BE1029673A1 - PROBIOTIC COMPOUND FOR IMPROVED ORAL HEALTH - Google Patents

Abstract

The present invention relates to a probiotic composition for oral use, wherein the composition Streptococcus Sp. and Lactobacillus Sp. wherein the composition comprises at least Streptococcus Salivarius, Lactobacillus Rhamnosus, Lactobacillus Reuteri and Lactobacillus Paracasei, wherein the composition further comprises sorbitol and xylitol.

Description

1 BE2021/5648

PROBIOTIC COMPOUND FOR IMPROVED ORAL HEALTH

TECHNICAL DOMAIN N

The present invention relates to a probiotic composition with antimicrobial properties for improving oral health.

STATE OF TECHNOLOGY

The oral cavity is home to a numerous and varied microbial flora. When the balance is disturbed and an imbalance occurs between the native bacteria, pathologies such as dental caries or periodontitis can arise. The beneficial effects of probiotics are achieved in part through modulation of the host's existing microbial flora, achieving a balanced and healthy relationship between microbes and host.

S. salivarius BLIS K12 is a natural human commensal isolated from the oral cavity of a healthy New Zealand child. S. salivarius is a naturally occurring bacteria mainly found on the back of the tongue and in the pharynx of humans. S. salivarius establishes itself in the oral cavity of humans within two days of birth. The content of S. salivarius in smears from newborns is 10% of the total number of isolated streptococci, increasing to 25-30% after one month. S. salivarius produces two antimicrobial bacteriocins that have been shown to inhibit the growth of oral pathogens.

The use of probiotic compositions for use in the oral cavity is known in the art.

US 9 155 766 describes probiotic compositions containing a mixture of at least two probiotic bacteria including Streptococcus salivarius K12® (BLIS K12®) and at least one Lactobacillus bacteria. The Streptococcus salivarius

K12® (BLIS K12®) may consist of Streptococcus salivarius K12® (BLIS K12®)

BAA1024. The Lactobacillus bacteria may be selected from bacteria known to be suitable for oral administration, e.g. bacteria known to improve the recipient's health, especially the recipient's oral health.

2 BE2021/5648

EP 3 453 394 describes probiotic compositions which can be administered to improve or maintain oral and/or intestinal health. For example, a toothpowder, toothpaste, mouthwash, and/or probiotic composition may be administered to improve or maintain oral and/or intestinal health.

A problem unrelated to the formation of caries is the development of unpleasant or bad breath (halitosis). Halitosis is considered to be the result of unwanted microorganisms colonizing the oral cavity. However, so far no single microorganism has been identified as the primary cause of oral malodors. However, it seems clear that the formation of caries and halitosis are independent processes, since oral malnourishment can also occur in the absence of teeth and caries can occur in the absence of halitosis.

EP3 424 516 shows micro-organisms or fragments thereof as sensory neutral oral care agents, in particular for the prevention of tartar, as anti-caries agents and/or anti-oral fresheners. EP '516 further relates to compositions containing microorganisms or fragments thereof for the reduction of mutans streptococci. Such compositions can be used in oral care compositions, for example for caries prophylaxis, or for the prophylaxis of tartar or oral malodors. They may also or instead be used for the prevention or treatment of mouth odour.

Although a large number of probiotic compositions are known, each composition has unique characteristics and particular health benefits. However, there is always a need for improved formulations of probiotic compositions, especially formulations that result in one or more of improved oral health, improved oral bioavailability, improved shelf life, reduced incidence of side effects, and additional improvements, as well as methods of administration or uses thereof.

The present invention aims at solving at least some of the above-mentioned problems or disadvantages.

3 BE2021/5648

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a probiotic composition for oral use according to claim 1, wherein the composition Streptococcus Sp. and

Lactobacillus Sp. includes. What is special about the present invention is that the composition contains at least Streptococcus Salivarius, Lactobacillus Rhamnosus, Lactobacillus Reuteri and Lactobacillus Paracasei. The specific combination of the probiotic components is extremely effective for the inhibition and/or elimination of pathogens in the oral cavity.

Probiotics can help keep the oral flora in balance. The idea is that large numbers of these good bacteria displace the harmful bacteria in the mouth.

The inventors unexpectedly discovered that the specific combination of

Streptococcus Salivarius, Lactobacillus Rhamnosus, Lactobacillus Reuteri and

Lactobacillus Paracasei significantly reduces the number of pathogenic bacteria compared to the compositions described in the prior art.

According to the present invention, the ratio between the different probiotic strains is important. The composition must contain a minimum of 2 CFU/g

Streptococcus Salivarius, minimum 5 CFU/g Lactobacillus Rhamnosus, minimum 2

CFU/g Lactobacillus Reuteri and a minimum of 2 CFU/g Lactobacillus Paracasei.

This ratio ensures an optimal antibacterial effect and is able to significantly reduce the number of pathogenic microorganisms. This improves the oral health of the user.

In a second aspect, the present invention relates to a probiotic composition for use for improving oral hygiene according to claim 13. This has the advantage, among others, that a composition comprising Streptococcus Salivarius,

Lactobacillus Rhamnosus, Lactobacillus Reuteri and Lactobacillus Paracasei and further comprising sorbitol and/or xylitol is capable of significantly reducing the number of pathogenic bacteria over the compositions described in the prior art. Consequently, the probiotic composition prevents and/or delays the formation of dental plaque, the occurrence of gingivitis, the occurrence of bad odors and the occurrence of dental caries. This use results in improved oral hygiene for the user.

4 BE2021/5648

DESCRIPTION OF THE FIGURES

Figure 1 shows a fluorescence activated cell sorting (FACS) profile in a control condition.

Figure 2 shows a fluorescence activated cell sorting (FACS) profile in a control condition of an embodiment of the present invention.

Figure 3 shows a histogram with a comparison between control condition, the composition according to the present invention and a composition described in the prior art.

Figure 4 shows a fluorescence activated cell sorting (FACS) profile with a comparison between control condition, the composition according to the present invention and a composition described in the prior art.

DETAILED DESCRIPTION

Unless defined otherwise, all terms used in the description of the invention, including technical and scientific terms, have the meaning as commonly understood by those skilled in the art of the invention. For a better appreciation of the description of the invention, the following terms are explicitly explained. "A", "the" and "the" in this document refer to both the singular and the plural unless the context clearly dictates otherwise. For example, "a segment" means one or more than one segment.

