsin D.

11. The binding microorganism or fragment thereof according to claim 6, wherein the binding is resistant to protease treatment with pronase E, proteinase K, trypsin and/or chymotrypsin and preferably also resistant to protease treatment with elastase, thrombin, aminopeptidase I, carboxypeptidase, domtrypain, endoproteinase, papain, cathepsin B, pepsin, gastrixin, chymosin and/or cathepsin D.

12. A binding microorganism or fragment thereof according to embodiment 8, wherein the binding is resistant to heat treatment, where the heat treatment is carried out in saturated steam at 2 bar pressure at 121° C. for 20 minutes.

13. The binding microorganism or fragment thereof according to claim 1, wherein the binding depends on the presence of Ca ²⁺ ions at a concentration of at least 2 mM, more preferably at least 1 mM, most preferably at least 0.05 mM.

14. The binding microorganism or fragment thereof according to claim 1, in which the binding depends on the presence of Ca ²⁺ ions at a concentration of 0.05-500 mm, more preferably 1-100 mm, most preferably 2-30 mm.

15. A binding microorganism or fragment thereof according to embodiment 12 or 13, wherein the binding is resistant to heat treatment, where the heat treatment is carried out in saturated steam at 2 bar pressure at 121°C for 20 minutes.

16. The binding microorganism or fragment thereof according to embodiment 12 or 13, wherein the binding is resistant to protease treatment with pronase E, proteinase K, trypsin and/or chymotrypsin and preferably also resistant to protease treatment with elastase, thrombin, aminopeptidase I, carboxypeptidase, domtrypain, endoproteinase, papain, cathepsin B, pepsin, gastrixin, chymosin and/or cathepsin D.

17. The binding microorganism or fragment thereof according to claim 1, wherein the binding occurs at a pH of 4.0-9.0, preferably 4.0-7.0, more preferably 4.2-5.0 and most preferably 4.5 .

18. The binding microorganism or fragment thereof according to claim 16, wherein the binding is resistant to heat treatment, where the heat treatment is carried out in saturated steam at a pressure of 2 bar at a temperature of 121°C for 20 minutes.

19. The binding microorganism or fragment thereof according to claim 16, wherein the binding is resistant to protease treatment with pronase E, proteinase K, trypsin and/or chymotrypsin and preferably also resistant to protease treatment with elastase, thrombin, aminopeptidase I, carboxypeptidase, domtrypain, endoproteinase, papain, cathepsin B, pepsin, gastrixin, chymosin and/or cathepsin D.

20. The binding microorganism or fragment thereof according to claim 16, wherein the binding depends on the presence of Ca ²⁺ ions at a concentration of at least 2 mM, more preferably at least 1 mM, most preferably at least 0.05 mM.

21. The binding microorganism or its fragment according to claim 1, where the binding is independent of the presence of magnesium ions, preferably is resistant to heat treatment, where the heat treatment is carried out in saturated steam at a pressure of 2 bar at a temperature of 121°C for 20 minutes.

22. The binding microorganism or fragment thereof according to claim 1, in which the binding takes place in the presence of saliva, and preferably is resistant to heat treatment, where the heat treatment takes place in saturated steam at a pressure of 2 bar at a temperature of 121°C for 20 minutes.

23. The binding microorganism or fragment thereof according to claim 1, wherein the binding is resistant to heat treatment, is resistant to protease treatment and occurs in the presence of saliva.

24. The binding microorganism or fragment thereof according to any of the preceding embodiments, wherein the Streptococcus mutans strain is selected from the group consisting of Streptococcus mutans serotype c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923), Streptococcus mutans serotype f (NCTC 11060 ), Streptococcus sobrinus DSMZ 20742, Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ 20562, Streptococci ferus DSMZ 20646 and Streptococcus macacae DSMZ 20714, and preferably where the specific binding of the binding microorganism or fragment thereof can be assayed as follows:

(a) growing said binding microorganism to stationary phase, or, if the fragment is to be tested, obtaining such a fragment,

(b) mixing said binding microorganism or fragment with mutans Streptococcus grown to stationary phase,

(c) incubating the mixture obtained in step (b) under conditions allowing the formation of aggregates from said microorganism and said Streptococcus, and

(d) detection of aggregates by precipitation.

