JP7763473B2 - Oral hygiene products that kill oral bacteria, including periodontal disease bacteria, and inactivate viruses - Google Patents

Description

The present invention relates to oral hygiene products that inhibit the growth of and kill various bacteria present in the oral cavity that cause oral diseases such as periodontal disease and tooth decay, and also have the effect of inactivating viruses.In particular, the present invention relates to oral hygiene products that contain oral hygiene additives whose active ingredients are natural foodstuffs and extracts thereof, the safety of which has been confirmed through long years of human consumption or ingestion.

The human oral cavity is home to not only various opportunistic resident bacteria that cause various oral diseases, but also periodontal disease bacteria and "caries-related bacteria," which are bacteria that cause tooth decay by dissolving tooth tissue with acid.

Furthermore, the human oral cavity, throat including the trachea, and nasal cavities (collectively referred to in this application as the "oral cavity") are the digestive organs through which food and drink are first introduced into the body, and are also the entrance to the respiratory organs through which air (oxygen) is taken into the body. Therefore, they are the first places where bacteria, including various molds, and viruses are introduced into the body from the outside world.

Periodontal disease, the most serious oral disease, is said to affect approximately 80% of Japanese adults today. Periodontitis is often referred to as pyorrhea, a condition in which the periodontal tissues that support the teeth (gums, cementum, periodontal ligament, and alveolar bone) become inflamed by various bacteria, resulting in a variety of symptoms. When inflammation occurs only in the gums due to the multiple causative bacteria that cause periodontal disease, it is called gingivitis, while when the inflammation spreads to the periodontal tissues, such as the periodontal ligament and alveolar bone, it is called periodontal disease.

A glycoprotein, a component of saliva in the mouth, forms a highly adhesive film called a pellicle on the surface of the teeth. At least 500 species of bacteria lurking in the mouth, including Streptococcus mutans, a cavity-causing bacterium, and Porphyromonas gingivalis (Pg), a periodontal disease bacterium, adhere to this pellicle, forming bacterial clumps. These bacterial clumps, known as dental plaque, adhere to teeth if brushing and removal of oral bacteria are neglected. It is said that approximately 1 billion bacteria lurk in 1 mg of dental plaque.

Furthermore, it has been found that Streptococcus mutans, one of the bacteria that causes tooth decay, can be transmitted orally. However, the source of infection for periodontal disease bacteria such as Porphyromonas gingivalis (Pg), which causes periodontal disease, is not entirely clear, and in addition to oral infection, it has been pointed out that food-borne infection via the digestive tract, and droplet and airborne infection via the respiratory tract, including the mouth, nasal cavity, throat, and airways, are also possible.

Periodontal disease occurs when plaque accumulates in the periodontal pockets, which are the grooves between the gums and teeth. Bacteria such as Porphyromonas gingivalis (Pg) multiply and release toxins, causing inflammation of the surrounding gums and destroying the periodontal pockets, deepening them.

In the early stages of periodontal disease, similar to gingivitis, slight bleeding may occur when brushing your teeth, but it often progresses without you realizing it is periodontal disease, and by the time you notice it, the periodontal tissues - the gums, cementum, periodontal ligament, and alveolar bone - have been destroyed, resulting in the teeth no longer being able to support themselves and causing them to fall out.

In recent years, research papers from around the world have pointed out that periodontal bacteria not only cause oral diseases in the teeth, gums, and jaw area, which consists of the upper and lower jaws, as mentioned above, but also contribute to diseases such as diabetes, heart disease, and respiratory diseases, as well as Alzheimer's disease, the main cause of dementia, and premature birth.

Furthermore, research studies have examined the oral hygiene status of seriously ill patients and those who died from the novel coronavirus disease (COVID-19), which has become a global pandemic since 2020, and have found that a significant number of them had severe periodontal disease.

For this reason, it has been pointed out that viruses that cannot replicate themselves (nm-level size) may not directly infect human cells, but may instead infect and host bacteria that are orders of magnitude larger than viruses (μm-level) with their own genes, allowing the viral genes to multiply within the host bacteria, and then infecting the human body via these bacteria.

For these reasons, in order to suppress the growth of bacteria that cause periodontal disease and tooth decay in the oral cavity, it is extremely important to simultaneously kill these bacteria and inactivate the viruses within them. Simply suppressing or killing only the bacteria that cause periodontal disease temporarily will easily allow periodontal disease bacteria and caries-related bacteria to revive via various viruses that reside in or invade the oral cavity, making it difficult to suppress the growth of or kill these oral bacteria.

The oral cavity is connected to the nostrils and throat, which are respiratory organs, and is the first organ through which air (oxygen) is breathed into the body (lungs). It is also the first place susceptible to invasion by foreign substances from the outside world, such as various bacteria and viruses, including mold. The oral cavity is also connected to the esophagus, and food and drink ingested into the oral cavity travels through the esophagus to the stomach. Nutrients from food digested in the stomach are absorbed primarily in the small intestine, and water is absorbed in the large intestine.

Humans become infected with respiratory diseases such as pneumonia and bronchitis when the bacteria or viruses that cause them attach to the mucous membranes of the throat, nose, throat, or trachea through breathing, causing infection and the onset of the disease.

Furthermore, digestive diseases such as food poisoning in humans occur when viruses such as norovirus or mold that have become ingested in food or drink are taken up by the body's digestive organs via the mouth and esophagus.

For this reason, in order to prevent oral diseases, it is extremely important not only to suppress the growth and kill the bacteria that cause periodontal disease and tooth decay, but also to inactivate various viruses that have entered the oral cavity and trachea in order to maintain human health.

It has been confirmed that viral infections such as food poisoning caused by epidemic influenza, parainfluenza, norovirus, Middle East Respiratory Syndrome (MERS), viral acute bronchitis and pneumonia, measles, and more recently the novel coronavirus (SARS-CoV-2) that causes the novel coronavirus disease (COVID-19), which was recognized as a pandemic by the WHO in the spring of 2020, spread through droplets of saliva and nasal mucus in the mouth of an infected person when they cough or sneeze.

In addition, viruses can survive for a relatively long time when attached to objects or the surface of the human body in cold environments (low temperature and humidity). In particular, the low temperature of the human respiratory organs, including the throat, dries out the cilia in the lining of the upper respiratory tract and reduces ciliary activity (upper respiratory tract barrier function), making person-to-person infection more likely to spread than in warm environments. Therefore, it is extremely important to constantly suppress the growth of bacteria in the oral cavity and inactivate viruses therein.

In addition, from the perspective of preventing bacterial and viral infections, it has been proven through use cases and various experiments around the world that wearing a mask is effective in minimizing the range of virus droplets released into the air when coughing or sneezing, and in reducing the amount of droplets released into the outside atmosphere. Frequent hand washing and gargling are also important.

As mentioned above, removing bacteria, including mold, from the oral cavity and inactivating viruses not only prevents periodontal disease and tooth decay, but also respiratory diseases including pneumonia, diabetes, heart disease, and the worsening of diseases caused by viral infections.

In recent years, various dentifrices (dental pastes, liquid toothpastes, or dental rinses) have been sold that claim to remove mold and bacteria from the oral cavity and prevent periodontal disease and tooth decay. Furthermore, functional food compositions and pharmaceutical compositions for oral antibacterial use that have antibacterial effects against normal oral bacteria that cause periodontal disease, caries such as tooth decay, and opportunistic infections have also been proposed (see, for example, Patent Documents 1 and 2).

Japanese Patent Application Laid-Open No. 2020-62006 Japanese Patent Application Laid-Open No. 2008-182931 Japanese Patent Application Publication No. 9-48715

However, many of the dentifrices (dental pastes, liquid toothpastes, dental rinses) currently sold widely in Japan and overseas contain, for example, 1,3,3-trimethyl (1,8-thioneol), thymol, methyl salicylate, cetylpyridinium chloride, and other antiseptics that suppress the growth of bacteria that cause periodontal disease, as well as dipotassium glycyrrhizinate, which has the effect of soothing inflammation in the gums and mouth.

In addition, toothpastes (dental pastes, liquid toothpastes) containing antibiotics that inhibit certain bacteria that cause periodontal disease and tooth decay are also available on the market. However, these toothpaste agents with antibacterial properties are essentially disinfectants that are not ideal for daily intake into the body. If you brush your teeth after every meal using toothpaste containing such antibacterial ingredients, the ingredients will be absorbed into the body from the mouth via the esophagus and trachea, posing a long-term risk of causing unforeseen consequences to the human body.

Furthermore, if antibiotics that have an inhibitory effect on some of the bacteria that cause periodontal disease and tooth decay are used for a long period of time, bacteria and viruses that are resistant to those antibiotics will inevitably emerge, making them extremely difficult to remove and sterilize.

Furthermore, the oral antibacterial composition described in Cited Document 1, which has antibacterial activity against periodontal disease, caries-related bacteria, and resident oral bacteria that cause opportunistic infections, contains a mastic component as its main functional component, is a resin used in chewing gum and the like, and is an aromatic substance dissolved in a polyhydric alcohol resin acid ester, and is not itself a composition suitable for daily ingestion.

Furthermore, the substance disclosed in Patent Document 2 that is said to have the effect of inhibiting periodontal disease bacteria is an antibiotic that can cause resistance to some oral bacteria that cause periodontal disease, including microorganisms belonging to the Bifidobacterium genus. It is also difficult for many people to take such antibiotics on a daily basis, including the substance described in Reference 1. Furthermore, there are concerns about the safety and durability of the bactericidal activity of such antibiotics when taken on a daily basis.

Furthermore, Patent Document 3 lists many compositions extracted from red and brown algae extracts that exhibit antibacterial activity against Volphyromonas gingivalis. However, the test method described therein involves adding a bacterial strain cultured overnight to a liquid medium containing the composition, stirring the mixture, and culturing it in an anaerobic environment at a temperature of 37 degrees Celsius for three days. The growth inhibitory effect on the test bacteria was then confirmed by measuring the absorbance at a specific wavelength. Considering the daily time it takes to gargle (several tens of seconds) or brush teeth (several minutes to 10 minutes), the short-term use of 15 seconds or 30 to 60 seconds targeted by the present invention does not effectively inhibit or kill the growth of bacteria that cause oral disease, nor does it inactivate viruses.