When "about" or "around" is used in this document for a measurable quantity, a parameter, a time period or moment, and the like, it means variations of +/-20% or less, preferably +/-10% or less. less, more preferably +/-5% or less, even more preferably +/-1% or less, and even more preferably +/-0.1% or less than and of the quoted value, to the extent that such variations from are applicable in the described invention. However, it should be understood that the value of the quantity using the term "about" or "around" is itself specifically disclosed.

The terms “include”, “comprising”, “consisting of”, “consisting of”, “comprising”, “containing”, “containing”, “contain”, “containing” are synonyms and are inclusive or open terms meaning the indicate the presence of what follows, and which do not exclude or preclude the presence of other components, features, elements, members, steps, known or described in the art.

Quoting numeric intervals by the endpoints includes all integers, fractions, and/or real numbers between the endpoints, including those endpoints. “Antimicrobial activity” as described in this document refers on the one hand to the inhibiting effect of the composition on microbial growth in the oral cavity and on the other hand to the antiseptic effect of the composition, preventing microbial contamination in the oral cavity.

The term "streptococci" (Streptococcus) according to the present invention are a genus of gram-positive facultative anaerobic cocci bacteria. Reproduction is by cell division along a single axis in this bacterial genus.

As a result, they grow in a chain shape or as pairs. Among the streptococci there are both beneficial and pathogenic bacteria for humans and animals. They mainly occur in the nasopharynx and on the skin, where any infection manifests itself.

A number of species that fall within the genus include:

Streptococcus agalactiae Streptococcus anginosus Streptococcus equisimilis

Streptococcus gordonii Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus sanguis

Streptococcus salivarius Streptococcus sobrinus Streptococcus suis

Streptococcus thermophilus Streptococcus vestibularis Enteroccus faecalis.

The term "Lactobacillus" according to the present invention is a bacterial genus of gram-positive, facultative anaerobic rods. The lactobacilli belong to the group of lactic acid bacteria and all species are capable of converting sugars into lactic acid. Some species also make acetic acid and carbon dioxide.

Lactobacilli occur naturally in decaying plant or animal material, but also in the human oral cavity, vagina, small and large intestine, where they form an important part of the intestinal flora. Lactobacillus species are widely used in the production of fermented foods such as cheese, yoghurt, salami, wine, sourdough, kefir, geuze, sauerkraut, olives, tempeh and many traditional fermented products. Various types are also used today as

6 BE2021/5648 probiotics to improve the intestinal flora. A number of species that fall within the genus include:

Lactobacillus acidophilus Lactobacillus casei Lactobacillus delbrueckii Lactobacillus fermentum Lactobacillus plantarum Lactobacillus reuteri Lactobacillus rhamnosus

Lactobacillus paracasei.

Improving and/or maintaining oral health can improve and/or maintain overall health. Scientific studies have shown that improving the integrity of the oral flora can benefit the upper respiratory tract (e.g. by preventing infections). In addition, while links between oral/periodontal health and cardiac and peripheral vascular health have been discovered, the link between improvement of oral flora and/or eradication of certain oral organisms leading to benefits for cardiac and vascular health has not previously been proven.

Bacteria are everywhere and not all bacteria are bad bacteria. The danger is mainly in certain specific pathogenic bacteria and especially when they grow too much and cause inflammation. Today, about 2/3 of adults suffer from oral inflammation. This is worrying, especially since the majority of those people do brush their teeth regularly and see a dentist on a regular basis. A probiotic composition that people can use to prevent bacterial growth could limit the consequences of this bacterial growth. Antibiotics temporarily eliminate the bacteria in the oral cavity, but over time, the bacteria just come back. In fact, because a lot of good bacteria were also removed by the antibiotic, the pathogenic bacteria get the chance to multiply undisturbed.

Metal ions and/or the corresponding metal salts can have antibacterial properties, for example by inhibiting certain bacterial enzymes important for the survival of the bacteria (bactericidal properties) or by reducing the formation of bacterial biofilms (bacteriostatic properties). However, taking care of an optimal balance in the oral flora provides a more long-acting solution.

The term "fungus" according to the present invention is a simple plant organism. Fungi are divided into different classes, for example candida, blastomycetes and trichophytes. In general, fungi thrive best in a dark, moist, warm environment. Most fungal infections affect the skin; the skin in skin folds in particular is affected. Most come

7 BE2021/5648 athlete's foot for. Some types of mold are able to invade the body and multiply, for example, in the mouth or lungs; for example, the fungus monilia, which settles in the mouth (and also in the vagina), causes thrush. Severe diseases cause the actinomycetes; they affect, among other things, the jaw and lead to the formation of purulent fistulas.

In a first aspect, the invention relates to a probiotic composition for oral use, wherein the composition Streptococcus Sp. and Lactobacillus Sp. includes.

What is special about the invention is that the composition contains at least Streptococcus

Salivarius, Lactobacillus Rhamnosus, Lactobacillus Reuteri and Lactobacillus Paracasei.

The streptococci family has several "bad" strains. Streptococcus mutans is responsible for tooth decay, S. pneumoniae causes certain pneumonias, S. pyogenes causes scarlet fever, etc. S. salvarius, on the other hand, is accepted as a good bacteria. It is particularly abundant in the oral flora and also in the intestine. The inventors found that the antibiotic power of S. salivarius goes beyond its streptococcal relatives. In general, a bacterium's bacteriocins are directed against a specific set of targets, often species of the same family. It was discovered that bacteriocins from S. salivarius can kill staphylococcus aureus, listeria and even some enterococci - disease-causing bacteria that are increasingly resisting antibiotic treatments.”

L. rnamnosus is a type of bacteria that lives in the intestines. It belongs to the genus Lactobacillus, a species of bacteria that produces the enzyme lactase. This enzyme breaks down the sugar lactose - found in dairy - into lactic acid. Bacteria from this genus, such as L. rhamnosus, are considered probiotics. This bacteria is uniquely adapted to survive in acidic and basic conditions in your body, and can also attach to and colonize your intestinal walls.

Such traits give L. rhamnosus a better chance of survival – and thus potentially offer longer-term benefits. L. rhamnosus can improve overall digestive health, treat diarrhea, the symptoms of

Relieve Irritable Bowel Syndrome (IBS) and strengthen the intestines. It can also protect against tooth decay and urinary tract infections. Probiotic bacteria such as L. rhamnosus have antimicrobial properties, which can help fight these harmful bacteria.