25. The binding microorganism or fragment thereof according to any one of embodiments 1-22, wherein the microorganism or fragment thereof capable of binding to each of strains selected from the group consisting of Streptococcus mutans serotype c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923) and Streptococcus mutans serotype f (NCTC 11060).

26. Binding microorganism or fragment thereof according to embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, wherein said microorganism or fragment thereof is unable to bind to at least one, preferably at least two, and more preferably at least three, and even more preferably all, microorganisms selected from the group , consisting of Streptococcus salivarius ssp. thermophilus, Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus salivarius DSMZ 20567.

27. Binding microorganism or fragment thereof according to embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24 or 25, where the binding microorganism belongs to the Lactobacillaceae family, preferably the Lactobacillus genus, the Paralactobacillus genus, the Pediococcus genus or the Sharpea genus, more preferably the Lactobacillus paracasei species, the Lactobacillus rhamnosus species, the Lactobacillus casei species, the Lactobacillus zeae species, and most preferably is any of the strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 or DSMZ 16673, or a mutant or derivative thereof.

28. The binding microorganism or fragment thereof according to embodiment 26, wherein the microorganism or fragment thereof capable of binding to each of strains selected from the group consisting of Streptococcus mutans serotype C (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923 ) and Streptococcus mutans serotype f (NCTC 11060), and not capable of binding to a microorganism selected from the group consisting of Streptococci Salivarius ssp. thermophilus, Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus salivarius DSMZ 20567.

29. A binding microorganism or fragment thereof according to embodiment 26 or 27, wherein said microorganism or fragment thereof, after heat treatment in saturated steam at a pressure of 2 bar at a temperature of 121°C for 20 minutes, retains the ability to bind Streptococcus mutans selected from the group consisting of Streptococcus mutans serotype c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923), Streptococcus mutans serotype f (NCTC 11060), Streptococcus sobrinus DSMZ 20742, Streptococcus ratti DSMZ 20564, Streptococ cus cricetus DSMZ 20562, Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ 20714 in the presence of at least 0.05 mM calcium ions and in the presence of saliva and regardless of treatment with pronase E, proteinase K, trypsin and/or chymotrypsin, where under these conditions the thermally treated microorganism or its fragment does not capable of binding to a microorganism selected from the group consisting of Streptococcus salivarius ssp.thermophilus, Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus salivarius DSMZ 20567 .

30. Binding microorganism according to any of the embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30, where the binding microorganism is in an inactivated form obtained or resulting from heat inactivation, lyophilization or spray drying, where heat inactivation is preferably achieved by

- autoclaving the cells of said binding microorganism at 121° C. for 20 minutes in the presence of saturated steam at an atmospheric pressure of 2 bar, or

- freezing these cells for at least 1 hour at -20°C.

31. Inactivated binding microorganism in accordance with option 29, obtained or obtained by thermal inactivation at a temperature of ≥55°C, preferably at a temperature of ≥65°C, even more preferably at a temperature of 95-121°C and most preferably at a temperature of 121°C

32. An inactivated binding microorganism according to embodiment 30 obtained or obtained by thermal inactivation for ≥15 minutes, preferably 15-120 minutes, preferably 15-30 minutes, most preferably 20 minutes.

33. An inactivated binding microorganism according to embodiment 29, 30 or 31, obtained or obtained by thermal inactivation in saturated steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably 2 bar, and most preferably by thermal inactivation in saturated steam at a pressure of 2 bar at a temperature of 121°C for 20 minutes.

34. A binding microorganism fragment according to any of the previous embodiments, wherein the fragment is a lysate or membrane fragment of the binding microorganism.

35. The binding microorganism or fragment thereof according to any of the embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 as a sensory neutral oral hygiene product.