The present invention aims to provide a long-term oral hygiene additive and oral hygiene product containing natural food ingredients and extracts thereof as active ingredients, whose safety has been confirmed through long-term human consumption and ingestion. These ingredients inhibit the growth of and kill multiple types of bacteria present in the oral cavity, respiratory tract, and upper digestive tract that cause oral diseases such as tooth decay and periodontal disease. They are also effective in inactivating various viruses, including epidemic influenza, parainfluenza, acute viral bronchitis and pneumonia, measles, and the novel coronavirus (SARS-CoV-2) that causes COVID-19. Furthermore, they are remarkably effective when left in the oral cavity for a short period of time, such as 15 to 60 seconds.

An additional objective of the present invention is to provide oral hygiene products that promote good saliva secretion, activate ciliary activity to eliminate viruses and bacteria that have entered the larynx and upper respiratory tract, and enhance the upper respiratory tract barrier function, while also sterilizing oral bacteria and inactivating viruses using natural foods or extracts thereof whose safety has been confirmed.

There are various problems to be solved by the oral hygiene product of the present invention, but they can be summarized as achieving the following objectives (1) to (3):
(1) When eaten or drunk on a daily basis, the texture and taste are not strange, and rather, the food contains umami components and is itself nutritious. Furthermore, even when ingested after coming into contact with sensitive mucous membranes in the human body, such as the throat, nostrils, oral cavity, or respiratory organs, including the bronchi, it does not cause any pain, discomfort, or problems with taste or smell.
(2) It is not a drug that kills specific bacteria, but an oral hygiene product that is safe even when used continuously. It enters the digestive organs such as the esophagus and stomach and intestines through the oral cavity, inactivating various viruses and sterilizing various bacteria that cause periodontal disease and dental caries by remaining in the oral cavity for only a short period of time, such as 30 to 60 seconds.
(3) It is not a therapeutic drug or medicine for treating a specific disease, and there is no risk of health safety issues even if it is taken/used for a long period of time. In addition, there is no risk of resistant viruses or bacteria developing.

Therefore, the present invention provides a toothpaste and oral hygiene products for the mouth, mouthwash, nasal cavity or throat, which comprise seaweed such as kelp, wakame, and Ulva, and/or one or more extracts from said seaweed, and which have the effect of inhibiting the growth of and killing Porphyromamonas gingivalis, which causes periodontal disease . The seaweed such as kelp, wakame, and Ulva, and/or the extracts from said seaweed, are particularly kelp powder, fucoidan powder, laminaran powder and iodine powder, and are characterized in that they are left in the mouth, nasal cavity or throat for at least 30 seconds before use .

Here, the toothpaste includes paste, liquid, and powder types, and the oral hygiene products and mouthwashes include dental rinses, mouthwashes, and oral inflammation medications.

Furthermore, the nasal hygiene products include nasal drops and nasal sprays, and the throat hygiene products include lozenges and throat sprays that are dissolved in the mouth.

These oral hygiene products include throat lozenges, chewing gum, and pacifier foods that remain in the mouth for a certain period of time to stimulate saliva production.

Each of the oral hygiene products described above stimulates saliva secretion and interacts with saliva by remaining in the mouth, nostrils, or throat for at least 30 to 60 seconds, enhancing their inactivation effect against viruses. Furthermore, these oral hygiene products not only kill bacteria that cause periodontal disease and dental caries, but also bactericidal bacteria such as E. coli and Staphylococcus aureus, thereby effectively preventing viruses that are unable to replicate in their own cells from infecting the human body via bacteria.

The viscous polysaccharide fucoidan is a storage polysaccharide found in seaweed and mushrooms, along with arginine, sodium alginate, laminaran, mannuronic acid, and guluronic acid. It is easily extracted with water, and most of its components are viscous components. It is a highly safe extract that has long been known to have anti-tumor, anti-thrombotic, and anti-hypertensive effects.

Mannitol is a type of sugar alcohol. It is a white, sweet, water-soluble crystal. It is widely found in nature, and is found in large amounts in foods such as kelp and mushrooms, and is also used as a food additive.

Furthermore, tests have confirmed that this oral hygiene product is even more effective when one or more of the following are added: citric acid, baking soda, iodine, aminolevulinic acid, L-glutamic acid, phloroglucinol (a polyphenol), and beta-carotene.

Citric acid and baking soda are nutrients that have been confirmed to be safe, and their refreshing acidity and carbon dioxide-generating action stimulate saliva secretion and they themselves have antibacterial properties. Furthermore, the refreshing acidity and mild irritation of citric acid and baking soda activate the mucous membranes and cilia that line the inner walls of the airways from the throat to the lungs.

Furthermore, saliva itself has bactericidal and antiviral effects, and it is believed that these bactericidal and antiviral effects of saliva are achieved by the bound sialic acid in saliva adsorbing bacteria and viruses before they reach receptors on the cell surfaces of the throat and upper respiratory tract, thereby preventing infection. In other words, since bacterial infections and the like are suppressed according to the amount of bound sialic acid in sublingual saliva, the intake of citric acid and baking soda, in combination with the products extracted from kelp or seaweed belonging to the brown algae and seaweed extracts of the present invention, suppresses the infectivity of bacteria and the like.

Furthermore, published test results have shown that when appropriate amounts of citric acid or baking soda are ingested, saliva secreted from multiple sites, such as the parotid gland, sublingual gland, and submandibular gland, increases the secretion of sublingual saliva, which is secreted from the palate of the tongue. This sublingual saliva is known to have stronger antibacterial and antiviral effects than other types of saliva.

The aminolevulinic acid added to this oral hygiene product is a starting material for the porphyrin synthesis pathway and is a substance used in the plastids of prokaryotes and eukaryotes. Recently, cell culture experiments using viruses with 5-aminolevulinic acid (5-ALA) have confirmed its effectiveness in inhibiting bacterial and viral infections. In the present invention, even more significant bacterial growth and viral infection suppression effects have been confirmed when used in combination with seaweed and its extracts.

In addition, the polyphenols added to this oral hygiene product are bitter and pigmenting components found in most plants. Similar to vitamin C and vitamin E, they are generally known to have strong antioxidant properties, converting harmful substances such as active oxygen into harmless substances and helping to prevent lifestyle-related diseases such as arteriosclerosis. However, when used in combination with seaweed and its extracts, this oral hygiene product has been shown to have a significant bacterial and viral inhibitory effect.

Beta-carotene not only has a bactericidal effect against oral bacteria, but is also converted into vitamin A, which helps maintain the health of the skin and mucous membranes in the body and contributes to the growth and differentiation of mucosal cells. Beta-carotene is also known to have antioxidant and immunostimulatory effects.

The seaweed extract in this oral hygiene product is produced by dissolving dried seaweed powder in saline or physiological saline, and the particle size of this dry powder is approximately 5 μm. The weight ratio of the dried seaweed powder to the saline or physiological saline solution is approximately 2%, and its extremely effective effects have been confirmed. Oral hygiene products using seaweed or its extract with the above particle size and weight ratio do not cause any discomfort when actually used in the oral cavity.

The oral hygiene product of the present invention inhibits bacterial growth and kills multiple types of bacteria present in the oral cavity, throat, respiratory tract, and upper digestive tract that cause oral diseases such as tooth decay and periodontal disease when used for approximately 30 to 60 seconds.It is also effective in inactivating various viruses, including epidemic influenza, parainfluenza, viral acute bronchitis and pneumonia, measles, and the novel coronavirus (SARS-CoV-2) that causes novel coronavirus disease (COVID-19).This makes it possible to provide oral hygiene products whose active ingredients are natural food ingredients and extracts thereof, the safety of which has been confirmed through human consumption over many years.

The results of a test (1) on the effectiveness of the oral hygiene product (oral cavity, throat including respiratory tract, and nostrils) according to the present invention against PED virus, a coronavirus whose morphology is similar to that of SARS-CoV-2, are shown. The following shows the results of a test (2) on the effectiveness of kelp, a type of seaweed that is the main ingredient of the oral hygiene product of the present invention, as well as the main components of seaweed, alginic acid, fucoidan, and iodine, and a mixture of alginic acid, fucoidan, and iodine, against the novel coronavirus SARS-CoV-2. The results of a test (3) on the effectiveness of the oral hygiene product of the present invention, in which alcohol is added as the main ingredient, against the PED virus are shown. The results of tests (4) on the effectiveness of the seaweeds kelp and wakame, which are the main ingredients of the oral hygiene product of the present invention, as well as the components of seaweed, iodine, arginine, and fucoidan, and the combined component of iodine, arginine, and fucoidan, against the PED virus are shown. The test results (5) show the bactericidal effect of the main component of the oral hygiene product of the present invention on Escherichia coli. 1 shows the test results (5) of the bactericidal effect of the main component of the oral hygiene product of the present invention on Staphylococcus aureus. The main ingredients of the oral hygiene product of the present invention, L-glutamic acid, β-carotene, Ulva, red wine (containing 14% alcohol and polyphenols), laminaran, and fucoidan, are shown in Table 6 as test results for their effectiveness against PED virus. The test results (7) of the effectiveness of the main component of the oral hygiene product of the present invention against influenza viruses are shown. The results of a test (8) on the effectiveness of the main ingredient of the oral hygiene product of the present invention against SARS-CoV-2, the new coronavirus, are shown. The results of a test (9) on the effects of kelp powder, a type of seaweed, its main component fucoidan powder, gyokuro tea leaf powder, and a mixture thereof, which constitute the oral hygiene product of the present invention, on Porphimonas gingivalis, a bacterium that causes periodontal disease, are shown. The results of a test (10) on the effects of kelp powder, a type of seaweed that constitutes the oral hygiene product of the present invention, its main component fucoidan powder, gyokuro tea leaf powder (gyokuro powder), and a mixture of these on Porphimonas gingivalis, a bacterium that causes periodontal disease, are shown.

The main ingredient of the oral hygiene product of the present invention is derived from a product extracted from kelp or seaweed belonging to the brown algae family, and seaweed extract.

Therefore, before describing the detailed description of the preferred embodiment of the invention, we will briefly describe kelp and its components.

Kelp belongs to the seaweed family, which is a general term for algae that grow in the sea. Unlike seaweed, algae do not flower, but reproduce through spores. Most seaweed is edible.