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Lactobacillus reuteri (L. reuteri) is a well-studied probiotic bacteria that can colonize a wide range of mammals. In humans, L. reuteri is found in various places in the body, including the gastrointestinal tract, urinary tract, skin and breast milk. The abundance of L. reuteri varies between different individuals. There are several beneficial effects of it

L. reuteri detected. First, L. reuteri can produce antimicrobial molecules such as organic acids, ethanol and reuterine. Second, L. reuteri may benefit the host's immune system. For example, some L. reuteri strains can reduce the production of pro-inflammatory cytokines and promote the development and function of regulatory T cells. Third, by strengthening the intestinal barrier, colonization with L. reuteri may reduce microbial translocation from the intestinal lumen to the tissues. Remarkably, the decline in the abundance of L. reuteri in humans over the past few decades has been correlated with an increase in the number of inflammatory disease cases over the same period.

Lactobacillus paracasei is a type of lactic acid bacteria commonly found in the human intestinal tract and mouth, as well as in foods such as yogurt and naturally fermented vegetables and milk. L. paracasei has been shown to exhibit specific differences from other Lactobacillus species, particularly in its ability to withstand higher temperatures. L. paracasei has been shown to stimulate the immune system. In addition to its immune-stimulating properties

L. paracasei also help inhibit harmful pathogens such as

Shigella dysenteriae, Staphylococcus aureus, Cronobacter sakazakii, Escherichia coli, and Candida albicans. Yeast infections such as Candida were also inhibited. Lactobacillus paracasei also helps with dental health by inhibiting the growth and proliferation of bacteria associated with gingivitis.

The inventors found that the specific combination of the four probiotic bacterial strains was able to achieve a significant reduction in the number of pathogenic bacteria in the oral cavity. This is the result of the synergistic effect caused by the sophisticated combination. As stated earlier, antibiotics are very efficient at eliminating bacteria in the oral cavity, but over time, the bacteria come back. Moreover, because a lot of good bacteria were also removed by the antibiotic, the pathogenic bacteria get the chance to multiply again undisturbed. The present invention offers a better and longer lasting solution.

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According to the present invention, the composition comprises a minimum of 2 CFU/g

Streptococcus Salivarius, minimum 5 CFU/g Lactobacillus Rhamnosus, minimum 2

CFU/g Lactobacillus Reuteri and a minimum of 2 CFU/g Lactobacillus Paracasei. These ratios are important to ensure optimal functioning of the composition. The probiotic composition comprises approximately 45.4%

Lactobacillus Rhamnosus and the remaining bacteria each comprise about 18.2% (+18.2% Streptococcus Salivarius/+18.2% Lactobacillus Reuteri /+18.2%

Lactobacillus Paracasei). Preferably, a probiotic composition can include about 25% of each bacteria (+25% Lactobacillus Rhamnosus/ +25%

Streptococcus Salivarius/+25% Lactobacillus Reuteri/+25% Lactobacillus

Paracasei). Preferably, a probiotic composition may contain about 30%

Lactobacillus Rhamnosus and the remaining bacteria each comprise approximately 23% (+23% Streptococcus Salivarius/+23% Lactobacillus Reuteri/+23% Lactobacillus

Paracasei). Preferably, a probiotic composition may contain about 40%

Lactobacillus Rhamnosus and the remaining bacteria each comprise approximately 20% (+20% Streptococcus Salivarius/+20% Lactobacillus Reuteri/+20% Lactobacillus

Paracasei). Preferably, a probiotic composition may contain about 50%

Lactobacillus Rhamnosus and the remaining bacteria each comprise approximately 16.6% (+16.6% Streptococcus Salivarius/+16.6% Lactobacillus Reuteri /+16.6%

Lactobacillus Paracasei). Preferably, a probiotic composition may contain about 35% Lactobacillus Rhamnosus and the remaining bacteria each comprise about 21.7% (+21.7% Streptococcus Salivarius/+21.7% Lactobacillus Reuteri/+21.7%

Lactobacillus Paracasei).

In one embodiment, the composition comprises between 1 and 10 CFU/g

Streptococcus Salivarius, between 1 and 25 CFU/g Lactobacillus Rhamnosus, between 1 and 10 CFU/g Lactobacillus Reuteri and between 1 and 10 CFU/g Lactobacillus Paracasei.

Preferably, the composition comprises between 1 and 5 CFU/g Streptococcus

Salivarius, between 1 and 15 CFU/g Lactobacillus Rhamnosus, between 1 and 5 CFU/g

Lactobacillus Reuteri and between 1 and 5 CFU/g Lactobacillus Paracasei. Preferably, the composition comprises between 1.5 and 3.5 CFU/g Streptococcus Salivarius, between 1.5 and 10 CFU/g Lactobacillus Rhamnosus, between 1.5 and 3.5 CFU/g

Lactobacillus Reuteri and between 1.5 and 3.5 CFU/g Lactobacillus Paracasei. More preferably, the composition comprises between 1.5 and 2.5 CFU/g Streptococcus

Salivarius, between 2.5 and 7.5 CFU/g Lactobacillus Rhamnosus, between 1.5 and 2.5

CFU/g Lactobacillus Reuteri and between 1.5 and 2.5 CFU/g Lactobacillus Paracasei.

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In one embodiment, in addition to the probiotic components, the composition also comprises sorbitol.

Sorbitol, also called D-sorbitol, 50-70-4, E420, and D-glucitol, is a carbohydrate.

It belongs to a category of sugar alcohols called polyols.

Commercially, sorbitol is used to retain moisture, add sweetness and texture to products, as well as aid digestion and oral health. Second, the sweetener is often added to foods for people with diabetes. Third, unlike table sugar, sugar alcohols such as sorbitol do not contribute to the formation of caries or tooth decay. This is one of the reasons why they are often used to sweeten sugar-free chewing gum and liquid medicines. The “Food and Drug

Administration” (FDA) has even recognized that sugar alcohols such as sorbitol may benefit oral health. In the present invention, sorbitol works! additionally as a catalyst to improve the functioning of the probiotic bacteria.

In one embodiment, the composition comprises between 25 and 750 g/kg of sorbitol.

Preferably the composition comprises between 50 and 700 g/kg sorbitol, between 100 and 600 g/kg sorbitol, between 200 and 550 g/kg sorbitol, between 300 and 550 g/kg sorbitol, more preferably between 400 and 550 g/kg kg sorbitol, preferably between 450 and 550 g/kg sorbitol. This amount of sorbitol is optimal to act as a catalyst in the composition, strengthening the action of the probiotic bacteria. Preferably, the ratio between sorbitol and probiotic bacteria is between 1:3 and 1:20. More preferably between 1:4 and 1:18. More preferably between 1:4 and 1:17. Again, this ratio ensures an optimal reinforcing effect of the bacteria.