36. Binding microorganism or fragment thereof according to any of the embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 as an anti-tartar, sensory-neutral anti-caries and/or as an anti-bad breath .

37. The binding microorganism or fragment thereof according to embodiment 35 as a sensory neutral anti-tartar agent, a sensory neutral anti-caries agent, and/or a sensory neutral anti-malodor agent.

38. The binding microorganism or fragment thereof according to any one of embodiments 34, 35 or 36, wherein said binding microorganism or fragment thereof is an isolated and purified binding microorganism or fragment thereof.

39. A composition containing a binding microorganism or fragment thereof according to any of the embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, as an oral oral care agent, preferably as an anti-tartar agent, an anti-caries agent and/or an anti-malodorous agent.

40. A composition containing a binding microorganism or a fragment thereof according to any of the embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, as a sensory neutral oral hygiene product, preferably as a sensory neutral anti-tartar agent, a sensory neutral anti-cavity agent and/or a sensory neutral anti-malodor agent.

41. A composition containing a binding microorganism or fragment thereof according to any of the embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, in the amount

- sufficient to prevent or reduce the intensity of bad breath and/or

- sufficient to prevent caries or delay the formation of caries and/or

- sufficient to prevent the formation of tartar or slow down the formation of tartar.

42. A composition containing a binding microorganism according to any of the embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36 or 37, in concentration

- 10 ² -10 ¹³ cells/mg, preferably 10 ² -10 ¹² cells/mg, even more preferably 10 ² -10 ⁸ cells/mg or

- 10 ² -10 ¹³ cells/ml, or

- ≥0.01 wt. % relative to the total weight of the composition, preferably 0.01-10 wt. % and more preferably 0.025-2 wt. % or

- ≥0.01 mg dry weight/g total composition, preferably 0.01-100 mg dry weight/g total composition and more preferably 0.025-2 mg dry weight/g total composition.

43. A composition containing a fragment of a binding microorganism according to any of the options for performing 32, 33, 34, 35, 36 or 37, at a concentration

- ≥0.01 wt. % relative to the total composition, preferably 0.01-10 wt. % and more preferably 0.025-2 wt. % or

- ≥0.01 mg dry weight/g total composition, preferably 0.01-100 mg dry weight/g total composition and more preferably 0.025-2 mg dry weight/g total composition.

44. The composition according to any one of the embodiments of 38, 39, 40, 41 or 42, containing the additional microorganism in a viable, thermally inactivated or lyophilized form, where the thermally inactivated form is obtained or obtained by treatment in saturated steam at a pressure of 1-5 bar , preferably 1-3 bar, more preferably 2 bar, and most preferably by thermal inactivation in saturated steam at a pressure of 2 bar at a temperature of 121°C for 20 minutes.

45. The composition of embodiment 43, wherein the additional microorganism is an anti-malodorous microorganism at a concentration sufficient to prevent, modify, or reduce bad breath, preferably capable of promoting the growth of Streptococcus salivar, but not promoting the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

46. The composition according to embodiment 44, wherein the anti-malodorous microorganism is a Lactobacillus acidophilus species, and preferably any of Lactobacillus strains DSMZ 19825, DSMZ 19826, or DSMZ 19827.

47. Composition according to any of the embodiments 38, 39, 40, 41, 42, 43, 44 or 45,

- where the composition is intended for use in a human or animal, and is a toothpaste, dentifrice, tooth powder, oral gel, mouth rinse, denture material, aerosol mouth flavor, lozenge, oral tablets, chewing gum, mouthwash, dental floss, chewing product, or food, feed, or beverage additives, or

- where the composition is in the form of a powder, tablet, film preparation, solution, aerosol, granule, tablet, suspension, emulsion, capsule, syrup, liquid, elixir, extract, tincture or liquid extract, leaf food product, bottled food product, canned food, autoclaved food or liquid food or

- where the composition is a food or drink selected from the group consisting of chewing gum, spray, drink, candy, baby food, ice cream, frozen dessert, sweet salad dressing, milk preparation, cheese, cottage cheese, yogurt, acidified milk, cream for coffee, whipped cream, butter, cheese, processed milk and skimmed milk, meat product - preferably ham, sausage, and hamburger, fish meat, flour products, egg products - preferably egg rolls and egg curds, confectionery products - preferably biscuits , jellies, snacks and chewing gum, bread, pasta, marinated, smoked products, dried fish, condiments.