Seaweed is classified by color into species such as cyanobacteria, diatoms, green algae, brown algae, and red algae, with kelp belonging to the brown algae category. However, the color of kelp depends on the depth of the water where it grows, in other words, the amount of sunlight that reaches it; it is green in shallow waters, brown in deeper areas, and red in areas with the least amount of light. Many of the brown algae that kelp belongs to include wakame, hijiki, mozuku, and mekabu, which are used as food. Green laver belongs to the green algae category, while funori and amanoiri belong to the red algae category.

Kelp, a brown seaweed, is a species of seaweed that has been identified in 14 genera and 45 species in Japan. Of these, kelp, a cold-water brown seaweed, is found along the Pacific coast north of Miyagi Prefecture and throughout Hokkaido, with Hokkaido being the main production area. Most of the domestic kelp production is harvested in Hokkaido.

Kelp contains arginine, alginic acid, and fucoidan, a unique mucilaginous polysaccharide found only in seaweed. Arginine is known to have blood pressure lowering properties, while fucoidan is known to have anti-thrombosis and cancer-preventing properties.

Fucoidan is a type of sulfated polysaccharide. It is a dietary fiber found in large amounts in the mucilage of brown algae such as kelp, wakame (including mekabu, a part of it), and mozuku. Similar substances have also been found in animals such as sea cucumbers, which have antiviral properties and are described in the aforementioned Patent Document 1.

The arginine, alginic acid, and fucoidan contained in kelp are compounds consisting of tens to hundreds of thousands of L-fucose (polysaccharide) molecules linked together via A1-2 and A1-4 bonds, with an average molecular weight of approximately 200,000. Fucoidan is classified into U-fucoidan, which contains glucuronic acid; F-fucoidan, which consists only of sulfated fucose; and G-fucoidan, which contains galactose.

Unlike mushrooms (such as agaricus) and other polysaccharide (sugar chain) components, L-fucose has a sulfate group bound to it. This L-fucose is found in large amounts in brown algae (such as mozuku, mekabu, kombu, akamoku, and Sargassum species such as seaweed), and is often described as the sticky component of seaweed.

In contrast, arginine is an acidic polysaccharide composed of two types of uronic acid called mannuronic acid (M) and guluronic acid (G). The hardness and elasticity of arginine vary greatly depending on the ratio of (M) and (G) bonds, so it is used in a variety of applications, including as an ingredient in puddings, jellies, ice cream, and jams, for emulsifying yogurt and cheese, and for artificial salmon roe. In kelp, it exists in a jelly state, bound to calcium and magnesium.

Arginine is known to have the following physiological actions (1) to (3).
(1) Blood pressure lowering effect Excessive salt intake disrupts the balance of sodium and potassium ions in the blood, causing blood vessels to constrict and resulting in an increase in blood pressure. In particular, when arginine bound to potassium is ingested with food, the arginine separates the potassium with gastric acid. It then travels to the intestines, where it binds with the sodium ions contained in the food and is carried out of the body. Meanwhile, potassium separated from arginine becomes potassium ions, is absorbed by the intestines, and has the function of expelling sodium from the blood. In this way, arginine has a dual function of lowering blood pressure.
(2) Activating the activity of digestive enzymes When arginine is taken with food, it promotes digestion by increasing the activity of digestive enzymes such as amylase and protease in the intestine.
(3) Ability to remove harmful substances When harmful substances and pollutants accumulate in the body, they can cause various abnormalities and diseases. In an experiment in which arginine was given to laboratory animals contaminated with radioactive strontium, it was reported that arginine had the effect of excreting the radioactive element from the body.

By the way, kelp is rich in dietary fiber, the main components of which are arginine and fucoidan, which have a different chemical structure than the dietary fiber found in vegetables and grains.

As mentioned above, these two polysaccharides are contained in the slimy components of kelp. In addition to dietary fiber, dried kelp also contains amino acids that create umami and mannitol, which has a sweet taste. It also contains minerals such as magnesium and calcium, as well as iodine, making it an excellent food for nutritional supplementation.

Fucoidan, a slippery substance found in kelp and other seaweed, is said to have numerous biological activities, including anticoagulant effects, cell adhesion inhibitory effects, anti-inflammatory effects, cell protection from viral infections, and antitumor effects.

Iodine is an essential mineral for the human body, found mainly in kelp and other foods, and has long been used as the main ingredient in mouthwash to reduce inflammation in the back of the mouth and trachea. However, the amount of iodine required by the human body is said to be 0.095-0.13 mg per day (equivalent to 40-60 mg in kelp), so if you are consuming it on a daily basis, you must be careful not to exceed this limit.

Fucoidan, a water-soluble dietary fiber and slimy component found in kelp, is said to slow the movement of ingested food from the stomach to the small intestine. When digestion slows down in the stomach, fucoidan uses sulfate groups to stimulate the stomach lining.

The antiviral or virus inactivating agent containing the product extracted from kelp or seaweed and seaweed extract of the present invention may be part of this function of fucoidan.

In other words, it is possible that the fucoidan and its components contained in kelp stimulate the mucosal immune function in the intestines, thereby contributing to an increase in the defensive power of immune cells throughout the body. For example, when fucoidan is held by M cells present in the human body, lymphocytes (NK cells, T cells, B cells) attack it, NK cells, a type of lymphocyte involved in immunity, are activated, and when activated lymphocytes enter the bloodstream, they promote the secretion of antibodies, thereby boosting immunity.

Furthermore, in the present invention, it is preferable to produce the product by adding citric acid to seaweed and seaweed extracts. Here, citric acid is an organic compound found in citrus fruits and is a hydroxy acid. It has a refreshing sour taste and is widely used as a food additive, so there are no safety issues.

Citric acid also inhibits phosphofructokinase activity in the glycolytic pathway and is one of the factors that regulate the influx from glycolysis to the citric acid cycle. Because it indirectly breaks down lactic acid in muscles in the citric acid cycle, it was once said to have a post-exercise fatigue-reducing effect. This is because citric acid also forms a chelate complex with calcium, which is considered a fatigue-causing substance. This calcium binding is generally important in the trade-off with reducing acidosis in lactic acid breakdown, and so it may be effective in reducing fatigue.

Depending on age, approximately 1,000 to 1,500 mL of saliva is secreted per day. It has also been confirmed that the amount of saliva secreted decreases as people get older. This makes elderly people more susceptible to viral and bacterial infections, and if they do become infected, they are more likely to develop serious symptoms.

For this reason, what is extremely important in relation to the present invention is that the sour taste of citric acid stimulates saliva secretion when eaten.

Most of the water in saliva is secreted by the parotid and submandibular glands, with additional saliva from minor salivary glands (palatine, lingual, buccal, labial, molar, and Ébner's glands). At rest, 60-70% of saliva comes from the submandibular gland. Saliva from the submandibular and sublingual glands is excreted from the area under the tongue (floor of the mouth).

On the other hand, when we eat citric acid, its taste stimulating effect promotes saliva secretion. Saliva has been shown to have functions such as digestion, oral self-cleaning, protecting the oral mucosa, regulating pH, and even antibacterial and antiviral effects. In this way, the large amount of saliva produced by citric acid and the antiviral effects of saliva suppress the amount of virus and the prolonged infectiousness of the virus.

Furthermore, in the present invention, it is preferable to produce the product by adding baking soda to seaweed and seaweed extracts. As mentioned above, baking soda is a highly safe food additive that has traditionally been consumed as baking powder, a weakly alkaline food ingredient used to make cookies, pancakes, etc. rise.

Baking soda, made in accordance with the provisions of the Food Sanitation Act, is sometimes used as a medicine as an antacid to treat excess stomach acid. Because gastric juice contains hydrochloric acid, the sodium bicarbonate that makes up baking soda breaks down, producing carbon dioxide bubbles. These bubbles stimulate the taste buds and stomach, promoting the secretion of more saliva and gastric juices.

I. [Confirmation Test (1)]
In light of the above, the applicant of the present application first conducted a test (1) using an external testing institution to examine the inactivation effect of the seaweed constituting the oral hygiene product for the mouth, nasal passages or throat according to the present invention on the PED virus, which is the coronavirus most similar in form to SARS-CoV-2.

Note that I. [Confirmation Test (1)] to VIII. [Confirmation Test (8)], which will be described in order below in this application, were commissioned by the applicant to external, authoritative testing organizations, and this application publishes the reports on the first through eighth test results from those testing organizations without any manipulation or evaluation of the test results. However, because the virus reduction numbers for the test materials showing the confirmation test results are presented in logarithmic notation to the base 10, a table has been added in which the logarithmic values are converted to decimal notation to make the numbers easier to understand.

(A) Test Materials: Seaweeds and Seaweed Extracts Test Material I: Seaweeds and seaweed extracts were dissolved in physiological saline to prepare a 2% solution.

Test Material II: Seaweeds and seaweed extracts were dissolved in boiling water (100°C) in physiological saline to a concentration of 2%, and then cooled before use. Sterile phosphate buffer solution was used as the control material.

(B) Test Microorganisms The test microorganism (virus) used was the P-5V strain of PED virus (Porcine epidemic diarrhea virus).

This PED virus (Porcine epidemic diarrhea virus) is commonly known as the swine infectious disease virus. The pathogenic PED virus belongs to the Alphacoronavirus genus of the Coronaviridae family. It has spikes that protrude radially (like a crown or corona) from the surface of its envelope, and its genome is positive-sense single-stranded RNA. It is the type of coronavirus that is most similar to the novel coronavirus (COVID-19) that caused the 2020 pandemic.

It has been scientifically proven that viruses belonging to the Alphacoronavirus genus of the Coronaviridae family have corona-like spikes that protrude radially from the surface of the envelope, at the tips of which they attach to the surface of cell membranes that make up the human body, allowing the virus to penetrate the human body and ultimately lead to infection.

Therefore, since the inactivation effect against PED virus, which belongs to the Alphacoronavirus genus of the Coronaviridae family, was confirmed in the above-mentioned confirmatory test of the present application, it is expected that there is a very high possibility that the same inactivation effect will also be exerted against the novel coronavirus (COVID-19).

Vero cells were used to culture PED virus (Porcine epidemic diarrhea virus). Vero cells are derived from kidney epithelial cells of African green monkeys and are a cell line used for cell culture. Along with Hela cells, they are one of the most commonly used cell lines.