In one embodiment, the composition includes an anti-caking agent such as silicon dioxide or tricalcium phosphate. Silicon dioxide is found naturally in many plants, such as: green leafy vegetables, beets, peppers, brown rice, oats and alfalfa. Silicon dioxide is also added to many foods and supplements. As a food additive, it serves as an anti-caking agent to prevent clumping. In supplements, it is used to prevent the various powdered ingredients from sticking together. It also binds metals, enhances antioxidant activity and strengthens the structure of canned vegetables. Finally, it is used as an anti-caking agent and as an abrasive in toothpaste. Preferably the composition comprises an anti-caking agent such as silicon dioxide or

11 BE2021/5648 tricalcium phosphate in an amount between 1 and 20 g/kg. Preferably between 3 and 17 g/kg, between 4 and 16 g/kg, between 5 and 15 g/kg, between 6 and 14 g/kg, between 7 and 13 g/kg, between 8 and 12 g/kg. More preferably, the composition comprises an anti-caking agent such as silicon dioxide or tricalcium phosphate in an amount between 9 and 11 g/kg.

In one embodiment, the composition comprises a lubricant such as magnesium stearate, croscarmellose sodium or sodium carboxymethyl cellulose.

Magnesium stearate is generally added to many foods, medicines and cosmetics. In medicines and vitamins, it primarily serves as a lubricant. Magnesium stearate is an additive mainly used in medicine. It is considered a "flux". It prevents the individual ingredients in a composition from sticking together. It helps improve consistency and quality control of formulations. Preferably, the composition comprises a lubricant such as magnesium stearate in an amount between 1 and 20 g/kg. Preferably between 3 and 17 g/kg, between 4 and 16 g/kg, between 5 and 15 g/kg, between 6 and 14 g/kg, between 7 and 13 g/kg, between 8 and 12 g/kg. More preferably, the composition comprises a lubricant such as magnesium stearate in an amount between 9 and 11 g/kg.

In one embodiment, in addition to the probiotic components, the composition also includes xylitol. Preferably, the composition comprises between 15 and 450 9/kg xylitol. Preferably the composition comprises between 20 and 400 g/kg xylitol, between 50 and 400 g/kg xylitol, between 60 and 400 g/kg xylitol, between 70 and 400 g/kg xylitol, more preferably between 100 and 350 g/kg kg xylitol, preferably between 200 and 350 g/kg xylitol.

Xylitol is also categorized as a sugar alcohol. Chemically, sugar alcohols combine properties of sugar molecules and alcohol molecules. Their structure allows them to stimulate the taste receptors for sweetness on your tongue. Xylitol has a similar sweetness to regular sugar, but contains 40% fewer calories. While some plaque on your teeth is normal, excess plaque triggers your immune system to attack the bacteria in the plaque. This can lead to inflammatory gum diseases such as gingivitis.

These oral bacteria feed on glucose from food, but they cannot use xylitol. Replacing sugar with xylitol thus reduces the available fuel for the harmful bacteria. In the present invention works

12 BE2021/5648 xylitol additionally, just as is the case for sorbitol, additionally as a catalyst to improve the functioning of the probiotic bacteria.

In one embodiment, the composition comprises probiotic bacteria in lyophilized form or in the form of live bacteria. Freeze-drying, also known as lyophilization, is primarily used to remove water from sensitive — usually organic — products without damaging them.

Such products can be brought into a state suitable for permanent storage in this way and then reconstituted by replenishing the water. Examples of freeze-dried products are antibiotics, bacteria, serums, vaccines, diagnostic drugs, proteinaceous and biotechnological products, cells and tissues, chemicals. The product to be dried is frozen under atmospheric pressure. Then, in a first drying phase — defined as primary drying phase — the water (in the form of ice) is removed by sublimation; in the second phase — secondary drying phase — it is removed via desorption. Freeze-drying is done under vacuum. Alternatively, the bacteria may also be added to the composition as live bacteria.

In a preferred form, the probiotic composition is formulated as a capsule, tablet, powder, mouthwash, chewable tablet, gel, paste, lozenge, or powder.

According to a further embodiment, the composition is a powder, more preferably a water-soluble powder. The oral composition according to the present invention may consist of a powder, which upon dissolution in water turns into a mouthwash with an oral probiotic composition. To this end, the powder according to the present invention is preferably soluble in water. Preferably, the powder is soluble under atmospheric pressure and at room temperature.

In addition, the powder is preferably hydrophilic. More preferably, the powder is highly hydrophilic.

The powder can reduce caries by reducing the amount of harmful bacteria in the mouth and/or increasing the good flora in the mouth, improving the health of the gastrointestinal tract (for example, the health of the mouth and/or the intestines), and /or improve user health, in some implementations. The powder may reduce inflammation (e.g. local}, inhibit and/or treat gingivitis, inhibit acid vein erosion, inhibit and/or treat periodontal disease, and/or otherwise improve oral health. Gargling and/or rinsing the mouth with mouthwash may remove food particles from the mouth (which are released, for example, when brushing with toothpaste), the

13 BE2021/5648 mouthwash spread through the mouth, reduce the number of unwanted bacteria and micro-organisms in the mouth and/or reduce inflammation. However, the composition may be administered via any suitable means of administration for oral administration, such as tablets (e.g., lozenges, chewable tablets and/or tablets for swallowing). The effect can be obtained by allowing the probiotics to dissolve in the probiotic composition in the mouth (e.g. by dissolving a tablet, chewing a tablet, circulating the probiotic in the mouth, etc.). The composition can optionally be be swallowed after being placed in the mouth.

According to a preferred form, the recommended daily dose of the powder is between 1 and 25 grams, preferably between 2 and 20 grams, more preferably between 3 and 10 grams, more preferably between 3 and 7 grams. The powder can be taken once or several times during the day. A daily dose ensures the optimal effect of the composition.

According to a preferred form, the composition comprises the following elements: - between 1 and 10 CFU/g Streptococcus Salivarius; - between 1 and 25 CFU/g Lactobacillus Rhamnosus; - between 1 and 10 CFU/g Lactobacillus Reuteri; - between 1 and 10 CFU/g Lactobacillus Paracasei; - between 25 and 750 g/kg of sorbitol - between 15 and 450 g/kg of xylitol; - between 1 and 20 g/kg of magnesium stearate; - between 1 and 20 g/kg silicon dioxide.