48. A composition according to any one of embodiments 38, 39, 40, 41, 42, 43, 44, or 45, wherein the composition is a food or feed composition.

49. A food or feed composition according to embodiment 47, wherein the composition is for infants or a pet, preferably a dog, cat, rat, mouse, hamster, guinea pig, or monkey.

50. The pet food or food composition of embodiment 48, wherein the composition is a pet chew, and preferably in the form of a bone, roll, donut, staple, pretzel, figure eight, or chip.

51. The composition of any one of embodiments 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, wherein the composition is a cosmetic, pharmaceutical, or veterinary composition.

52. Food or feed composition for pets,

comprising an inactivated binding microorganism in a thermally inactivated or lyophilized form, wherein the inactivated microorganism is capable of binding to a strain of Streptococcus of the mutans Streptococcus group, and wherein the binding occurs in the presence of saliva and is resistant to calcium dependent heat treatment, wherein the heat treatment takes place in saturated water vapor at pressure 2 bar at 121°C for 20 minutes,

and where the Streptococcus mutans strain is selected from the group consisting of Streptococcus mutans serotype c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923) and Streptococcus mutans serotype f (NCTC 11060),

and wherein the inactivated binding microorganism is unable to bind to a microorganism selected from the group consisting of Streptococcus salivarius ssp. thermophilus, Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus salivarius DSMZ 20567,

and where the binding microorganism is present in a sensory neutral amount

- sufficient to prevent or reduce the intensity of bad breath and/or

- sufficient to prevent caries or delay the formation of caries and/or

- sufficient to prevent the formation of tartar or slow down the formation of tartar.

53. The pet food composition according to embodiment 51, wherein the concentration of inactivated binding microorganisms is

- 10 ² -10 ¹³ cells/mg, preferably 10 ² -10 ¹² cells/mg, even more preferably 10 ³ -10 ⁸ cells/mg or

- 10 ² -10 ¹³ cells/ml, or

- ≥0.01 wt. % relative to the total composition, preferably 0.01-10 wt. % and more preferably 0.025-2 wt. % or

- ≥0.01 mg dry weight/g total composition, preferably 0.01-100 mg dry weight/g total composition and more preferably 0.025-2 mg dry weight/g total composition

54. Food or feed composition for pets containing a fragment in accordance with option 33, at a concentration

- ≥0.01 wt. % relative to the total composition, preferably 0.01-10 wt. % and more preferably 0.025-2 wt. % or

- ≥0.01 mg dry weight/g total composition, preferably 0.01-100 mg dry weight/g total composition and more preferably 0.025-2 mg dry weight/g total composition.

55. Use of a binding microorganism or fragment thereof according to any one of embodiments 1-37 as a sensory neutral oral hygiene agent, preferably as a sensory neutral antitartar agent and/or a sensory neutral anti-caries agent and/or a sensory neutral agent against bad breath.

56. The use of a binding microorganism or fragment thereof according to any of the embodiments 1-37 for the production of any

- a medicinal product for the prevention or treatment of tartar formation,

- a medicinal product for the prevention or treatment of caries,

- medicines for the prevention or treatment of bad breath,

wherein the binding microorganism and/or fragment thereof is in a sensory neutral amount.