(C) Setting of Groups For the test groups, 0.1 mL of the virus solution was added to 1 mL of the above-mentioned test material (a 2% solution of seaweed and seaweed extract dissolved in physiological saline), and the sensitization time was set to 1 minute after the start of the test.

For the control group, 0.1 mL of virus solution was simply added to 1 mL of phosphate buffer without adding any of the above test materials, and the sensitization times were 0 and 1 minute after the start of the test.

(D) Test method: The test was carried out with reference to "Virus Experiments, General Theory, Revised Second Edition, Maruzen Co., Ltd., Virus Neutralization Test Method."

(E) Test Procedure (E-1) Preliminary Test:
Prior to the test, the effects of the test materials on cultured cells (cytotoxicity) were investigated.

The test material was serially diluted 10-fold with phosphate buffer and then inoculated into cultured cells. The highest concentration showing normal cell condition after culture was confirmed, and the virus concentration to be used in the test was determined. As a result, poor cell growth was confirmed in the 100-fold solution of the test material for cytotoxicity. Therefore, it was determined that the test material and virus solution mixture must be diluted 100-fold or more before inoculation into the cells. For this reason, the virus concentration added was set at 10 ⁶ TCID ₅₀ /mL or more.

(E-2) Main test: Mixing of test solutions According to the test category, 1 mL each of the test material and phosphate buffer solution was taken, and the virus solution was added to the concentration determined in the preliminary test.

After adding the virus solution, the mixture was left to stand at room temperature (25°C) for the specified time.

(E-3) Main Test: Cell Inoculation and Bacterial Count Measurement After sensitization for each test category, the mixture was serially diluted 10-fold, and 100 μL of each was inoculated onto cells cultured on a 96-well plate.

After culturing at 37°C in 5% carbon dioxide gas for five days, the cultured cells were observed under a microscope, and the presence or absence of viral growth was confirmed by the appearance of CPE (cytopathic encephalopathy) in the cultured cells, and the viral concentration was calculated.

(F) Results Figure 1 shows the test results (1) of the effectiveness of the main components of the oral hygiene product according to the present invention against PED virus, a coronavirus whose morphology is similar to that of SARS-CoV-2. Table 1 summarizes the test results.

Note that the vertical axis in Figure 1, as well as the subsequent Figures 2 to 7, is expressed as an exponent of 10, meaning that each step down represents a tenth.

In the control group (comparison test group without test materials), no change in the viral load was observed from the start of the test until 1 minute after the start of the test (10 ^6.1 TCID ₅₀ /mL).

On the other hand, in the test group, the concentration was <10 ^3.5 TCID ₅₀ /mL (below the detection limit: a reduction of 99.7% or more) one minute after the start of treatment. This result of "below the detection limit: a reduction of 99.7% or more" indicates an astonishingly high virus inactivation effect.

(G) Discussion: This time, we conducted a test to see if the test material could inactivate the PED virus (Porcine Epidemic Diarrhea Virus), commonly known as the swine coronavirus. The results showed that the material had an astonishing viral inactivation effect of over 99.7% within one minute of contact.

As mentioned above, the genome of the PED virus (Porcine epidemic diarrhea virus) is a positive-stranded single-stranded RNA, and since it is the type of coronavirus most similar to the novel coronavirus (COVID-19), it was predicted that it would also have a significant inactivation effect on the novel coronavirus SARS-CoV-2.

II. [Confirmation test (2)]
Having confirmed the remarkable antiviral and virus inactivation effects of the seaweed in the oral hygiene product of the present invention against the PED virus, the applicant of the present application then conducted a test to confirm its effectiveness against the novel coronavirus "SARS-CoV-2." The details of this test are described below.

(A) Test materials: Seaweed and seaweed extracts Test material 1: 2% kelp powder with a particle size of 5 μm in physiological saline solution Test material 2: 2% kelp powder with a particle size of 5 μm in physiological saline solution (prepared by dissolving in boiling water)
Test material 3: 2% arginine powder in saline solution Test material 4: 2% fucoidan powder in saline solution (prepared by dissolving in boiling water)
Test material 5: 2% iodine powder in saline solution (prepared by dissolving in boiling water)
Test material 6: 2% (arginine + fucoidan + iodine) powder solution in physiological saline. Sterile phosphate buffer solution was used as a control material.

(B) Test Microorganisms The test microorganism (virus) used was SARS-CoV-02 (novel coronavirus). This SARS-CoV-02 is a human isolate that was isolated from saliva and cultured using Vero cells, and amplification of the SARS-CoV-2 gene was confirmed using real-time PCR (Ministry of Health, Labour and Welfare notification method).

The cultured cells, Vero cells, are an established cell line derived from the kidney epithelium of African green monkeys.

(C) Setting of Groups In the control group, 0.1 mL of the virus solution was added to 1 mL of phosphate buffer, and the sensitization time was set to 0 seconds and 60 seconds after the start of the test.

For the test group, 0.1 mL of virus solution was added to 1 mL of the above-mentioned test material, and the sensitization times were 15, 30, and 60 seconds after the start of the test.

(D) Test method: The test was carried out with reference to "Virus Experiments, General Theory, Revised Second Edition, Maruzen Co., Ltd., Virus Neutralization Test Method."

(E) Test Procedure (E-1) Preliminary Test:
Prior to the test, the effects of the test materials on cultured cells (cytotoxicity) were investigated.

The test material was serially diluted 10-fold with phosphate buffer and then inoculated into cultured cells. The highest concentration that showed a normal state of the cells after culture was confirmed, and the virus concentration to be used in the test was determined.

The results of the cytotoxicity tests are shown in the table below. Cell growth was confirmed even with a maximum 10x dilution of the test material. Therefore, it was determined that the test material and virus solution mixture should be diluted 10x or more before inoculation into the cells. The virus concentration added was set at ¹⁰ TCID ₅₀ /mL or more.

(E-2) Main test: Mixing of test solutions According to the test category, 1 mL each of the test material and phosphate buffer solution was taken, and the virus solution was added to the concentration determined in the preliminary test.

After adding the virus solution, the mixture was left to stand at room temperature (25°C) for the specified time.

(E-3) Main Test: Cell Inoculation and Bacterial Count The mixtures after sensitization for each test section were serially diluted 10-fold, and 100 μL of each was inoculated onto cells cultured on a 96-well plate.

After culturing the cells at 37°C in 5% carbon dioxide gas for five days, the cells were observed under a microscope to confirm the presence or absence of viral growth based on the appearance of CPE (cytopathic encephalopathy) in the cultured cells, and the viral concentration was calculated.

(F) Results Figure 2 shows the test results for the effectiveness of kelp, a type of seaweed that is the main ingredient of the oral hygiene product of the present invention, as well as the main components of seaweed, alginic acid, fucoidan, and iodine, and a mixture of alginic acid, fucoidan, and iodine, against the novel coronavirus SARS-CoV-2. Tables 2A, 2B, and 2C summarize the details of the test results shown in Figure 2. Table 2C is an easier-to-understand version of Table 2B.

In the control group, no change in the viral load was observed from the start of the test until 60 seconds after the start of the test (10 ^6.5 TCID ₅₀ /mL).

Meanwhile, in test area 1, the virus infectivity was reduced by 99.7% at 15 seconds and 99.9% at 60 seconds after the start; in test area 2, the virus infectivity was reduced by 99.7% at 15 seconds and 99.9% at 60 seconds; in test area 3, the virus infectivity was reduced by 59.3% at 15 seconds and 93.7% at 60 seconds; in test area 4, the virus infectivity was reduced by 97.5% at 15 seconds and 99.7% at 60 seconds; in test area 5, the virus infectivity was reduced by 90.0% at 15 seconds and 99.0% at 60 seconds; and in test area 6, the virus infectivity was reduced by 98.4% at 15 seconds and 99.8% at 60 seconds.

The viral infectivity titers for SARS-CoV-02 mentioned above were generally close to the detection limit, and this test confirmed an astonishingly high viral inactivation effect.

Furthermore, since both the seaweed and seaweed extract are 2% solutions and the particle size of the kelp powder is 5 μm, it has been found that the present invention can be used without discomfort when applied to oral hygiene products, including mouthwashes, cleansers for the oral cavity, nose, or throat.

(G) Discussion: We conducted a test to determine the inactivation effect of test materials on SARS-CoV-2. The results showed that a maximum contact time of 60 seconds resulted in an inactivation effect of 93.7-99.9%.

III. [Confirmation test (3)]
Furthermore, the applicant of the present application conducted a test to confirm the effect of adding alcohol to the main ingredient of the oral hygiene product of the present invention on the PED virus. The details are described below.

(A) Test materials: Seaweed and seaweed extracts Test material 1: 2% kelp powder with a particle size of 5 μm in physiological saline solution Test material 2: 2% kelp powder with a particle size of 5 μm in physiological saline solution (prepared by dissolving in boiling water)
Test material 3: 1% kelp powder particle size 5 μm saline solution (prepared by dissolving in boiling water)
Test material 4: 0.5% kelp powder particle size 5 μm saline solution (prepared by dissolving in boiling water)
Test material 5: 25% alcohol content barley shochu diluted 3 times with purified water (prepared by dissolving in boiling water)
Test material 6: 25% 5% alcohol kelp shochu diluted 3 times with purified water (prepared by dissolving in boiling water)
Sterile phosphate buffer was used as a control material.

(B) Test Microorganisms The test microorganism (virus) used was the P-5V strain of PED virus (Porcine epidemic diarrhea virus).

The cultured cells are Vero cells (a cell line derived from the kidney epithelium of African green monkeys).

(C) Setting of Groups In the test groups, 0.1 mL of the virus solution was added to 1 mL of the above-mentioned test material, and the sensitization time was set to 15 seconds, 30 seconds, and 60 seconds after the start of the test.

For the control group, 0.1 mL of virus solution was added to 1 mL of phosphate buffer solution, and the sensitization times were 0 seconds and 60 seconds after the start of the test.

(D) Test method: The test was carried out with reference to "Virus Experiments, General Theory, Revised Second Edition, Maruzen Co., Ltd., Virus Neutralization Test Method."