In a further aspect, the invention relates to a probiotic composition as described above for use of the probiotic composition for improving oral hygiene. According to a preferred form, the composition prevents and/or delays the formation of dental plaque, the occurrence of bad odors and the occurrence of dental caries. The invention relates to micro-organisms as oral care agents, in particular for the prevention of tartar, gingivitis, tooth sensitivity, as anti-caries agents and/or anti-oral odorants (halitosis). One of the main causes of tooth decay is caries. Due to the action of microorganisms colonizing the oral cavity, the tooth enamel is constantly weakened and eventually dissolved, leading to the formation of caries lesions. Such lesions, in turn, are suitable for

14 BE2021/5648 further colonization of micro-organisms, exacerbating the caries problem. Gingivitis or gingivitis is an inflammation of the gums.

Inflamed gums are often red, and can feel limp and swollen. It may bleed on a dental exam and may even bleed when brushing or eating. Healthy gums, on the other hand, have a pink color and lie firmly around the teeth. In addition, healthy gums do not bleed when brushing or eating. The inflammation in the gum line is caused by bacteria in the dental plaque. Dental plaque is a soft and sticky, barely visible layer of saliva proteins and bacterial mass (biofilm) on the teeth. Dental plaque can calcify into tartar that is firmly attached to the teeth. Gingivitis is not always clearly visible. Some characteristics that can occur are: red, flabby and swollen gums and/or bleeding gums when brushing. A bad taste or bad breath can also indicate gingivitis. Gingivitis rarely causes pain.

The inflammation in the gum line can spread towards the jaw bone.

As a result, the gums detach from the teeth and molars. Plaque forms again in the space that is created between the gums and the teeth, called the pocket. Because of this plaque, the inflammation moves even further to the depths and can spread to the bone and cause bone breakdown. This makes the pockets even deeper. This progressive inflammation with breakdown of fibers and jaw bone is called periodontitis. Once broken jaw bone cannot recover. A problem not directly related to caries formation is the development of oral malodors (halitosis). Halitosis is considered to be the result of unwanted microorganisms colonizing the oral cavity. However, so far no single microorganism has been identified as the primary cause of oral halitosis. However, it seems clear that caries formation and oral halitosis are independent processes, since oral halitosis can also occur in the absence of teeth and caries can occur in the absence of oral halitosis. Another problem of oral health is the formation of tartar. Calcified deposits on teeth are formed by microorganisms that colonize the tooth surfaces. Usually, within 7-10 days, calcified deposits on teeth have grown to the point that they are visible to the naked eye. Tartar forms a focal point that allows the colonization of the teeth by other microorganisms. This in turn can lead to the development of mouth odor, caries and inflammation of the gum tissue. There is therefore a need to prevent the development of tartar, for example by inhibiting the formation of tartar. The present invention substantially prevents and/or delays these problems.

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In a preferred form, the composition is taken daily. According to a further preferred form, the daily dose is between 1 and 25 grams. Preferably, the daily dose is between 2 and 20 grams, more preferably between 3 and 10 grams, more preferably between 3 and 7 grams. The composition can be taken once or several times during the day.

In a preferred form, the composition is compatible with the use of dental floss and/or a dental appliance. In the context of the present invention, the term "dental appliance" is understood to mean any appliance that can be placed in the oral cavity of a user and will perform a corrective, preventive and/or sterilizing function there. More specifically, in one embodiment, said dental appliance will include an orthodontic brace, a sports brace, a gingivitis brace, an orthodontic aligner, a temporary crown, a permanent crown, a bite plate, a denture, an antibacterial pad, a palatal splint, an antibacterial snoring device, a whitening shield, a bruxism splint, a coping, a fluoride shield, an individual impression tray, a michigan splint, a medication shield, a radiation protection splint, an occlusal splint, an interim prosthesis or similar device.

Dental floss, also known as floss or floss, consists of silk or nylon woven threads that are surrounded by a layer of wax. Dental floss serves to clean the interdental spaces (the spaces between two teeth) and prevent caries.

Preferably, the composition for use according to the present invention results in killing at least 70% of pathogenic bacteria within 24 hours of exposure. An exposure is defined here as the oral intake of the probiotic composition. The bacteriocidal effect of the composition results from the combination of the four selected probiotic bacteria in combination with sorbitol and/or xylitol added to the composition. The composition therefore provides a reduction in the number of live pathogenic bacteria through active killing, deprivation of sugars and/or nutrients, and competition in bacterial flora.

In a preferred form, the probiotic composition is bacteriocidal for pathogenic bacteria. Preferably, the composition kills at least 70% of pathogenic microorganisms within 24 hours of initial exposure. Preferably, the composition kills at least 20% of pathogenic microorganisms within 6 hours of initial exposure. An exposure is defined here as the oral intake of the probiotic composition. Since a healthy oral flora can

16 BE2021/5648 may be enhanced by the periodic use of antibacterial and antiseptic products, the use of oral compositions of the present invention may promote healthy oral flora, which may also promote healthy gut flora and/or provide other health benefits.

According to the present invention it is preferred that the concentration of viable pathogenic microorganisms is reduced by the treatment by at least 60%, 65% or 70%, and in particular by at least 75%, 80%, 85% % and more specifically by 86%, 87%, 88%, 89%, 90%.

According to one embodiment, the probiotic composition is manufactured in powder form, wherein a powder is soluble in water. According to a further embodiment, the probiotic composition is manufactured in tablet form, wherein a tablet comprises a melting tablet which is soluble in the mouth. The probiotic composition may also be provided in forms known in the art, such as, for example, a capsule, mouthwash, chewable tablet, gel, paste, lozenge.

Preferably, the probiotic composition can be dissolved in water and used as a mouthwash to rinse the mouth. Gargling and/or rinsing the mouth with mouthwash can remove food particles from the mouth (e.g. released when brushing with toothpaste), can spread the mouthwash throughout the mouth, can reduce the number of pathogenic microorganisms in the mouth and/or prevent and can reduce or counteract inflammation.

The composition for use can also be supplied as a tablet, thereby improving oral hygiene. The effect can be obtained by allowing the probiotics to dissolve in the probiotic composition in the mouth (e.g. by dissolving a tablet, chewing a tablet, and others). The composition may optionally be swallowed after being placed in the mouth.

In one embodiment, pathogenic bacteria belong to the genus Porphyromonas, Streptobacillus, Staphylococcus, Prevotella,

Fusobacterium, Aggregatibacter, Actinobacillus or Lactobacillus. The environment in the human mouth is suitable for the growth of characteristic microorganisms that occur there. It provides a source of water and nutrients, as well as a moderate temperature. Resistant microbes of the mouth adhere to the teeth and gums to resist mechanical lavage from the mouth to the stomach, where acid-sensitive microbes are destroyed by hydrochloric acid.