Claims (39)

1. A method for reducing bad breath in a dog, comprising administering to a dog in need thereof a dog food composition comprising a fragment of a microorganism belonging to the genus Lactobacillus in an effective amount, said fragment being a membrane fraction obtained by preparing a membrane preparation, as a sensory neutral oral care agent, wherein the microorganism is capable of binding to a microorganism from the mutans Streptococci group, wherein the microorganism is in a thermally inactivated form, and wherein the binding (i) resistant to heat treatment for at least 20 minutes at more than 95°C, and/or (ii) resistant to protease treatment, wherein the protease is selected from the group consisting of pronase E, proteinase K, trypsin and chymotrypsin, and/or (iii) calcium dependent, and/or (iv) formed in a pH range between 4.5 and 8.5, and/or (v) formed in the presence of saliva, and/or (vi) regardless of magnesium, and moreover, the specified fragment is used as a sensory neutral agent against bad breath, and wherein the composition is administered in an amount effective to reduce bad breath in said dog, and wherein the microorganism is selected from the group consisting of Lactobacillus strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673, moreover, the composition contains 3 g of a microorganism fragment per 1 kg of the base composition, moreover, the base composition includes beef, brown rice, rape seeds, linseed meal, sunflower seeds, buckwheat seeds, barley and millet. 2. The method according to p. 1, where the composition has a protein content of 26 wt. %, fat content 14 wt. % and fiber content of 5 wt. %, in each case based on the weight of the composition, or has a protein content of 20 wt. %, fat content 12 wt. % and fiber content of 5 wt. %, in each case based on the weight of the composition. 3. A method for reducing bad breath in a dog, comprising administering to a dog in need thereof a dog food composition comprising a fragment of a microorganism belonging to the genus Lactobacillus in an effective amount, said fragment being a membrane fraction obtained by preparing a membrane preparation, as a sensory neutral oral care agent, wherein the microorganism is capable of binding to a microorganism from the mutans Streptococci group, wherein the microorganism is in a thermally inactivated form, and wherein the binding (i) resistant to heat treatment for at least 20 minutes at more than 95°C, and/or (ii) resistant to protease treatment, wherein the protease is selected from the group consisting of pronase E, proteinase K, trypsin and chymotrypsin, and/or (iii) calcium dependent, and/or (iv) formed in a pH range between 4.5 and 8.5, and/or (v) formed in the presence of saliva, and/or (vi) regardless of magnesium, and moreover, the specified fragment is used as a sensory neutral agent against bad breath, and wherein the composition is administered in an amount effective to reduce bad breath in said dog, and wherein the microorganism is selected from the group consisting of Lactobacillus strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673, moreover, the composition contains 3 g of a microorganism fragment per 1 kg of the base composition, moreover, the base composition includes soybean meal, brown rice, rapeseed, linseed meal, sunflower seeds, buckwheat seeds, barley and millet. 4. The method according to p. 3, where the composition has a protein content of 20 wt. %, fat content 12 wt. % and fiber content of 9 wt. %, in each case based on the weight of the composition. 5. A method for reducing bad breath in a dog, comprising administering to a dog in need thereof a dog food composition comprising a fragment of a microorganism belonging to the genus Lactobacillus in an effective amount, said fragment being a membrane fraction obtained by preparing a membrane preparation, as a sensory neutral oral care agent, wherein the microorganism is capable of binding to a microorganism from the mutans Streptococci group, wherein the microorganism is in a thermally inactivated form, and wherein the binding (i) resistant to heat treatment for at least 20 minutes at more than 95°C, and/or (ii) resistant to protease treatment, wherein the protease is selected from the group consisting of pronase E, proteinase K, trypsin and chymotrypsin, and/or (iii) calcium dependent, and/or (iv) formed in a pH range between 4.5 and 8.5, and/or (v) formed in the presence of saliva, and/or (vi) regardless of magnesium, and moreover, the specified fragment is used as a sensory neutral agent against bad breath, and wherein the composition is administered in an amount effective to reduce bad breath in said dog, and wherein the microorganism is selected from the group consisting of Lactobacillus strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673, moreover, the composition contains 3 g of a microorganism fragment per 1 kg of the base composition, wherein the base composition includes chicken, chicken meal, barley, peas, brown rice, shredded extruded whole soybeans, and chicken fat. 6. The method according to p. 5, where the composition has a protein content of 28 wt. %, fat content 15 wt. % and a fiber content of 3.5 wt. %, in each case based on the weight of the composition.

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