(E) Test Procedure (E-1) Preliminary Test:
Prior to the test, the effects of the test materials on cultured cells (cytotoxicity) were investigated.

The test material was serially diluted 10-fold with phosphate buffer and then inoculated into cultured cells. The highest concentration that showed a normal state of the cells after culture was confirmed, and the virus concentration to be used in the test was determined.

The results of the cytotoxicity tests are shown in the table below. Cell growth was confirmed even with a maximum 10x dilution of the test material. Therefore, it was determined that the test material and virus solution mixture should be diluted 10x or more before inoculation into the cells. The virus concentration added was set at ¹⁰ TCID ₅₀ /mL or more.

(E-2) Main test: Mixing of test solutions According to the test category, 1 mL each of the test material and phosphate buffer solution was taken, and the virus solution was added to the concentration determined in the preliminary test.

After adding the virus solution, the mixture was left to stand at room temperature (25°C) for the specified time.

(E-3) Main Test: Cell Inoculation and Bacterial Count The mixtures after sensitization for each test section were serially diluted 10-fold, and 100 μL of each was inoculated onto cells cultured on a 96-well plate.

After culturing the cells at 37°C in 5% carbon dioxide gas for five days, the cells were observed under a microscope to confirm the presence or absence of viral growth based on the appearance of CPE (cytopathic encephalopathy) in the cultured cells, and the viral concentration was calculated.

(F) Results The test results for PED virus are shown in Figure 3 and Tables 3A, 3B, and 3C. Table 3C is an easier-to-understand representation of Table 3B.

In the control group, no change in the viral load was observed from the start of the test until 60 seconds after the start of the test (10 ^6.7 TCID ₅₀ /mL).

On the other hand, in test area 1, the virus infectivity was reduced by 99.7% at 15 seconds and 99.9% at 60 seconds after the start, in test area 2 by 99.7% at 15 seconds and 99.9% at 60 seconds, in test area 3 by 99.8% at 60 seconds, in test area 4 by 97.4% at 60 seconds, in test area 5 by 93.6% at 60 seconds, and in test area 6 by 97.4% at 60 seconds after the start.

(G) Discussion: We conducted a test to determine the inactivation effect of test materials on PED virus (porcine infectious coronavirus). The results showed that a maximum contact time of 60 seconds resulted in an inactivation effect of 93.6 to 99.9%.

IV. [Confirmation Test (4)]
Furthermore, the applicant of the present application has conducted tests to confirm the effects on PED virus of the seaweeds kelp and wakame, which are the main ingredients of the oral hygiene product of the present invention, as well as the main components of seaweeds, iodine, arginine, and fucoidan, and also the mixed component of iodine, arginine, and fucoidan. The details of these tests are described below.

(A) Test materials: Seaweed and seaweed extracts Test material 1: 2% kelp powder in physiological saline solution Test material 2: 2% kelp powder in physiological saline solution (prepared by dissolving in boiling water)
Test material 3: 2% kelp powder aqueous solution Test material 4: 2% wakame powder saline solution Test material 5: 2% iodine powder saline solution Test material 6: 2% arginine powder saline solution Test material 7: 2% fucoidan powder saline solution Test material 8: 2% (iodine + fucoidan + arginine powder) saline solution Sterile phosphate buffer solution was used as a control material.

(B) Test Microorganisms The test microorganism (virus) used was the P-5V strain of PED virus (Porcine epidemic diarrhea virus) (a pig-infectious coronavirus).

The cultured cells are Vero cells (a cell line derived from the kidney epithelium of African green monkeys).

(C) Setting of Groups In the test groups, 0.1 mL of the virus solution was added to 1 mL of the above-mentioned test material, and the sensitization time was set to 15 seconds, 30 seconds, and 60 seconds after the start of the test.

For the control group, 0.1 mL of virus solution was added to 1 mL of phosphate buffer solution, and the sensitization times were 0, 15, 30, and 60 seconds after the start of the test.

(D) Test method: The test was carried out with reference to "Virus Experiments, General Theory, Revised Second Edition, Maruzen Co., Ltd., Virus Neutralization Test Method."

(E) Test Procedure (E-1) Preliminary Test:
Prior to the test, the effects of the test materials on cultured cells (cytotoxicity) were investigated.

The test material was serially diluted 10-fold with phosphate buffer and then inoculated into cultured cells. The highest concentration that showed a normal state of the cells after culture was confirmed, and the virus concentration to be used in the test was determined.

The results of the cytotoxicity tests are shown in the table below. Even at a maximum dilution of 100 times the test material, poor cell growth was confirmed. Therefore, it was determined that the test material and virus solution mixture should be diluted 100 times or more before inoculating the cells. The virus concentration added was set at 10 ⁶ TCID ₅₀ /mL or more.

(E-2) Main test: Mixing of test solutions According to the test category, 1 mL each of the test material and phosphate buffer solution was taken, and the virus solution was added to the concentration determined in the preliminary test.

After adding the virus solution, the mixture was left to stand at room temperature (25°C) for the specified time.

(E-3) Main Test: Cell Inoculation and Bacterial Count The mixtures after sensitization for each test section were serially diluted 10-fold, and 100 μL of each was inoculated onto cells cultured on a 96-well plate.

After culturing the cells at 37°C in 5% carbon dioxide gas for five days, the cells were observed under a microscope to confirm the presence or absence of viral growth based on the appearance of CPE (cytopathic encephalopathy) in the cultured cells, and the viral concentration was calculated.

(F) Results The test results for PED virus are shown in Figure 4 and Table 4A, Table 4B, and Table 4C. Table 4C is a table that makes Table 4B easier to understand.

In the control group, no change in the viral load was observed from the start of the test until 60 seconds after the start of the test (10 ^6.5 TCID ₅₀ /mL).

On the other hand, the virus infectivity reduction was 99.8% at 30 seconds after the start in test area 1, over 99.9% at 30 seconds after the start in test area 2, 99.7% at 60 seconds after the start in test area 3, 98.4% at 60 seconds after the start in test area 4, 98.4% at 60 seconds after the start in test area 5, 59.3% at 60 seconds after the start in test area 6, 99.0% at 60 seconds after the start in test area 7, and 99.5% at 60 seconds after the start in test area 8.

(G) Discussion: We conducted a test to determine the inactivation effect of test materials on PED virus (porcine infectious coronavirus). The results showed that a maximum contact time of 60 seconds resulted in an inactivation effect of 59.3 to 99.9%.

V. [Confirmation Test (5)]
Furthermore, the applicant of the present application conducted a test to confirm the bactericidal effect of the main component of the oral hygiene product of the present invention against Escherichia coli and Staphylococcus aureus, the details of which are described below.

(A) Test materials: Seaweed and seaweed extracts Test material 1: 2% kelp powder in physiological saline solution Test material 2: 2% kelp powder in physiological saline solution (prepared by dissolving in boiling water)
Sterile saline was used as a control material.

(B) Test Microorganisms Escherichia coli (ATCC117775) and Staphylococcus aureus (ATCC6538) were used as test microorganisms.

The above microorganisms were pre-cultured in a nutrient medium and adjusted to a concentration of about 10 ⁸ CFU/mL with sterilized purified water to prepare a test bacterial solution.

(C) Setting of Groups In the test groups, 0.1 mL of the test bacterial solution was added to 10 mL of the control material, and the sensitization time was set to 30 seconds and 60 seconds after the start of the test.

For the control group, 0.1 mL of test bacteria solution was added to 10 mL of test material, and the sensitization times were 0, 30, and 60 seconds after the start of the test.

(D) Test method: The test was carried out with reference to JIS Z 2801 (antibacterial processed products - antibacterial test method, disinfection effect) and the carbolic acid coefficient method.

(E) Test Procedure (E-1) Microbiological Test Method (Measurement of Bacterial Count in Test Solution)
The test solution was diluted appropriately with sterile physiological saline and cultured on nutrient agar medium and each selective medium (Escherichia coli: desoxycholate agar medium, Staphylococcus aureus: egg yolk-added mannitol salt medium). The culture was carried out under aerobic conditions at 35°C for 24 to 48 hours, and the number of colonies that grew after the culture was counted to obtain the bacterial count.

(E-2) Test Method The test material and the control material were placed in a sterilized test tube, and 0.1 mL of the test bacteria solution was added to 10 mL of the material and mixed well.

In accordance with the test settings, the number of remaining viable bacteria was measured immediately after mixing and after reacting at room temperature for a certain period of time according to the microbiological testing method.

(F) Results [E. coli]
The test results are shown in Table 5A.

In the control area, the number remained the same from the start to the end of the test, at 640,000 CFU/mL. 60 seconds after the start of the test, the number in test area 1 was 360,000 CFU/mL (a 43.7% decrease), and in test area 2 it was 320,000 CFU/mL (a 50.0% decrease).

[Staphylococcus aureus]
The test results are shown in Table 5B.

In the control area, the number remained roughly the same from the start to the end of the test, at 2,300,000 CFU/mL. 60 seconds after the start of the test, the number in test area 1 was 1,100,000 CFU/mL (a 52.1% decrease), and in test area 2 it was 1,600,000 CFU/mL (a 30.4% decrease).

(G) Discussion As a result of the test, a 43.7% reduction in Escherichia coli was confirmed 60 seconds after contact with Test Material 1, and a 50.0% reduction was confirmed with Test Material 2. Furthermore, a 52.1% reduction in Staphylococcus aureus bacteria was confirmed 60 seconds after contact with Test Material 1, and a 30.4% reduction was confirmed with Test Material 2.

VI. [Confirmation Test (6)]
Furthermore, the applicant conducted tests to confirm the effects of L-glutamic acid, β-carotene, red wine (containing 14% alcohol and polyphenols), laminaran, and fucoidan, which are additives added to the main component of the oral hygiene product of the present invention, on the PED virus. The details are described below.

(A) Test materials: seaweed, seaweed extracts, and additives. Test material 1: 2% L-glutamic acid powder in physiological saline (prepared as a boiling solution).
Test material 2: 2% beta-carotene powder in saline solution (prepared by dissolving in boiling water)
Test material 3: 2% Ulva microparticle powder in physiological saline solution (prepared by dissolving in boiling water)
Test material 4: 14% alcohol red wine solution Test material 5: 1% kelp 5μm powder 14% alcohol red wine solution (prepared by dissolving in boiling water)
Test material 6: 1% laminaran powder in saline solution (prepared by dissolving in boiling water)
Test material 7: Fucoidan aqueous solution. Sterile phosphate buffer solution was used as a control material.