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Oral bacteria have evolved mechanisms to sense their environment and avoid or adapt to the host. Bacteria occupy the ecological niche formed by both the tooth surface and the gingival epithelium. However, a highly efficient innate defense system continuously controls bacterial colonization and prevents bacterial invasion of local tissues. There is a dynamic balance between dental plaque bacteria and the host's innate defense system. Of particular interest is the role of oral pathogenic microorganisms in the two major dental diseases: dental caries and periodontal disease. These pathogenic bacteria mainly belong to the genus

Porphyromonas, Streptobacillus, Staphylococcus, Prevotella, Fusobacterium,

Aggregatibacter, Actinobacillus or Lactobacillus. In addition, research has linked poor oral health to the resulting ability of the oral microbiota to enter the body and affect the heart and cognitive function.

In what follows, the invention is described by means of. non-limiting examples or figures illustrating the invention, and which are not intended or construed to limit the scope of the invention.

EXAMPLES

Example 1

Example 1 concerns an evaluation of the number of live and dead bacteria in a culture taken by means of a sample taken from the mouth of a patient. The analysis was done using Propidium lodide and SYBR-green. The analysis is done using fluorescence activated cell sorting (FACS).

bacterial strains

Porphyromonas gingivalis bacteria were used in this study. The bacteria were isolated through a sample taken from a patient.

Growing conditions

For culturing bacterial cells, Gibco® cell culture base medium became powder

M199 (without NaCO3) (Life Technologies, Carlsbad, CA) used as a chemically defined but rich medium without the addition of complex components buffered with phosphate. Additional nitrilotriacetic acid was added to prevent precipitation. Therefore, 800 mL of ultrapure water, 16 mL of 0.25 mM nitrilotriacetic acid solution (Sigma-Aldrich, Steinheim, Germany) in 0.6 M NaOH, and 25 mL of sodium potassium phosphate buffer (1.5 M NaH2PO4/K2HPO4, pH 7.0; Carl Roth) were ,

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Karlsruhe, Germany) mixed with the amount of powder indicated by the supplier and made up to 1 L with ultrapure water. Bacterial cells were cultured in shake flasks in 20 ml culture medium in 250 ml wide neck Erlenmeyer flasks in a humidified orbital shaking incubator (Kuehner, Birsfelden, Switzerland) at 37°C, rotation speed 200 rpm, eccentric radius 1.25 cm and relative humidity of 85% .

To optimize viability staining, cultures were prepared. The cells were harvested, centrifuged at 3,522 g for 10 min at 4°C (Heraeus® Multifuge 1S-R; Thermo Scientific, Waltham, WA), and washed with PBS (8 g/L NaCl, 0.2 g/L KCl , 1.15 g/L NaH2PO4, 0.2 g/L K2HPO4;

Karl Roth). Subsequently, the suspension was centrifuged again (3,522 g, 10 min, 4°C), and the cells were resuspended in 20 ml of fresh culture medium and cultured for 5 hours. Samples were taken from exponential and stationary growth phases at 2 or 5 hours, respectively.

For evaluation of viability in mixed cultures, cultures were prepared using cultures of the species as inoculums as described above. In contrast, the cultures were grown to the exponential growth phase for 1.5 hours. The cells were harvested and mixed in 50 ml of pre-warmed fresh culture medium for the mixed culture. The inoculation volume was adjusted for each species to an initial concentration of 0.5 x 107 cells/ml based on optical density (OD650) measurements (OD at 650 nm; Photometer Ultraspec 3000; Amersham Biosciences, Otelfingen, Switzerland). The cultures were then grown for 32 hours. In addition, pure cultures of each species were prepared to serve as controls in the same manner. All cultures were performed in three biological replicates.

Sample preparation before staining

Harvested cells were centrifuged at 4,700 g for 10 min at 4°C (Heraeus Fresco; Thermo Scientific) and resuspended in sodium chloride (NaCl) phosphate buffer. This buffer was prepared according to the conditions described by

Müller et al. (27) (0.4 M Na2HPO4/NaH2PO4, 0.15 M NaCl, pH 7.2; Carl Roth). For optimization of viability staining, the OD650 of the samples before staining was adjusted to 0.03 by dilution with NaCl-phosphate buffer to obtain defined cell concentrations for each species in the range of 107-108 cells/ml. Samples from 32 h cultures were diluted as necessary to an OD650 in the range of 0.01 to 0.04.

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NaCl-phosphate buffer was supplemented with 0.05 mg/ml glutaraldehyde (GTA) (50% grade |; Sigma-Aldrich), except for the buffer in the optimization of the staining of P. gingivalis in culture.

Viability Staining Procedure

Cell suspensions were stained with SYBR Green| (Life Technologies, Carlsbad, CA) and Propidium lodide (PI) (Sigma-Aldrich). Both dyes were added to the suspensions simultaneously and incubated in the dark at room temperature for 20 min. For staining, a 1 mg/ml working solution of PI was prepared in ultrapure water and stored at 4°C before use. SYBR Green | was used directly from the commercial stock solution after thawing (10,000 x concentrate in

DMSO, stored at -20°C) or after dilution in ultrapure water.

Flow cytometry

Flow cytometric analyzes were performed with a CyFlow® space flow cytometer (Partec, Münster, Germany) equipped with a 16 mW 375 nm UV diode laser and a 20 mW 488 nm argon solid state laser. Forward (FSC) and side scatter (SSC) were collected at 488 nm excitation. SSC was set as a discriminator to reduce electronic background noise during the analysis. SYBR Green I and Pl fluorescence were excited by a 488-nm laser and collected by a 527/30-nm bandpass filter or a 630-nm long pass filter, respectively. All signals were amplified logarithmically (four decades). The sampling rate was set to less than 1,000 particles per second.

The total number of particles was set to 20,000 events. Degassed ultrapure water was used as jacket fluid. The data were acquired with FloMax software (version 2.70; Partec). Stained cell suspensions were analyzed immediately after dye incubation. If necessary, samples were diluted with sheath fluid prior to staining.

Flow Cytometric Data Analysis

Data analysis, gating and compensation were performed with FlowJo software (version 7.6.4; Tree Star, Ashland, OR). The geometric mean value of the fluorescence intensity (MFI) was expressed as relative fluorescence units (RFU). SYBR Green I and Pl fluorescence signals were compensated for spectral overlap. Therefore, single-stained controls of SYBR Green | and PI (isopropanol-treated cells) processed according to the dye concentration used. Compensation

20 BE2021/5648 was determined and applied for data analysis on a day-to-day basis for the respective species and the applied concentration.

All gates used for population discrimination were set manually based on control samples. For viability determination, manually set gates were applied for each species based on the SYBR Green I/PI fluorescence of positive controls in culture. Only fluorescence signals from dead cells were detected. Viability gates were defined and applied separately for each exponential and stationary growth phase.

Results

The results are presented in figures 1 to 4.

Figure 1 shows a fluorescence activated cell sorting (FACS) profile in an untreated control condition. Porphyromonas gingivalis bacteria were cultured in basal LB medium for 6 hours, and dissociated, filtered and resuspended in 2 mL of PBS. Propidium lodide (concentration 0.5 uM) and SYBR-green (concentration 1 uM) were added to the solution. The bacteria were incubated for 30 minutes with Propidium lodide and SYBR-green. At least 200,000 cells were analyzed by fluorescence activated cell! sorting (FACS). Propidium lodide only stains cells with a compromised cell wall and which are moribund/dead. Propidium lodide emits light with a maximum wavelength of 617 nm and is detected in the red spectrum (in the settings of the FACS engine this is indicated in the PerCP-Cy5.5 channel). SYBR-green stains all cells (dead and alive). SYBR-green emits light with a maximum wavelength of 497 nm and is detected in the green spectrum (in the settings of the FACS machine this is indicated in the FITC channel). Consequently, dying/dead cells show a higher intensity in the PerCP-Cy5.5 channel (in the ordinate), and living cells show a higher intensity in the FITC channel (in the abscissa). Figure 1 shows that the majority of Porphyromonas gingivalis bacteria show a higher intensity in the FITC channel and therefore represent live bacteria (Live segment). Untreated bacteria are therefore alive and healthy. Segments 2-4 represent different degrees of cell death (low to high). Segment 1 represents debris.

Figure 2 shows a fluorescence activated cell sorting (FACS) profile for a condition treated with the composition described in of the present invention.

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Porphyromonas gingivalis bacteria were cultured in basal LB medium and exposed for 6 hours to the composition of the present invention (2

CFU/g Streptococcus Salivarius, minimum 5 CFU/g Lactobacillus Rhamnosus, minimum 2 CFU/g Lactobacillus Reuteri, minimum 2 CFU/g Lactobacillus Paracasei, sorbitol), and dissociated, filtered, and resuspended in 2 mL of PBS. Propidium lodide (concentration 0.5 uM) and SYBR-green (concentration 1 uM) were added to the solution. The bacteria were incubated for 30 minutes with

Propidium lodide and SYBR green. A minimum of 200,000 cells were analyzed by fluorescence activated cell sorting (FACS). Propidium lodide only stains cells with a compromised cell wall and which are moribund/dead. Propidium lodide emits light with a maximum wavelength of 617 nm and is detected in the red spectrum (in the settings of the FACS engine this is indicated in the PerCP-Cy5.5 channel). SYBR-green stains all cells (dead and alive). SYBR-green emits light with a maximum wavelength of 497 nm and is detected in the green spectrum (in the settings of the FACS machine this is indicated in the FITC channel). Consequently, dying/dead cells show a higher intensity in the PerCP-Cy5.5 channel (in the ordinate), and living cells show a higher intensity in the FITC channel (in the abscissa). Figure 2 shows that a significant proportion of Porphyromonas gingivalis bacteria exhibit higher intensity in the PerCP-Cy5.5 channel. These bacteria therefore have a higher absorption of

Propidium lodide and consequently represent dead bacteria (segment 4).

Segments 2-4 represent different degrees of cell death (low to high). Segment 1 represents debris. Bacteria are visible in segments 2 and 3.

These bacteria are dying. Only a limited number of live bacteria (Live segment) are present.

Figure 3 shows a histogram with a comparison between control condition, the composition according to the present invention and a composition described in the prior art. The histogram represents the quantification per condition of the number of bacteria in the different segments in the cell death analysis using

Propidium lodide and SYBR-green during the FACS analysis. In the control condition (Porphyromonas gingivalis bacteria in basal medium) more than 95% of the bacteria are alive (segment Live). In contrast, in the condition exposed to the composition of the present invention, more than 50% of the bacteria are dead (segment 4). In addition, about 25% of the bacteria are strongly moribund (segment 3) and 20% of the bacteria are weakly moribund (segment 2). Only a fraction of the bacteria are alive (Live segment). In a third condition

22 BE2021/5648 exposed to a composition according to the state of the art, only a fraction of the bacteria are dead (segment 4) or very moribund (segment 3).

In addition, about 50% of the bacteria are weakly moribund (segment 2) and 20% of the bacteria are cellular debris (segment 1). A significant proportion of the bacteria are alive (Live segment).

Figure 4 shows a fluorescence activated cell sorting (FACS) profile with a comparison between control condition, the composition according to the present invention and a composition described in the prior art. Porphyromonas gingivalis bacteria were cultured in basal LB medium and exposed for 6 hours to control medium, the composition of the present invention (2 CFU/g

Streptococcus Salivarius, minimum 5 CFU/g Lactobacillus Rhamnosus, minimum 2

CFU/g Lactobacillus Reuteri, minimum 2 CFU/g Lactobacillus Paracasei, sorbitol), and a composition according to the prior art. The bacteria were dissociated, filtered and resuspended in 2 mL of PBS. Propidium lodide (concentration 0.5 uM) and

SYBR-green (concentration 1 µM) was added to the solution. The bacteria were incubated for 30 minutes with Propidium lodide and SYBR-green.

A minimum of 200,000 cells were analyzed by fluorescence activated cell sorting (FACS). Propidium lodide only stains cells with a compromised cell wall and which are dying/dead. Propidium lodide emits light with a maximum wavelength of 617 nm and is detected in the red spectrum (in the settings of the FACS engine this is indicated in the PerCP-Cy5.5 channel).

SYBR-green stains all cells (dead and alive). SYBR-green emits light with a maximum wavelength of 497 nm and is detected in the green spectrum (in the settings of the FACS machine this is indicated in the FITC channel).

Consequently, dying/dead cells show a higher intensity in the PerCP-Cy5.5 channel (in the ordinate) and living cells show a higher intensity in the FITC channel (in the abscissa). The control condition showed that most of the

Porphyromonas gingivalis bacteria show a higher intensity in the FITC channel and therefore represent live bacteria (Live segment). Untreated bacteria were therefore alive and healthy. Segments 2-4 represent different degrees of cell death (low to high). Segment 1 represents cellular debris. The condition according to the present invention showed that a significant part of the Porphyromonas gingivalis bacteria show a higher intensity in the

PerCP-Cy5.5 channel. These bacteria consequently had a higher uptake of

Propidium lodide and consequently represent dead bacteria (segment 4).