(B) Test Microorganisms The test microorganism (virus) used was the P-5V strain of PED virus (Porcine epidemic diarrhea virus) (a pig-infectious coronavirus).

The cultured cells are Vero cells (a cell line derived from the kidney epithelium of African green monkeys).

(C) Setting of Groups In the test groups, 0.1 mL of the virus solution was added to 1 mL of the above-mentioned test material, and the sensitization time was set to 15 seconds, 30 seconds, and 60 seconds after the start of the test.

For the control group, 0.1 mL of virus solution was added to 1 mL of phosphate buffer solution, and the sensitization times were 0 seconds and 60 seconds after the start of the test.

(D) Test method: The test was carried out with reference to "Virus Experiments, General Theory, Revised Second Edition, Maruzen Co., Ltd., Virus Neutralization Test Method."

(E) Test Procedure (E-1) Preliminary Test:
Prior to the test, the effects of the test materials on cultured cells (cytotoxicity) were investigated.

The test material was serially diluted 10-fold with phosphate buffer and then inoculated into cultured cells. The highest concentration that showed a normal state of the cells after culture was confirmed, and the virus concentration to be used in the test was determined.

The results of the cytotoxicity tests are shown in Table 6A. Cell growth was poor even at a maximum concentration of 10 times the test material. Therefore, it was determined that the test material and virus solution mixture should be diluted 10 times or more before inoculation into the cells. The virus concentration added was set at ¹⁰ TCID ₅₀ /mL or more.

(E-2) Main test: Mixing test solutions According to the test category, 1 mL of each test material and phosphate buffer solution was taken, and the virus solution was added to the concentration determined in the preliminary test. After adding the virus solution, the mixture was allowed to stand at room temperature (25°C) for the specified time.

(E-3) Main Test - Cell Inoculation After sensitization for each test section, the mixture was serially diluted 10-fold, and 100 μL of each was inoculated onto cells cultured in a 96-well plate.

After culturing the cells at 37°C in 5% carbon dioxide gas for five days, the cells were observed under a microscope to confirm the presence or absence of viral growth based on the appearance of CPE (cytopathic encephalopathy) in the cultured cells, and the viral concentration was calculated.

(F) Results The test results are shown in Figure 6 and Tables 6A, 6B, and 6C. Table 6C is a table that makes Table 6B easier to understand.

In the control group, no change in the viral load was observed from the start of the test until 60 seconds after the start of the test (10 ^6.5 TCID ₅₀ /mL).

In test area 1, the virus infectivity was reduced by 37.5% at 15 seconds and 90.0% at 60 seconds after the start of testing; in test area 2, the virus infectivity was reduced by 75.3% at 15 seconds and 90.0% at 60 seconds; in test area 3, the virus infectivity was reduced by 59.3% at 15 seconds and 93.7% at 60 seconds; in test area 4, the virus infectivity was reduced by 37.5% at 15 seconds and 99.7% at 60 seconds; in test area 5, the virus infectivity was reduced by 99.7% at 15 seconds and 99.9% at 60 seconds; in test area 6, the virus infectivity was reduced by 95.9% at 15 seconds and 99.3% at 60 seconds; and in test area 7, the virus infectivity was reduced by 98.4% at 15 seconds and 99.8% at 60 seconds.

(G) Discussion: We conducted a test to determine the inactivation effect of test materials on PED virus (porcine infectious coronavirus). The results showed that a maximum contact time of 60 seconds resulted in an inactivation effect of 90.0 to 99.9%.

VII. [Confirmation Test (7)]
Furthermore, the applicant has conducted the following tests to confirm the effectiveness of the main ingredients of the oral hygiene product of the present invention against influenza viruses, the details of which are described below.

(A) Test materials: Seaweed and seaweed extracts Test material 1: 2% kelp powder with a particle size of 5 μm in physiological saline solution Test material 2: 2% kelp powder with a particle size of 5 μm in physiological saline solution (prepared by dissolving in boiling water)
Sterile phosphate buffer was used as a control material.

(B) Test Microorganisms The test microorganism (virus) used was influenza virus (swine influenza virus H1N1 IOWA strain).

The cultured cells are MDCK cells (a cell line derived from canine kidney).

(C) Setting of Groups In the test groups, 0.1 mL of the virus solution was added to 1 mL of the above-mentioned test material, and the sensitization time was set to 15 seconds, 30 seconds, and 60 seconds after the start of the test.

For the control group, 0.1 mL of virus solution was added to 1 mL of phosphate buffer solution, and the sensitization times were 0 seconds and 60 seconds after the start of the test.

(D) Test method: The test was carried out with reference to "Virus Experiments, General Theory, Revised Second Edition, Maruzen Co., Ltd., Virus Neutralization Test Method."

(E) Test Procedure (E-1) Preliminary Test:
Prior to the test, the effects of the test materials on cultured cells (cytotoxicity) were investigated.

The test material was serially diluted 10-fold with phosphate buffer and then inoculated into cultured cells. The highest concentration that showed a normal state of the cells after culture was confirmed, and the virus concentration to be used in the test was determined.

As a result, poor cell growth was confirmed in all 10-fold solutions of the test materials. Therefore, it was found that the test required diluting the mixture of test material and virus solution by 10 times or more before inoculating the cells. The virus concentration added was set to ¹⁰ TCID ₅₀ /mL or more.

(E-2) Main test: Mixing test solutions According to the test category, 1 mL of each test material and phosphate buffer solution was taken, and the virus solution was added to the concentration determined in the preliminary test. After adding the virus solution, the mixture was allowed to stand at room temperature (25°C) for the specified time.

(E-3) Main Test: Cell Inoculation After sensitization for each test category, the mixture was serially diluted 10-fold, and 100 μL of each was inoculated onto cells cultured in a 96-well plate.

After culturing the cells at 37°C in 5% carbon dioxide for five days, the culture supernatant from each well was collected, and the presence or absence of viral growth was confirmed by hemagglutination, and the viral concentration was calculated.

(F) Results The test results are shown in Figure 7 and Tables 7A and 7B.

In the control group, no change in the viral load was observed from the start of the test until 60 seconds after the start of the test (10 ^6.9 TCID ₅₀ /mL).

In test area 1, the virus infectivity was reduced by 99.3% at 15 seconds and 99.9% at 60 seconds after the start of testing, while in test area 2 the virus infectivity was reduced by 99.3% at 15 seconds and 99.9% at 60 seconds.

(G) Discussion: We conducted a test to determine the inactivation effect of test materials on influenza viruses. The results showed that contact for 15 to 60 seconds resulted in an inactivation effect of 99.3 to 99.9%.

VIII. [Confirmation Test (8)]
Based on the results of the above-mentioned [Confirmation Test (1)] to [Confirmation Test (7)], the applicant further conducted Confirmation Test (8) to confirm in more detail the inactivation effect of kelp and its extracts against viruses, including SARS-CoV-2.

Figure 8 shows the test results (8) of the effectiveness of the main ingredient of the oral hygiene product of the present invention against SARS-CoV-2, a novel coronavirus.

(A) Test materials: Seaweed and seaweed extracts Test material 1: 2% kelp powder, particle size 5 μm, in physiological saline solution Test material 2: 2% kelp powder, particle size 5 μm, in physiological saline solution Test material 3: 2% kelp powder, particle size 5 μm, in physiological saline solution (prepared by boiling)
Test material 4: 2% kelp powder particle size 5 μm saline solution (prepared by boiling water solution)
Test material 5: 2% kelp powder particle size 5 μm saline solution (prepared by boiling water solution)
Test material 6: 2% kelp powder particle size 5 μm physiological saline solution (prepared by boiling water solution)
Test material 7: 2% kelp powder particle size 5 μm saline solution (prepared by boiling water solution)
Test material 8: 2% laminaran powder in saline solution Test material 9: 2% phloroglucinol powder in saline solution (prepared by boiling)
Test material 10: 2% Eriocheir powder in saline solution (prepared by dissolving in boiling water)
As a control material, sterilized phosphate buffer solution was used.

By the way, the above-mentioned "Test Materials 1," "Test Materials 2," and "Test Materials 3" to "Test Materials 7" are the same, but this was done to confirm the variation in effects under the same test conditions and to understand the reliability of the test results.

(B) Test Microorganisms SARS-CoV-02 (novel coronavirus) was used as the test microorganism (virus). SARS-CoV-02 is a human isolate that was isolated and cultured from saliva using Vero cells, and amplification of the SARS-CoV-2 gene was confirmed using real-time PCR (Ministry of Health, Labour and Welfare Notification Method). The cultured cells used here were Vero cells (a cell line derived from the kidney epithelium of African green monkeys).

(C) Setting of Groups In the control group, 0.1 mL of the virus solution was added to 1 mL of phosphate buffer, and the sensitization time was set to 0 seconds and 60 seconds after the start of the test.

For the test group, 0.1 mL of virus solution was added to 1 mL of the above-mentioned test material, and the sensitization times were 15, 30, and 60 seconds after the start of the test.

(D) Test method: The test was carried out with reference to "Virus Experiments, General Theory, Revised Second Edition, Maruzen Co., Ltd., Virus Neutralization Test Method."

(E) Test Procedure (E-1) Preliminary Test:
Prior to the test, the effects of the test materials on cultured cells (cytotoxicity) were investigated.

The test material was serially diluted 10-fold with phosphate buffer and then inoculated into cultured cells. The highest concentration that showed a normal state of the cells after culture was confirmed, and the virus concentration to be used in the test was determined.

The results of the cytotoxicity tests are shown in the table below. Cell growth was confirmed even with a maximum 10x dilution of the test material. Therefore, it was determined that the test material and virus solution mixture should be diluted 10x or more before inoculation into the cells. The virus concentration added was set at ¹⁰ TCID ₅₀ /mL or more.

(E-2) Main test: Mixing of test solutions According to the test category, 1 mL each of the test material and phosphate buffer solution was taken, and the virus solution was added to the concentration determined in the preliminary test.

After adding the virus solution, the mixture was left to stand at room temperature (25°C) for the specified time.