Segments 2-4 represent different degrees of cell death (from low to

23 BE2021/5648 high). Segment 1 represents cellular debris. Bacteria were visible in segments 2 and 3. These bacteria are dying. Only a limited number of live bacteria (Live segment) were present. The prior art condition showed that a very limited proportion of the Porphyromonas gingivalis bacteria show a higher intensity in the PerCP-Cy5.5 channel. These bacteria consequently have a higher uptake of Propidium lodide and consequently represent dead bacteria (segment 4) or strongly dying bacteria (segment 3).

In addition, about half of the bacteria are weakly moribund (segment 2) and one-fourth of the bacteria are cellular debris (segment 1). A significant proportion of the bacteria were alive (Live segment).

Conclusions

The composition according to the present invention achieved a significant improvement in the bacteriocidal activity of a probiotic composition compared to the current state of the art. The number of pathogenic bacteria was dramatically reduced in the condition treated with the composition of the present invention, with more than half of the pathogenic bacteria dead after exposure to the composition. This was in contrast to the control condition where most of the bacteria were alive, and to the prior art composition where only a portion of the bacteria were moribund.

Example 2

A probiotic composition according to the present invention was used to prevent tooth decay.

The composition consisted of: - between 7 CFU/g Streptococcus Salivarius; - between 16 CFU/g Lactobacillus Rhamnosus; - between 7 CFU/g Lactobacillus Reuteri; - between 7 CFU/g Lactobacillus Paracasei; - between 50 g/100g of sorbitol - between 30,875 g/100g of xylitol; - between 1 g/100g magnesium stearate; - between 1g/100g silicon dioxide.

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The composition was formulated as a powder. The powder was then dissolved in water, 5 g of powder being dissolved in 10 ml of water. The dissolved composition was used as a mouthwash to rinse the oral cavity.

The number of subjects (8 subjects in total) was divided into 4 controls and 4 experimental subjects. The subjects were randomly assigned to males and females between the ages of 25 and 45 years.

Control and experimental subjects were blinded to the compound administered. The subjects took 5 grams of the compound twice a day (morning and evening). The powder was dissolved in water and the mouth was rinsed for 2 minutes. This was repeated for 7 consecutive days.

On day 8 after the start of the experiment, the subjects' mouths were examined by a licensed dentist.

Conclusions

Fewer signs of dental plaque were seen in the subjects receiving the composition of the present invention compared to the controls. In addition, signs of incipient tooth decay were detected in control subjects. However, none of the participants were diagnosed with tooth decay in the experimental subjects. No objective evaluation of the occurrence of bad breath (halitosis) was possible within the current set-up, but the overall evaluation of the participants receiving the composition of the present invention was more positive compared to the control subjects.

Claims (18)

25 BE2021/5648 CONCLUSIONS A probiotic composition for oral use, wherein the composition contains bacteria selected from Streptococcus Sp. and Lactobacillus Sp. characterized in that the composition comprises at least 2 CFU/g Streptococcus Salivarius, at least 5 CFU/g Lactobacillus Rhamnosus, at least 2 CFU/g Lactobacillus Reuteri and at least 2 CFU/g Lactobacillus Paracasei. Probiotic composition according to claim 1, characterized in that the composition is between 1 and 10 CFU/g Streptococcus Salivarius, between 1 and 25 CFU/g Lactobacillus Rhamnosus, between 1 and 10 CFU/g Lactobacillus Reuteri and between 1 and 10 CFU /g includes Lactobacillus Paracasei. A probiotic composition according to claim 1 or 2, characterized in that the composition comprises sorbitol. A probiotic composition according to any one of claims 1-3, characterized in that sorbitol is present in the composition in an amount between 25 g/kg and 750 g/kg of composition 5. Probiotic composition according to any one of the preceding claims 1-4, characterized in that the composition comprises an anti-caking agent such as silicon dioxide or tricalcium phosphate, preferably in an amount between 1 and 20 g/kg. 6. Probiotic composition according to any one of the preceding claims 1-5, characterized in that the composition comprises a lubricant such as for instance magnesium stearate, preferably in an amount between 1 and 20 g/kg. A probiotic composition according to any one of claims 1-6, characterized in that xylitol is present in the composition in an amount between 15 g/kg and 450 g/kg of composition A probiotic composition according to any one of the preceding claims 1-7, characterized in that the bacteria are present in the composition in lyophilized form or as live bacteria. 26 BE2021/5648 A probiotic composition according to any one of claims 1-8, characterized in that the composition is formulated as a capsule, tablet, powder, mouthwash, chewable tablet, gel, paste, lozenge, or powder. 10. Probiotic composition according to any one of the preceding claims 1-9, characterized in that the composition is a powder, more preferably a water-soluble powder. A probiotic composition according to any one of claims 1-8, characterized in that the recommended daily dose of the powder is between 1 and 25 grams, preferably between 2 and 20 grams, more preferably between 3 and 10 grams. grams, more preferably between 3 and 7 grams. A probiotic composition according to any one of the preceding claims 1-11, characterized in that the composition comprises: - between 1 and 10 CFU/g Streptococcus Salivarius; - between 1 and 25 CFU/g Lactobacillus Rhamnosus; - between 1 and 10 CFU/g Lactobacillus Reuteri; - between 1 and 10 CFU/g Lactobacillus Paracasei; - between 25 and 750 g/kg of sorbitol - between 15 and 450 g/kg of xylitol; - between 1 and 20 g/kg of magnesium stearate; - between 1 and 20 g/kg silicon dioxide. A probiotic composition according to any one of claims 1 to 12 for use in improving oral hygiene. A probiotic composition according to any one of claims 1 to 12 for the treatment and/or prevention of the formation of dental plaque, the occurrence of gingivitis, the occurrence of tooth sensitivity, the occurrence of bad odors in the oral cavity and/or dental caries. A probiotic composition for use according to claim 13 or 14, characterized in that the composition is taken daily. A probiotic composition for use according to any one of the preceding claims, characterized in that a daily dose is between 1 and 25 grams. 27 BE2021/5648 A probiotic composition for use according to any one of the preceding claims, characterized in that the composition is used together with dental floss or a dental appliance. A probiotic composition for use according to any one of the preceding claims, characterized in that the composition is bacteriocidal for pathogens in the oral cavity, pathogens being pathogenic bacteria of the genus Porphyromonas, Streptobacillus, Staphylococcus, Prevotella, Fusobacterium, Aggregatibacter, Actinobacillus and/or or Lactobacillus, and/or pathogenic fungi of the genus Candida, Cladosporium, Aspergillus, Fusarium, Glomus, Alternaria, Penicillium, and/or Cryptococcus.