(E-3) Main Test: Cell Inoculation and Bacterial Count The mixtures after sensitization for each test section were serially diluted 10-fold, and 100 μL of each was inoculated onto cells cultured on a 96-well plate.

After culturing the cells at 37°C in 5% carbon dioxide gas for five days, the cells were observed under a microscope to confirm the presence or absence of viral growth based on the appearance of CPE (cytopathic encephalopathy) in the cultured cells, and the viral concentration was calculated.

(F) Results The test results for SARS-CoV-02 are shown in Figure 8, Table 8A, Table 8B, Table 8C, and Table 8D. Tables 8C and 8D are an easier-to-understand version of Table 8B.

In the control group, no change in the viral load was observed from the start of the test until 60 seconds after the start of the test (10 ^6.3 TCID ₅₀ /mL).

On the other hand, in test area 1, the results were 99.3% at 15 seconds and 99.9% at 60 seconds after the start, in test area 2 it was 99.6% at 15 seconds and 99.9% at 60 seconds, in test area 3 it was 99.7% at 15 seconds and 99.7% at 60 seconds, in test area 4 it was 99.3% at 15 seconds and 99.9% at 60 seconds, in test area 5 it was 99.6% at 15 seconds and 99.9% at 60 seconds, and in test area 6 it was 99.6% at 15 seconds and 99.9% at 60 seconds. In test area 1, the virus infectivity was reduced by 99.3% in 15 seconds and 99.9% in 60 seconds; in test area 7, the virus infectivity was reduced by 99.6% in 15 seconds and 99.9% in 60 seconds; in test area 8, the virus infectivity was reduced by 90.0% in 15 seconds and 96.0% in 60 seconds; in test area 9, the virus infectivity was reduced by 96.0% in 15 seconds and 98.4% in 60 seconds; and in test area 10, the virus infectivity was reduced by 99.0% in 15 seconds and 99.6% in 60 seconds.

The detailed results of I. [Confirmation Test (1)] to VIII. [Confirmation Test (8)] above are summarized in Tables 9 to 12D.

Table 9 summarizes the virus inactivation effect of each seaweed component.

As shown in Table 9, the main components of the seaweed that constitute the main ingredient of the oral hygiene product of the present invention have been confirmed to have a significant inactivation effect (reduction in virus count) on the novel coronavirus "SARS-CoV-2" that causes COVID-21, and the "PED virus," which is similar to "SARS-CoV-2." Based on this, it is expected that the same significant inactivation effect will also be exerted on mutated strains of the novel coronavirus "SARS-CoV-2."

Table 10 summarizes the virus inactivation effect (virus reduction rate) for SARS-CoV-2, PEDV (PED virus), and influenza virus. Additionally, the number of tests was increased for saline solution containing 2% kelp powder with a particle size of 5 μm, and saline solution containing a boiled solution of the same 2% kelp powder with a particle size of 5 μm, and the variation in the inactivation effect was confirmed.

As shown in Table 10, similar to Table 10 above, it has been confirmed that the major components of the seaweed that constitute the main ingredient of the oral hygiene product of the present invention have a significant inactivation effect (drastically reduced virus count) against SARS-CoV-2, the novel coronavirus that causes COVID-21, influenza viruses, and PED viruses similar to SARS-CoV-2. Based on this, it is easy to predict that they will also have a similarly significant inactivation effect against various types of influenza viruses, such as the Hong Kong and Soviet strains, and various mutant strains of SARS-CoV-2.

Table 11 summarizes the inactivation effect of saline solution containing 2% powder microparticles of several types of seaweed (kelp, wakame, sea lettuce, and sea lettuce) on the PED virus.

As shown in Table 11, it was confirmed that it has a significant inactivation effect (dramatic reduction in virus count) on the PED virus, which is similar to the novel coronavirus SARS-CoV-2 that causes COVID-21.

Table 12A summarizes the inactivation effect of seaweed powder, the main ingredient of the oral hygiene product of the present invention, on PED viruses, which are similar to SARS-CoV-2.

As shown in Table 12A, similar to Table 10 above, it has been confirmed that seaweed powder, which is the main ingredient of the oral hygiene product of the present invention, has a significant inactivation effect (dramatic reduction in virus count) on the PED virus, which is similar to SARS-CoV-2.

Table 12B summarizes the inactivation effects of the major ingredients and various additives that make up the seaweed, which is the main ingredient of the oral hygiene product of the present invention, on the novel coronavirus "SARS-CoV-2" that causes COVID-21 and the "PED virus," which is similar to "SARS-CoV-2."

As shown in Table 12B, it has been confirmed that the main ingredients and various additives that make up the seaweed, which is the main ingredient of the oral hygiene product of the present invention, have a significant inactivation effect (drastically reduced virus count) on the novel coronavirus "SARS-CoV-2" that causes COVID-21 and the "PED virus," which is similar to "SARS-CoV-2."

Table 12C summarizes the inactivation effect of alcoholic beverages containing kelp components on the PED virus, which is similar to the novel coronavirus SARS-CoV-2 that causes COVID-21.

As shown in Table 12C, as has long been known, it has been confirmed that wine containing alcohol and kelp components has a significant inactivation effect (drastically reducing the number of viruses) on the PED virus, which is similar to the novel coronavirus SARS-CoV-2 that causes COVID-21.

Table 12D summarizes the results of the bactericidal effect of seaweed powder, the main ingredient of the oral hygiene product of the present invention, against Escherichia coli and Staphylococcus aureus.

As shown in Table 12D, it was confirmed that the seaweed powder, which is the main ingredient of the oral hygiene product of the present invention, has a significant bactericidal effect against Escherichia coli and Staphylococcus aureus. From this, it can be understood that the seaweed powder, which is the main ingredient of the oral hygiene product of the present invention, has a bactericidal effect against other bacteria that cause food poisoning.

Table 13 summarizes the inactivation effect (dramatic reduction in virus count) of the major ingredients and additives that make up the seaweed, which is the main ingredient of the oral hygiene product of the present invention, on the novel coronavirus "SARS-CoV-2" that causes COVID-21 and the "PED virus," which is similar to "SARS-CoV-2."

As shown in Table 13, it has been confirmed that the main ingredients and additives that make up the seaweed, which is the main ingredient of the oral hygiene product of the present invention, have a significant inactivation effect (drastically reduced virus counts) on the novel coronavirus "SARS-CoV-2" that causes COVID-21, and the "PED virus," which is similar to "SARS-CoV-2."

Table 13 also summarizes the inactivation effect of seaweed powder, the main ingredient of the oral hygiene product of the present invention, and its ingredients against viruses such as SARS-CoV-2, the novel coronavirus that causes COVID-21, PED virus, which is similar to SARS-CoV-2, and norovirus, as well as the bactericidal effect against Escherichia coli and Staphylococcus aureus.

As shown in Table 13, it has been confirmed that the seaweed powder and its components, which are the main ingredients of the oral hygiene product of the present invention, not only have a significant inactivating effect on viruses such as SARS-CoV-2, the novel coronavirus that causes COVID-21, PED virus, which is similar to SARS-CoV-2, and norovirus, but also have a bactericidal effect on Escherichia coli and Staphylococcus aureus.

(G) Discussion: We conducted a test to determine the inactivation effect of test materials on SARS-CoV-2. The results showed that a maximum contact time of 60 seconds resulted in an inactivation effect of 96.0 to 99.9%.

IX. [Confirmation Test (9)]
Based on the detailed test results of the above-mentioned [Confirmation Test (1)] to [Confirmation Test (8)], the applicant of the present application conducted "Confirmation Test (9) for inhibiting the growth of and killing the causative bacteria that cause periodontal disease or dental caries," which is the problem that the present invention aims to solve.

Figure 9 shows the results of a test (9) on the effectiveness of kelp powder, a type of seaweed that constitutes the oral hygiene product of the present invention, its main component fucoidan powder, gyokuro tea leaf powder (gyokuro powder), and a mixture of these against Porphimonas gingivalis, a bacteria that causes periodontal disease.

(A) Test materials: Seaweed and seaweed extracts Test material 1: 2% kelp powder with a particle size of 5 μm in physiological saline solution (prepared by boiling water)
Test material 2: Fucoidan water (prepared by dissolving in boiling water)
Test material 3: 2% fucoidan powder in physiological saline solution (prepared by boiling water)
Test material 4: 2% gyokuro powder in physiological saline solution (prepared by boiling water solution)
Test material 5: 2% gyokuro powder + kelp powder particle size 5 μm physiological saline solution (prepared by boiling water solution)
Test material 6: 2% gyokuro powder + 2% fucoidan powder particle size 5 μm physiological saline solution (prepared by boiling water solution)
2% kelp powder particle size 5μm saline solution (prepared by boiling water solution)
Sterile saline was used as a control material.

(B) Test microorganism (bacteria)
Porphyromonas gingivalis ATCC33277 was used as the donor microorganism (bacteria), and this test microorganism (bacteria) was pre-cultured in a blood medium and adjusted to a concentration of approximately 10 ⁸ cfu/mL with sterilized purified water to prepare a test bacterial solution.

(C) Setting of Groups In the control group, 0.1 mL of the test bacterial solution was added to 10 mL of the control material, and the sensitization time was set to 0, 30, and 60 seconds after the start of the test.

For test areas 1-6, 0.1 mL of test bacteria solution was added to 10 mL of test material, and the sensitization time was set at 30 and 60 seconds after the start of the test.

The sensitization times were set at 30 and 60 seconds based on the estimated time that the oral hygiene product according to the present invention, such as toothpaste, remains in the oral cavity.

(D) Test method: The test was carried out with reference to "JIS Z 2801 (antibacterial processed products, antibacterial testing method, bactericidal effect)" and the carbolic acid coefficient method.

(E) Test Procedure (E-1) Microbiological Test Method (Measurement of Bacterial Count in Test Solution)
The test solution was diluted appropriately with sterile saline and cultured on a blood agar medium. The culture was carried out under anaerobic conditions at 35°C for 10 days, and the number of colonies that grew after the culture was counted to determine the bacterial count.
(E-2) Test Method The test material and the control material were placed in a sterilized test tube, and 0.1 mL of the test bacteria solution was added to 10 mL of the material and mixed thoroughly.

According to the test settings, the number of remaining viable bacteria was measured immediately after mixing and after reacting at room temperature for a certain period of time according to the microbiological testing method.
(F) Results The test results of the above-mentioned confirmation test (9) are shown in Figure 9, Table 14, Table 15A, and Table 15B. Tables 15A and 15B are tables that make the test results shown in Table 14 easier to understand.

Test Results As shown in Table 14, in the control group, the number of CFU/mL was approximately the same from the start to the end of the test, at 1.2×10 ⁶ CFU/mL.

On the other hand, the results for test areas 1-6 were as follows 60 seconds after the start of the test:

Test area 1: 5.3 x 10 ⁵ CFU/mL (55.8% reduction)
Test area 2: 6.8 x 10 ⁵ CFU/mL (43.3% reduction)
Test area 3: 7.8 x 10 ⁵ CFU/mL (35.0% reduction)
Test area 4: 1.0 x 10 ⁶ CFU/mL (16.6% reduction)
Test area 5: 8.8 x 10 ⁵ CFU/mL (26.6% reduction)
Test area 6: 1.1 x 10 ⁶ CFU/mL (8.3% reduction)
The above values are the average values of three trials.

(G) Discussion As a result of the above test, the test materials were confirmed to be effective in reducing the number of Porphyromonas gingivalis bacteria, and it was determined that a reduction of 8.3 to 55.8% was achieved within 60 seconds of contact with each test material.

X. [Confirmation Test (10)]
Based on the test results of the above-mentioned [Confirmation Test (9)], the applicant further conducted a "Confirmation Test (10) on the inhibition of proliferation and sterilization of the causative bacteria that cause periodontal disease or dental caries," which is the problem that the present invention aims to solve.

Figure 10 shows the results of a test (10) on the effectiveness of kelp powder, a type of seaweed that constitutes the oral hygiene product of the present invention, its main component fucoidan powder, gyokuro tea leaf powder (gyokuro powder), and a mixture of these against Porphimonas gingivalis, a bacterium that causes periodontal disease.

(A) Test materials: seaweed, seaweed extracts, and additives. Test material 1: 5% kelp powder, particle size 5 μm, physiological saline solution (prepared by dissolving in boiling water). Test material 2: 0.5% isopropyl methylphenol physiological saline solution.
Test material 3: 2% iodine powder in saline solution (prepared by dissolving in boiling water)
Test material 4: 2% iodine powder in saline solution (prepared by dissolving in boiling water)
Test material 5: 4% fucoidan powder in saline solution (prepared by dissolving in boiling water)
Test material 6: 2% laminaran powder in saline solution
Test material 7: 2% phloroglucinol (polyphenol) powder in saline solution (prepared as 217488N3 solution for boiling water)
Test material 8: 2% sodium alginate powder in physiological saline solution (prepared by boiling water solution)
Test material 9: 2% erythritol powder in physiological saline solution (prepared by boiling water solution)
Test material 10: 2% mannitol powder in physiological saline solution (prepared as a boiling solution)
Sterile saline was used as a control material.

(B) Test microorganism (bacteria)
Porphyromonas gingivalis ATCC33277 was used as the donor microorganism (bacteria), and this test microorganism (bacteria) was pre-cultured in a blood medium and adjusted to a concentration of approximately 10 ⁸ cfu/mL with sterilized purified water to prepare a test bacterial solution.

(C) Setting of Groups In the control group, 0.1 mL of the test bacterial solution was added to 10 mL of the control material, and the sensitization time was set to 0, 30, and 60 seconds after the start of the test.

For test areas 1-10, 0.1 mL of test bacteria solution was added to 10 mL of test material, and the sensitization time was set at 30 and 60 seconds after the start of the test.

The sensitization times were set at 30 and 60 seconds because this is the estimated time that the oral hygiene product according to the present invention, such as toothpaste, will remain in the oral cavity.

(D) Test method: The test was carried out with reference to "JIS Z 2801 (antibacterial processed products, antibacterial testing method, bactericidal effect)" and the carbolic acid coefficient method.

(E) Test Procedure (E-1) Microbiological Test Method (Measurement of Bacterial Count in Test Solution)
The test solution was diluted appropriately with sterile saline and cultured on a blood agar medium. The culture was carried out under anaerobic conditions at 35°C for 10 days, and the number of colonies that grew after the culture was counted to determine the bacterial count.
(E-2) Test Method The test material and the control material were placed in a sterilized test tube, and 0.1 mL of the test bacteria solution was added to 10 mL of the material and mixed thoroughly.

According to the test settings, the number of remaining viable bacteria was measured immediately after mixing and after reacting at room temperature for a certain period of time according to the microbiological testing method.
(F) Results The test results of the above confirmation test (9) are shown in FIG. 10 and Table 16.

Test Results As shown in Table 16, in the control group, the number of CFU/mL was approximately the same from the start to the end of the test, at 1.2×10 ⁶ CFU/mL.

On the other hand, the results for test areas 1-6 were as follows 60 seconds after the start of the test:

Test area 1: 5.2 x 10 ⁵ CFU/mL (56.6% reduction)
Test area 2: 5.2 x 10 ⁵ CFU/mL (56.6% reduction)
Test area 3: 1.6 x 10 ⁵ CFU/mL (86.6% reduction)
Test area 4: 1.8 x 10 ⁵ CFU/mL (85.0% reduction)
Test area 5: 6.2 x 10 ⁵ CFU/mL (48.3% reduction)
Test area 6: 2.5 x 10 ⁵ CFU/mL (79.1% reduction)
Test area 7: 1.8 x 10 ⁵ CFU/mL (85.0% reduction)
Test area 8: 1.1 x ¹⁰⁶ CFU/mL (8.3% reduction)
Test area 9: 1.1 x ¹⁰⁶ CFU/mL (8.3% reduction)
Test area 10: 1.0 x 10 ⁶ CFU/mL (16.6% reduction)
The above values are the average values of three trials.

Table 17 is shown. Note that this table combines Tables 15A, 15B, and 16 for easier understanding.

(G) Discussion As a result of the above test, the test materials were confirmed to be effective in reducing the number of Porphyromonas gingivalis bacteria, and it was determined that a reduction of 8.3 to 55.8% was achieved within 60 seconds of contact with each test material.

The present invention inhibits the growth of and kills multiple types of bacteria present in the oral cavity, respiratory tract, and upper digestive tract that cause oral diseases such as tooth decay and periodontal disease. It is also effective in inactivating various viruses, including epidemic influenza, parainfluenza, acute viral bronchitis and pneumonia, measles, and the novel coronavirus (SARS-CoV-2) that causes COVID-19. Furthermore, it is remarkably effective when left in the oral cavity for just 15 to 60 seconds. This makes it possible to provide oral hygiene products whose active ingredients are natural food ingredients and extracts thereof, whose safety has been confirmed through human consumption and ingestion over many years.

What's important about the relationship between seaweed and seaweed extract, which are the main ingredients of the oral hygiene product of the present invention, is that the sour taste of citric acid stimulates saliva secretion when ingested. Furthermore, when ingesting citric acid, its taste stimulating effect promotes saliva secretion. Furthermore, saliva has digestive functions, oral self-cleaning functions, oral mucosal protection, pH regulation, and even antibacterial and antiviral effects.

The seaweed powder, which is the main ingredient of the oral hygiene product of the present invention, is prepared by dissolving it in saline or physiological saline (tested at a weight ratio of 0.9%). This makes it suitable for everyday use as a food additive, ideal for adding umami flavor to foods such as candy and kelp tea, as well as to prepared foods.

In addition, kelp typically contains around 5% fucoidan, a mucilaginous polysaccharide, and it is expected that the previously confirmed fucoidan (F, U, and G types) will promote the production of influenza-specific secretory Ig antibodies in the human trachea and other areas.

Furthermore, the oral hygiene product of the present invention is expected to have secondary effects against periodontal disease, a bacterial infection caused by periodontal bacteria in periodontal plaque that causes inflammation of the gums and destroys surrounding tissue.Recent research papers have shown that this is a contributing factor to diabetes, brain disease, and heart disease.

Claims (11)

A toothpaste and hygiene product for the oral cavity, mouthwash, nasal cavity or throat that contains kelp powder as an active ingredient and is to be left in the oral cavity, nasal cavity or throat for at least 30 seconds in order to prevent or reduce periodontal disease caused by Porphyromamonas gingivalis. 2. The sanitary product according to claim 1, wherein the physiological saline solution contains 5% by weight of kelp powder having a particle size of 5 μm . A toothpaste and hygiene product for the oral cavity, mouthwash, nasal cavity or throat that contains fucoidan powder as an active ingredient and is to be left in the oral cavity, nasal cavity or throat for at least 30 seconds in order to prevent or reduce periodontal disease caused by Porphymonas gingivalis . 4. The sanitary product according to claim 3 , wherein the physiological saline solution contains 4% by weight of fucoidan powder . A toothpaste and hygiene product for the oral cavity, mouthwash, nasal cavity or throat that contains laminaran powder as an active ingredient and is to be left in the oral cavity, nasal cavity or throat for at least 30 seconds to prevent or alleviate periodontal disease caused by Porphyromonas gingivalis. 6. The sanitary product according to claim 5 , containing 2% by weight of laminaran powder in a saline solution . A toothpaste and hygiene product for the oral cavity, mouthwash, nasal cavity or throat that contains iodine powder as an active ingredient and is to be left in the oral cavity, nasal cavity or throat for at least 30 seconds in order to prevent or alleviate periodontal disease caused by Porphymonas gingivalis . 8. The sanitary product of claim 7 , comprising 2% by weight of iodine powder in a saline solution . The hygiene product according to claim 2, 4, 6 or 8, wherein the toothpaste comprises a paste, liquid or powder, and the oral hygiene product and the mouthwash comprises a dental rinse, a mouthwash and an anti-inflammatory agent for oral cavity. The hygiene product according to claim 2, 4, 6 or 8, wherein the nasal hygiene product includes nasal drops and nasal spray liquid, and the throat hygiene product includes a troche or throat spray that is dissolved in the mouth for use . 9. The hygiene product according to claim 2, 4, 6 or 8 , wherein the oral or throat hygiene product includes throat lozenges, chewing gum or pacifier food that remain in the mouth for a certain period of time to stimulate saliva production .